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New sides of an old factor: TFIIA Function and Regulation

Een wetenschappelijke proeve op het gebied van de Natuurwetenschappen, Wiskunde en Informatica

PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen, op gezag van de Rector Magnificus Prof. dr. C.W.P.M. Blom, volgens besluit van het College van Decanen in het openbaar te verdedigen op maandag 31 october 2005 des namiddags om 1.30 uur precies

door

Torill Høiby

geboren op 23 November 1972 te Kongsvinger, Noorwegen

1

Promotor:

Prof. dr. H. G. Stunnenberg

Manuscriptcommissie:

Prof. dr. W. W. de Jong Prof. dr. H. T. Timmers (UMCU) Prof. dr. G. J. Adema

ISBN: 90-9019925-X Omslag: I. Høiby en F. Grytten

2

CONTENTS

CHAPTER 1

GENERAL INTRODUCTION

5

CHAPTER 2

TFIIA BEFORE AND AFTER

45

CHAPTER 3

THE CONVEX SURFACE OF TBP IS ESSENTIAL FOR TAC FORMATION

67

CHAPTER 4

CLEAVAGE AND PROTEASOME-MEDIATED DEGRADATION OF THE BASAL TRANSCRIPTION FACTOR TFIIA

85

CHAPTER 5

TFIIA IS A SUBSTRATE FOR TASPASE1, A CHROMATIN-ASSOCIATED PROTEASE

113

CHAPTER 6

REGULATION OF TFIIA DEGRADATION BY THE ARGININE tRNA TRANSFERASE

137

CHAPTER 7

SUMMARY AND GENERAL DISCUSSION

151

SAMENVATTING

163

ACKNOWLEDGEMENTS

169

CURRICULUM VITAE

171

3

4

1 GENERAL INTRODUCTION The eukaryotic transcription machinery

5

CHAPTER 1

1.1 Evolutionary perspectives of transcriptional regulation In the early nineties, the H. sapiens genome was estimated to encode around 100,000 genes, and this extensive number was used to explain the apparent difference in complexity between humans and simpler organisms like yeast (S. cerevisiae, 6,000 genes) and fruitfly (D. melanogaster, 13,000 genes). The estimated number of human genes has later consecutively decreased from 100,000 to 80,000 to 40,000. The latest reports from the human genome project (end 2003) estimate that a human genome contains 20,000-25,000 genes. Not only is this number four to five times lower than the original estimate but it also instigates the somewhat sobering idea that we are, from a genomic perspective, not far from either D. melanogaster or roundworm (C. elegans, 9,000 genes). Obviously, gene numbers alone do not reflect the sophistication of nature; other factors must explain how a modest increase in gene number from yeast to human can create such a different physiology. The human genome has been called “the blueprint of life” because it carries all information necessary for a single fertilized egg cell to transform into a human being. Though all cells in one organism contain identical “blueprints”, cells are able to develop and specialize because the transcription regulation machinery controls the precise expression of a subset of these genes in each cell. In effect, some genes (house-keeping genes) are expressed in all cells all the time, providing common routine metabolic functions. Other genes are expressed as a cell enters a particular pathway of differentiation or stage in the cell cycle and yet others are continuously expressed in a cell that has differentiated into a specialized state. Lastly, genes can be expressed as a direct consequence of a conditional change, for instance upon the presence of a hormone. Needless to say, one would expect a highly complex and dynamic machinery necessary to conduct such an elaborate task.

6

GENERAL INTRODUCTION

1.2 Historical preview Thirty years ago, Jacob and Monod described how genes are selectively switched on and off in bacterial cells1. According to their paradigm for gene regulation, trans-acting regulatory proteins interpret and mobilise specialized DNA sequences called cis-regulatory elements. Human cells share considerable functional and structural characteristics with simpler organisms, and genes of eukaryotic organisms are controlled largely the same way as in prokaryotic cells, although in eukaryotic cells cis-regulatory DNA sequences are named enhancers and silencers whereas trans-acting regulatory proteins are collectively termed transcription factors. The two largest eukaryotic RNA pol II subunits, Rpb1 and Rpb2, are homologous to the β and β’ subunits of bacterial polymerase, whereas a third subunit, Rpb3, is related to the α subunit of bacterial polymerase, and although there is no eukaryotic equivalent to the bacterial σ subunit family, structural and functional similarities between σ and certain general transcription factors have been postulated. In contrast to prokaryotes, multi-cellular eukaryotes comprise hundreds of different cell types, performing a specific role that contributes to the overall being of the organism. To facilitate this, eukaryotes possess, compared to prokaryotes, a huge number of transcription factors and accessory factors, cofactors, modifying enzymes and bridging factors.

1.3 The core promoter Core promoters are defined as the minimal DNA sequences required to recruit the appropriate RNA polymerase and initiate transcription2, and can be divided into core elements and regulatory elements. Core promoter elements define the site for

7

CHAPTER 1

TATA

Inr

DPE

Figure 1: A classical RNA pol II core promoter

assembly of the preinitiation complex (PIC) and a classical core promoter for RNA pol II can include a TATA box (at –20 to –30 relative to the transcription start site), an initiator element (at the transcription start site) and a downstream promoter element (DPE at +30 relative to the transcription start site) (figure 1). Regulatory elements control the rate of transcription initiation in a gene-specific manner, either from near-by start sites (upstream activating and repressing sequences) or from great distances (enhancers and silencers).

1.4 The basal transcription machinery Eukaryotic transcription is carried out by three polymerases; RNA polymerase I (RNA pol I) which is responsible for transcription of rRNA genes, RNA polymerase II (RNA pol II) transcribing all protein-coding genes and most snRNAs, and finally RNA polymerase III (RNA pol III), transcribing 5S rRNA, tRNAs and the U6 snRNA2,3. The earliest in vitro transcription experiments that were performed in the late 1970s and early 1980s showed that RNA pol II is unable to recognise promoter sequences and initiate RNA synthesis by itself4,5, and this observation provided the basis for characterizing the general transcription machinery3,6,7. Originally, the general transcription factors that reconstituted efficient selective transcription by purified RNA pol II in vitro were identified by fractionation of cellular extracts and include TFIIA, TFIID, TFIIE, TFIIF and TFIIH (see table 1). TFIID, which in fact is a multi-subunit complex comprising TATA-binding protein (TBP) and more than ten TBP-associated factors (TAFs) can bind directly to the TATA box and nucleates formation of the initiation competent complex8-10. In 8

GENERAL INTRODUCTION

addition to TBP binding to the TATA box, certain TAF subunits can interact directly with initiator or downstream promoter elements. Whereas TBP is sufficient for basal in vitro transcription, it must be complemented by the TAFs to respond to transcriptional activators (see 1.5). Table 1 Human class II general transcription initiation factors Factor Subunits TFIID TBP

Function

1

TAFs

Core promoter recognition TFIIB recruitment Core promoter recognition/selectivity (non-TATA elements), regulate TBP function, co-activator, kinase, ubiquitylase and acetylase activities Stabilising TBP-DNA interaction Anti-repressor and co-activator Stabilising TBP-DNA interactions, recruiting TFIIF/RNA polII, start site selection Recruiting RNA polII to DNA-TBP-TFIIB complex, destabilizing non-specific RNA polII-DNA interactions, facilitates RNA polII elongation RNA polymerase (pre-mRNA synthesis), recruiting TFIIE, CTD domain, interacts with Mediator complex, Elongator complex and processing factors TFIIH recruitment, TFIIH helicase ATPase and kinase modulating actitvity, Promoter melting Promoter melting, helicase, CTD kinase, role in nucleotide excision repair 2,3,11,12 Taken from

14

TFIIA

3

TFIIB

1

TFIIF

2

RNA pol II

12

TFIIE

2

TFIIH

9

TFIIA was initially identified as one of the general transcription factors but was later found to be dispensable for basal transcription2,11,13-18. Partially purified systems that were used to categorize the general transcription factors contained negative factors like NC1 or NC2 and may have necessitated the action of TFIIA to reverse their inhibitory effect on basal transcription3. TFIIA is now known to displace transcription repressors like NC2/Dr1-DRAP1, PC3/Dr2, HMG1 and BTAF1 and stabilize TBP-DNA association (see figure 2), and it can function as a co-activator for a number of activators. In highly purified systems, TFIIA is therefore dispensable because these systems are presumably devoid of regulators that are normally antagonized by TFIIA, in contrast to systems assessing crude extracts where TFIIA is essential for full transcriptional activation. However, TFIIA, like the TAFs, has many

9

CHAPTER 1

characteristics of a co-activator rather than a basal transcription factor. TFIIA is discussed in detail in chapter 2.

Figure 2: Crystal structure of TBP and TFIIA binding to the promoter19,20

TFIIB enters the PIC after TFIID and TFIIA and is a pre-requisite for RNA pol II recruitment in vitro21. It interacts with TBP and binds to DNA upstream and downstream of the TATA box22, is a target for a number of activators and is probably involved in start site selection23,24. TFIIB also interacts with and recruits RNA pol II and TFIIF. TFIIF is in many ways reminiscent of the bacterial σ factor; it binds tightly to RNA pol II and prevents non-specific binding of the polymerase to DNA, in addition to having an overall stabilizing effect on the PIC25-27. TFIIF also suppresses transient pausing of the polymerase and thus operates as an elongation factor28. Even with RNA pol II stably bound to the promoter, transcription can not be initiated before the last two general transcription factors, TFIIE and TFIIH, join the PIC and provide open complex formation, i.e. promoter melting. The function of TFIIE is closely linked to TFIIH function, recruits and stimulates its activity. TFIIH consists of nine subunits, including a DNA-dependent ATPase, two DNA helicases and a CTD kinase25-27. TFIIH is essential for promoter opening in vitro and both TFIIE and TFIIH are required for the ATP-dependent formation of the open promoter complex prior to formation of the first RNA phosphodiester bond. In addition, TFIIE, TFIIH and TFIIF cooperate to suppress promoter-proximal stalling, thus facilitating the transition of RNA pol II to productive elongation29.

10

GENERAL INTRODUCTION

A basal transcription factor was originally defined as “a factor that is important for initiation of transcription from a core promoter by RNA pol II”, whereas a general transcription factor was “essential for initiation of transcription at all promoters”30. From these definitions follows that all general transcription factors are basal factors, but the use of these terms today is more complicated than the original models prompted. Despite the name, it appears that many general transcription factors do not necessarily function at all genes in vivo and several other members of the transcription apparatus are as generally employed as the general transcription factors, as will be discussed later.

Figure 3 Assembly of the preinitiation complex on the promoter. Originally, the general transcription factors necessary for selective transcription by purified RNA pol II included TFIIA, TFIID, TFIIE, TFIIF and TFIIH. (See text for details)

1.4.1 The TAFII components of the TFIID complex Whereas purified TBP stimulates basal and not activated transcription in cellfree systems, the TFIID fraction mediates both due to the so-called TBP-associated factors (TAFs) (see table 2)

8,31-34

. In the context of TFIID, some TAFs can contact

initiator or downstream promoter elements50,51. These interactions are particularly important for promoters lacking a conventional TATA element; in this case the initiator element of the core promoter can be contacted by TFIID subunits dmTAF1 and dmTAF2, whereas the DPE has been shown to interact with dmTAF6 and dmTAF98,52-55. The TAFs are believed to be essential for linking the preinitiation complex to a diversity of transcription factors35, thereby defining the TAFs as coactivators. Direct contact has been demonstrated for example between the activator

11

CHAPTER 1

p53 and dmTAF9/6, VP16 and dmTAF9, SP1 and dmTAF4 and the nuclear estrogen receptor and hsTAF108,56-59. Table 2: The TBP-Associated factors Name

Complex

Characteristics

TAFs with enzymatic activities TAF1

TFIID

Protein kinase, phosphorylates Rap74 and TFIIA Acetylates H3 and H4 in vitro Contains Bromo domains and HMG boxes Contacts DNA and associates with TBP Cell cycle progression through G1/S boundary Repression of Apoptosis

BTAF1

B-TFIID

ATP-dependent inhibitor of TBP-mediated transcription

TAF3 TAF6

TFIID TFIID, hTFTC, ySAGA

TAF9

TFIID, hTFTC, ySAGA, hPCAF TFIID

Histone fold motif Histone fold motif Contacts TFIIEa, Rap74 and TBP Histone fold motif Contacts DNA, TFIIB, acidic activators Histone like Contacts VP16, TFIIB Histone fold motif Required for the HAT activity of SAGA Histone fold motif

Histone-like TAFs

TAF11 TAF12

TFIID, hTFTC, ySAGA, hPCAF TFIID

TAF13 Others TAF2

TFIID

TAF4

TFIID, hTFTC

TAF4b TAF5

TFIID, hTFTC, ySAGA

TAF7 TAF10

TFIID, hTFTC TFIID, hTFTC, ySAGA

TAF15

TFIID

TAF43

TFIID

Downstream promoter contact (specific) Contacts TBP Q-rich contacts DNA, hTFIIA, SP1, NFATp Specific for gonads WD40 repeats Contacts DNA and Rap74 promoter-specific and complex stabilizing function contacts DNA (hTAF55) and many activators contacts the estrogen receptor Progression through the G1/S boundary Repressor of Apoptosis RNA or ssDNA-binding domain contacts hRNAP might have a role in driving the preinitation complex in an open conformation and in RNA chain initiation

Taken from

8-10,35-49

TAF1 is the largest TAF and has historically been regarded as the key scaffold upon which the other TAFs assemble, because D. melanogaster, S. cerevisiae and human TAF1 bind both TBP and several other TAFs in vitro39. Amongst the many known protein domains harboured by TAF1 are two bromodomains40 that bind acetylated lysines, two kinase domains reported to phosphorylate Rap74 (TFIIF) and TFIIA37,47, a histone acetyl transferase domain that can acetylate histones H3, H4 and H2A41 and a Ubiquitin-activating/conjugating activity ubiquitylating histone H144. This considerable number of enzymatic activities is consistent with a broad role of

12

GENERAL INTRODUCTION

TAF1, which has been associated with regulation of the cell cycle, cell differentiation, cell proliferation and cell survival46,60,61. Studies have shown that the presence of TAF1 can decrease activated transcription in a promoter-dependent manner, and that TAF1 and (co-)activators like VP16 or TFIIA compete for binding to TBP38,62-65. It has been known for a time that TAFs are essential for basal transcription in vitro from TATA-less promoters33,35. In yeast, two active forms of TBP, one TAFdependent and one TAF-independent, have been shown to be involved in transcription66,67. These results suggested that the TAFs are not involved in transcription on all promoters, and genome-wide studies in yeast have estimated that TFIID pre-dominates at ~90% of the promoters68.

1.4.2 TAF-containing complexes without TBP According to their definition, TAFs exist in association with TBP, but the validity of the definition of TAFs has been challenged by the identification of the TBP-free-TAF containing complexes like hTFTC, hPCAF, hSTAGA and ySAGA42,49,69,70. These discoveries suggested that TBP might be dispensable for transcription at least on some promoters. Common to these complexes are intrinsic HAT activities, they contain homologues of ADA (adaptor) proteins and SPT (suppressor of Ty insertion) proteins and subsets of TAFs but no TBP. The hTFTC, ySAGA and hPCAF components were shown to be essential for in vitro and in vivo activated transcription, but not much is known about the mechanism of their function. They might change the acetylation state of chromatin by their intrinsic HAT activity, thereby potentiating initiation and activation of transcription (see 1.5.2.1), and secondly, they might contact TBP or other general transcription factors and coactivators such as p30036.

13

CHAPTER 1

Recent studies addressing the roles of TFIID and SAGA in transcriptional regulation in yeast found that mutation of shared SAGA and TFIID subunits affected as much as 99% of all yeast genes68. TFIID predominates at 90% of the measurable promoters and is responsible for transcribing house-keeping and non-regulated genes, whereas SAGA predominates at the remaining 10% at what appears to be largely stress-induced genes. Though some gene targets may be shared between the two complexes, they therefore appear to have separate roles in expression of certain sets of genes.

1.4.3 Diversity in the general transcription machinery Being involved in transcription by all three polymerases, TBP has been considered a universal transcription factor, but this dogma has been challenged by the recent discoveries of other TBP family members. Homologues of other basal transcription factors have subsequently been found as well, demonstrating that higher eukatyotes express cell and tissue-specific core promoter recognition factors that presumably exert their transcriptional effects at separate subsets of genes. The TBP family contains the apparently insect-specific TRF1 that acts in transcription of the tudor gene and the tRNA genes, and that associates with a novel set of associated factors (nTAFs)71,72. The TLF/TRF2 is found in all metazoan genomes examined so far, is required for spermatogenesis in mice73-75 and is essential for early embryogenesis in frogs, worms and fish76-78. A third member of the vertebrate TBP family, TBP2/TRF3, has an essential, specialized role in embryonic gene regulation in fish and X. laevis79,80. Some tissue-specific TAFs have also been identified; TAF4b is exclusively expressed in male germ cells and in granulosa cells of the ovary, and is essential for female fertility81. TAF1L is up-regulated during male germ cell

14

GENERAL INTRODUCTION

development to compensate for the silencing of its somatic counterpart on the X chromosome (i.e. TAF1)82 and dmTAF5 has a spermatocyte-specific homologue in the cannonball gene83. TFIIA also has a vertebrate homologue, the TFIIA-Like Factor (ALF) that is expressed predominantly in testis85-88. An additional complexity in the gene regulation by TFIIA may be gained by the association of uncleaved form of TFIIA αβ/γ in the complex TAC as discovered in P19 EC cells89. Figure 4: Diversity in the general transcription machinery. A variety of transcription complexes may compete for the same target genes or regulate distinct sets of genes. TBP can assemble with the TAFs into the TFIID complex, and some of the TAFs in TFIID can be replaced by variant TAFs. Conversely, both TAFs and related variant TAFs can participate in histone acetyl transferase (HAT) complexes such as TFTC. Both TFIIA and the unprocessed form of TFIIA in the TAC complex are capable of binding TBP. Similarly, TFIIA-like factor (ALF) can substitute for TFIIA in binding to TBP. TBP is not only found in TFIID and TAC, but also in the BTAF1 and NC2 complexes. So far TBP-like factor (TLF) has not been found in TFIID, TAC, BTAF1 or NC2. However, TLF can compete with TBP for TFIIA binding (modified from84).

The model emerging is one in which gene specificity is regulated not only by enhancer-binding factors but also by extensive variation within the basal transcription machinery. In contrast to S. cerevisiae, many of the general transcription factors in higher eukaryotes have diverged considerably, likely reflecting the greater complexity of gene regulatory pathways.

1.5 Activation and Repression of transcription Gene-specific transcriptional activation is generally effected by the binding of transcriptional activators to upstream activating sequences (see 1.3) where they recruit

15

CHAPTER 1

or regulate activities of co-activators and the basal transcription machinery. Regulation of transcription often involves interplay between activators and repressors25 and genes may be regulated by mixing and matching different types of activators and repressors in a coordinated fashion. The activators can function in several manners90; some (for example Gal4, Pho4, E2F-4 and Swi5) can bind to the packaged nucleosomes and perturb chromatin structure by recruiting chromatin remodelers activator, some (for example the E2Fs and SBF) can bind nucleosomes only after their conformational change. Many activators (for example Rap1) can recruit basal transcription factors directly. Co-activators are required for transcriptional activation but are dispensable for low-level basal transcription in vitro, and they do not bind DNA specifically91 as opposed to activators. Generally, co-activators are divided into four groups and the first group includes TFIIA and the TAFs that function by bridging activators and the preinitiation complex (1.4).

The second group is composed of the Mediator

complexes, the third group contains the histone modifying complexes and finally the fourth group includes the ATP-dependent remodelers (see below).

1.5.1 The Mediator Complex In the search for factors that could imitate the in vivo effects of activators in an in vitro setting, Kornberg and co-workers discovered the yeast Mediator complex that was required for stimulation of transcription in highly purified systems92,93. The Mediator complex transduces both negative and positive regulatory information from gene-specific activators and repressors to the basal transcriptional machinery94,95. The metazoan Mediator complexes have been reported to be holoenzymes with components ranging from DNA repair proteins to splicing and polyadenylation

16

GENERAL INTRODUCTION

factors96,97, in addition to homologues of the yeast SRB/Mediator proteins98. Purification methods generally produced two predominant complexes; one large complex variously named TRAP, DRIP, ARC, SMCC or NAT99-104 and a smaller complex named PC2/CRSP91,105. Both complexes were shown to mediate activatordependent transcription in vitro, and the NAT and SMCC complexes were additionally shown to repress activator-dependent as well as basal transcription106,107. The best-described metazoan Srb/Mediator-like complex is SMCC which was isolated from human cells through epitope-tagged components of the complex. SMCC contains 25 polypeptides including human homologues of yeast SRB10, SRB11, SRB7, MED6, MED7, NUT2 and RGR1 as well as TFIIB and subunits of RNA pol II. In addition, SMCC contains TRAP240, TRAP230, TRAP220 and several other subunits found in the thyroid-hormone-receptor-associated protein co-activator complex TRAP that was purified on its ability to mediate activation with thyroid hormone receptor100,101. The composition of metazoan (as well as yeast) Mediator thus varies according to the biochemical tools and the strategy used for its isolation, and the cell type and growth conditions as well as promoter-specific requirements may influence the composition. The functional significance of the range of documented Mediators is still unclear. This issue was recently addressed in a study where HeLa cell lines expressing FLAG-tagged subunits of several Mediator subunits were analysed by multi-dimensional protein identification technology108. All the isolated forms of Mediator were found to contain a consensus set of subunits, in addition to a variety of certain subunits, suggesting functional differences in the complexes. For example, a Mediator complex lacking the Med220 subunit is unable to serve as a co-activator at a subset of promoters109.

17

CHAPTER 1

Mediator was initially believed to function by recruiting the preinitiation complex to the promoter. This is supported by observations from yeast where the Mediator is required for stability of preinitiation complexes that are dependent on a functional activator protein66,67,110,111 and Mediator–dependent activators like Gal4 and GCN4112,113. The activator-dependent association of human Mediator to the promoter in vivo bridges the activator to members of the basal RNA pol II machinery114, and interaction of a number of activators with Mediator has directly been shown to stimulate preinitation complex assembly on the promoter115. On the other hand, in yeast the association of Mediator and RNA pol II occurs independently at the HO promoter and at other cell cycle regulated promoters like CLN1, CLN2 and PCL1114. This has lead to the suggestions that the Mediator performs different roles at different promoters. Several functions have been suggested; purified yeast Mediator stimulates the RNA pol II CTD-kinase activity of TFIIH, facilitating the transition from initiation to elongation106 and Mediator may contribute to recruiting other transcription co-activators like chromatin remodeling complexes, histone acetylase and methylase complexes and elongation factors116,117. Recently, it has been shown that Mediator functions at a post-recruitment step by enhancing the rate of transcription initiation by a preinitiation complex118. The mechanism behind is not clear but is suggested to be promoter melting or promoter clearance.

1.5.2 Chromatin as a substrate for transcriptional regulation An important factor for the overall outcome of transcription regulation is the structure in which the chromatin is stored in a eukaryotic cell. The basic unit of chromatin is the nucleosome particle, containing 147 bp of DNA wrapped nearly twice around an octamer of core histones119. The genetic material containing all

18

GENERAL INTRODUCTION

information necessary for development, differentiation and cell plasticity is thus packaged to fit the limited confines of the cell nucleus, and this dense, non-accessible substrate represents a significant barrier to the basal transcription machinery120,121. To facilitate access to DNA, eukaryotes have evolved multi-subunit protein complexes that alter chromatin structure by either covalently modifying nucleosomes or utilising ATP to remodel nucleosomes and mechanically restructure chromatin (SNF2-type ATPases)19,20,90,120-123.

1.5.2.1 Covalent modifications Histones are subjected to a large number of post-translational modifications, including acetylation, methylation and ubiquitylation of lysines, methylation of arginines and phosphorylation of serines, tyrosines and threonines. The best studied is acetylation, the transfer of an acetyl side chain from an acetyl-Coenzyme A moiety to a lysine. This transfer effectively neutralizes the positively charged lysine, possibly reducing its affinity for the DNA backbone and releasing the compactness of the nucleosomal arrays by disrupting the inter-nucleosomal interactions made by the histone tails. In addition, by establishing different combinations of histone tail modifications (the ‘histone code’), different proteins recognizing such signature through for example bromodomains (acetylated residues) or chromodomains (methylated residues) can be recruited121,124-126. The first activator of transcription found to possess histone acetyl transferase (HAT) activity was the yeast Gcn5p127,128, and this was later found to be the catalytic subunit of the yeast complexes ADA and SAGA129 (see table 3). Besides this, yeast contains many other HAT containing complexes, like the NuA3, NuA4 complexes and the SAS complex. As this multitude of different HAT complexes already suggests, HATs are required for a broad

19

CHAPTER 1

spectrum of biological functions ranging from transcriptional activation and silencing to DNA damage response and chromosome segregation69,99,128,130,131. Mammalian cells contain at least two proteins related to yGCN5; p300/CBPAssociated Factor (PCAF) and hGCN5. Both human proteins have been shown to play key roles in transcriptional activation, for example for the function of p53 mediated activation in response to DNA damage and MyoD-mediated activation during muscle differentiation132. The requirement of hPCAF as a histone acetyl transferase and transcriptional co-activator has been described, among others, for myogenesis133, growth factor signaling pathways134 and nuclear receptor mediated transcription135. In addition to acetylating histones, hPCAF has been shown to acetylate proteins like the HMG17136, HMGI(Y)137 and p53138 and the general transcription factors TFIIE and TFIIF139. Evidence is mounting arguing that the ‘histone code’ is not restricted to histones, but that it in fact includes many other proteins that are subjected to posttranslational modification signatures, introducing the more general term ‘protein code’132,140-142. Table 3: Representative HAT complexes and their composition HPCAF hTFTC ySAGA

yADA

yNuA4

yNuA3

PCAF

GCN5

GCN5

Esa1

Sas3

Ada2 Ada3

Ada3

Spt3

Spt3

PAF400 TAF5L TAF12 TAF6L TAF10 TAF9

TAF2 TAF4 TAF5 TAF6 TRRAP TAF5L TAF12 TAF10 TAF9

GCN5 Ada1 Ada2 Ada3 Ada5/Spt20 Spt3 Spt7 Spt8

Ada2 Ada3 Spt16

Tra1 TAF5 TAF12 TAF6 TAF10 TAF9 Sin4

Tra1 TAF14

Ahc1 Act3/ARP Act1 Ep11 Eaf3 Taken from

20

120

GENERAL INTRODUCTION

Other fundamental HAT-containing proteins are CBP and p300; these proteins are highly homologous and often considered a unity. CBP/p300 is ubiquitously expressed and has critical roles in cellular processes like heart, lung and small intestine formation, cell cycle control, differentiation and apoptosis143,144. Like hGCN5, CBP/p300 can act as a co-activator for a wide variety of transcription factors including nuclear receptors and other activators like c-Fos, c-Jun and c-Myb. CBP/p300 affects transcription by serving as molecular scaffolds to bridge activators to co-activators and RNA pol II, by acetylating histone tails or they can acetylate transcriptional activators to directly affect their activity145. Accumulating evidence indicates that CBP and p300 are not completely redundant but also have unique roles in vivo, may be due to association with different proteins or slightly different substrate specificities146. Besides acetylation, histone phosphorylation has been shown to have a role in transcriptional induction of immediate early genes in mammalian cells like c-Fos147. Phosphorylation of H4Ser1 may have a role during DNA damage148 and the DNA damage checkpoint kinase ATM is recruited to a DNA double-break where it phosphorylates H2AX in mammals121. Histone phosphorylation of H3Ser10 has a dual function and has been shown to be involved in both transcriptional activation and chromosome condensation during mitosis149-151. In line with the proposed complexity of the ‘histone code’, also histone methylation of lysine and arginine residues has been linked to both activation of transcription and silencing of chromatin152,153. Transcriptionally active euchromatin can

be

methylated

at

H3Lys4,

H3Lys36,

and

H3Lys79,

and

histone

methyltransferases move along with elongating RNA pol II complexes, thus spreading H3Lys36 methylation through transcribed open reading frames154. In contrast,

21

CHAPTER 1

transcriptionally repressed euchromatin can be methylated at H3Lys27 and H4Lys20, as well as H3Lys9154. Recently, several groups demonstrated that the yeast Set1 and Set2 enzyme complexes that mediate histone lysine methylation functionally interact with the PAF complex, a specific elongating RNA pol II complex155,156. In yeast, this interplay includes another covalent histone modification, namely ubiquitylation of lysine residues. It was shown that recruitment and activity of the Rad6-Bre1 ubiquitin ligase complex ubiquitylates H2BLys123 and this modification was required for sequential recruitment of the 19S proteasomal cap, the PAF complex and the various SET complexes. Histone ubiquitylation most likely can regulate gene transcription in a positive and negative fashion, depending on its genomic location and timing of occurrence. The ubiquitin hydrolase Ubp8 is a stable component of SAGA and biochemical and genetic evidence indicates that Ubp8 targets H2B for deubiquitylation prior to transcriptional initiation. The dynamic balance of H2B ubiquitylation/deubiquitylation was shown to be important for GAL1 transcription and further, this balance of ubiquitylation appears to set the balance of histone H3 methylation at Lys4 relative to Lys36157. Both phosphorylation and methylation of histone tails have been shown to regulate HAT activity and crosstalk among the different histone marks appears to be extensive, not only between adjacent modifications in the same histone tail, but also inter-nucleosomal158. Most known histone modifications can be enzymatically removed. Complementary to the HAT complexes, cells contain protein complexes like Sir2, Sin3/HDAC or the Mi-2/NuRD complex and the complexes containing the NCoR/SMRT that are able to deacetylate nucleosomes159,120. As with the HAT complexes, the different subunit compositions of the HDAC complexes imply that they are involved in separate biological functions and that they have different

22

GENERAL INTRODUCTION

substrate specificities. The HDAC complexes have been shown to be targeted to specific promoters by repressors, like the recruitment of yeast Sin3-Rpd3 complex by the transcription repressor Ume6p160. The Sin3/HDAC complex can be recruited by the Mad1 protein161, while the N-CoR/SMRT complex is recruited by unliganded nuclear hormone receptors162. N-CoR and SMRT can interact with nuclear receptors and have been implicated in repressing activities of factors like SRF, AP-1 and NFκB163 as well as the POU homeodomain factor Pit-1164,165 and the bHLH protein MAD161,165. HDACs can deacetylate core histones in vitro and in vivo166,167 and several observations suggest that the deacetylase activity of HDACs is responsible for the transcriptional repression; single amino acid mutations in the catalytic core of the HDACs RPD3 and HDAC1 demonstrated a direct correlation between its enzymatic activity and its repressive ability168-170. Additionally, nuclear receptor dependent repression can be inhibited by the deacetylase inhibitor trichostatin A130,171. It has been shown that whereas the Sin3/HDAC complex can deacetylate both histone H3 and H4, the N-Cor/SMRT complex only deacetylates H3, demonstrating that the different complexes have distinct histone tail specificities172. Analogous to HDACs, a histone demethylase LSD1 has recently been identified173. LSD1 is found to be a transcriptional co-repressor participating in the silencing of neuron-specific genes and has specificity for histone H3Lys4. Thus, like acetylation, methylation appears to be a dynamic process that is regulated by a balance between HATs and HDACs, methylases and demethylases, kinases and phosphatases and so on.

23

CHAPTER 1

1.5.2.2 ATP-dependent Chromatin Remodeling Complexes Complexes like ySWI/SNF, yRSC, dNURF, dCHRAC, dACF, dBrahma, hSWI/SNF, hNURD and hRSF represent the fourth and last group of co-activators174. These chromatin remodeling complexes function by promoting nucleosome disruption or displacement in an ATP-dependent manner and can alter the structure of large segments of chromatin. In yeast, the known nucleosome remodelers are grouped based on the functional domains present in their catalytic subunit resulting in three main groups; the SWI/SNF group is characterized by a C-terminal bromodomain (Swi2/Snf2 and Sth1), the ISWI group containing a SANT domain (Isw1 and Isw2), the Mi-2 group harbouring a chromodomain (Chd1), leaving Ino80p, Swr1p, Rad54p, Rdh54p and Yfr038wp116,119,120,122,129,175-177. Genome-wide expression studies using mutant of the well-characterized ySWI/SNF complex revealed that this complex is a key transcriptional regulator involved in activation or repression of 6% of yeast genes178 including a subset of highly inducible genes179. Furthermore, it is known that ySWI/SNF functionally collaborates with the SAGA complex to drive transcription of many (mitotic) genes, showing that ATP-dependent nucleosome remodelers and histone modifying complexes seamlessly cooperate. This intersection extends beyond mere recruitment of ATP-dependent chromatin remodelers by PTMs deposited by HATs and SETs, as examples have been described (such as the homothallic switching endonuclease HO180) where the ATP-dependent remodelers precedes and is required for recruitment of HATs prior to transcriptional activation. A diversity of transcription factors like the glucocortoid receptor, Myc, MyoD, HSF-1 and C/EBPb have been shown to recruit hSWI/SNF to specific promoters122,181. Further, the D. melanogaster SWI/SNF-like Brahma complex appears to be required for the Wingless signaling pathway and the E2F cell-cycle

24

GENERAL INTRODUCTION

regulator function182,183 and hSWI/SNF is required for full function of several activators, including the glucocorticoid receptor184, the estrogen receptor185 and the retinoid receptors186. Intriguingly, mammalian SWI/SNF complexes appear to be essential for mouse development187,188 and expression of key pRb-dependent cell cycle regulators189 and contain the known tumour suppressors Brg1 and Snf5. Mice heterozygous for Brg1 or Snf5/Ini1 deletions are prone to a variety of tumor types, including glandular epithelial tumors and malignant rhabdoid tumors187,188,190 and it has been shown that mutations in hSNF5 induce chromosome loss and polyploidy191. Other ATP-dependent chromatin remodelers play key roles in transcriptional regulation as well, as exemplified by the repressor Ume6p recruiting yeast Isw2p192 or the recruitment of Mi-2/NuRD by the DNA binding transcription factor Ikaros of the NuRD complex to heterochromatin regions upon T cell activation193, proposed to maintain an inactive chromatin state. Besides well-established roles in transcription regulation, ATP-dependent nucleosome remodelers play roles in a wide range of physiological functions, ranging from histone deposition (Isw2, Swr1) to DNA damage response (Ino80, Sth1, Rad54, Rdh54) and chromosome segregation (Sth1)119,131,160,176,177,188,190,191,194.

1.5.3 Other repressors of transcription Repressors of transcription can functionally be divided in two classes, and one class is functionally linked to chromatin and includes histones, histone-related proteins, histone deacetylases and histone demethylases (discussed in chapter 1.5.2). The other class operates through promoter elements and affects TBP binding, and includes for example BTAF1, which represses transcription at a subset of genes by binding TBP-DNA complexes and causing TBP to dissociate from the DNA43.

25

CHAPTER 1

BTAF1 (hTAFII170/Mot1) was identified in a screen for mutants with enhanced basal transcription and is an ATP-dependent inhibitor of TBP binding; however Mot1 has also been shown to function as an activator195. Another example is Dr1-DRAP1/NC2 which is a general negative regulator of class II and class III genes, and functions by preventing TBP binding to TFIIA and TFIIB and thus assembly into a preinitiation complex196.

1.5.4 The ubiquitin system and transcription Eukaryotes contain a highly conserved multi-enzyme system that covalently links ubiquitin to a variety of proteins with degradation signals (degrons) recognized by this system, followed by degradation of the tagged protein in the 26S proteasome. This system has mainly been associated with cellular degradation of discarded proteins, but an increasing compilation of data suggests that it also is linked to transcription and regulation thereof. A protein substrate, recognised through a degron, is conjugated to Ubiquitin through the mechanism of the three enzymes E1, E2 and E3197. UBR1, an E3 of the N-end rule pathway, can recognize both the so-called N-degrons198 and internal (nonN-terminal) degrons199. Most degrons are poorly defined, but amongst the known ones are N-end rule degrons200,201 cyclin destruction boxes202 and regions rich in proline, glutamic acid, serine and threonine (PEST sequences)203. Destabilising N-terminal amino acids may be generated through proteolysis and involve modifications like deamidation of asparagine and glutamine or arginylation of aspartic acid, glutamic acid or cysteines. The N-end rule has a hierarchical organization, where Asp (and Glu and Cys) are secondary destabilizing residues that must undergo conjugation by arginine-

26

GENERAL INTRODUCTION

tRNA transferase (ATE1) to Arg, one of the primary destabilising residues to be recognized by the ubiquitin ligase204-206.

Figure 5: The N-end rule pathway for the type 1 substrates Asp and Glu. N-terminal residues are denoted by single letter abbreviations for amino acids. The ovals represent the rest of a protein substrate. Primary 204-206 destabilising residues are recognised by functionally overlapping E3s that include UBR1 and UBR2 .

Ubiquitin-dependent pathways have been shown to play major roles in numerous processes, including cell differentiation, the cell cycle progression, embryogenesis, apoptosis, signal transduction, DNA repair and stress responses197,207. In addition, several pieces of evidence suggest that the transcription process is closely linked to the cellular protein degradation machinery. The largest subunit of RNA pol II is ubiquitylated during transcription in vitro208,209 and the yeast 19S proteasome is required for efficient transcription elongation by RNA pol II210. Ubiquitin mediated processing of p105211,212, IκBα (168), the SREBP family, VP16, the estrogen receptor and Ci-155213-217, has been shown to regulate the transcriptional activity of these factors. Importantly, many transcriptional regulators are conditionally short-lived proteins, and their cellular levels are largely determined by the rate of proteasomal degradation, rather than the rate of de novo synthesis218. Transcription-dependent degradation of transcription factors constitute a feedback mechanism to regulate the stability of transcription factors and, thereby, the dynamics of the transcriptional responses.

27

CHAPTER 1

1.6 The recruitment models The general transcription factors and RNA pol II were purified and identified as independent, chromatographically distinct factors, and reconstitution of transcriptional activity in vitro occurred by step-wise addition of these factors2,7,11, arguing that one functions after the other. This step-wise model was challenged by the purification of preassembled complexes (holoenzymes) from yeast and human containing subsets of GTFs, Mediator subunits and SWI/SNF chromatin remodeling complex subunits97,219,220. Collectively, these reports suggested that some transcription factors associate with RNA pol II prior to promoter binding. Later, this view was yet again questioned by reports of holoenzyme components being recruited to promoters in the absence of RNA pol II114,221, in addition to the relative abundance of the yeast general transcription factors being more compatible with the step-wise model222. This brings about a central question in eukaryotic transcription regulation; how is the recruitment of the multitude of necessary factors orchestrated? This issue has been addressed by a number of recent studies assessing the architecture of transcription components on various promoters in vivo over time. The now classical example of the HO promoter in yeast shows that the activator Swi5 transiently binds during telophase and recruits SWI/SNF, leading in turn to SAGA recruitment, histone modifications and binding of the activator SBF. Subsequent to that, and after Swi5 removal, SBF recruits the Mediator, followed by the binding of RNA pol II and the GTFs114. The mammalian IFN-β locus, on the other hand, is activated upon recruitment of GCN5, subsequent histone acetylation and binding of RNA pol II/CBP223,224. RNA Pol II/CBP in turn recruits hSWI/SNF, followed by remodeling and binding of TBP, TFIID recruitment and finally transcription initiation. As classical is the mammalian example of estrogen-responsive genes, demonstrating the

28

GENERAL INTRODUCTION

cycling of ER and p300, subsequent histone acetylation and binding of PBP, CBP, PCAF and RNA pol II upon estrogen stimulation225. These types of experiments have also led to a reconsideration of the common view that basal transcription factors and RNA pol II can only associate with nucleosomes after remodeling; on the α1-AT promoter, TBP and TFIIB bind long before the CBP/PCAF complex and the remodeling complex Brm are recruited to the promoter226. The important insight emerging from these studies is that the steps leading to chromatin remodeling and assembly of the preinitiation complex vary extensively, depending on the gene and its biological context. This variability may be interpreted as an additional level of regulation at a particular gene; any given promoter can dictate its individual recruitment patterns, and thereby obtain a more tailored regulation.

Figure 6: Eukaryotic transcription regulation. Mediator facilitates integration of multiple activities into the preinitiation complex formed at the eukaryotic promoter. The Mediator can be recruited by specific promoter and or enhancer bound activators.

Concluding remarks The compilation of data following the identification of RNA pol I, II and III almost four decades ago revealed that eukaryotic transcription is regulated in a manner far more complex than many anticipated. As in bacteria, gene transcription is achieved by factor recruitment, but the mechanism critically depends on the order of the recruited factors, the timing, the histone code and the selection of the available factors in the cell. Nevertheless, the composition of the general transcription 29

CHAPTER 1

machinery is largely conserved between yeast and man, as are the mechanisms by which it functions194. As far as we know at this point, all the difference appears to be gained by a more intricate use of this machinery in humans, the more effective use of activators and signaling pathways as well as differential splicing.

AIM AND OUTLINE OF THIS THESIS The aim of the research presented here was to gain insight into the function of TFIIA cleavage and thus the protein complex TAC. To accomplish this, we undertook to 1) identify the residues of TBP involved in formation of the TAC complex, 2) to identify the cleavage site of TFIIAαβ and 3) characterize it, to 4) identify the protease responsible for cleavage of TFIIA and ultimately 5) understand the function of cleavage of TFIIA. Chapter 1 is a summary of the eukaryotic transcription regulation. Data that has been obtained in the last four decades revealed that eukaryotic gene regulation requires distinct multi-protein complexes to modulate chromatin structure, bind to enhancers, bind to promoters, to communicate between the basal transcription machinery and activators, to modify the nucleosomal structure and to generate transcripts. Global ChIP-on-chip profiling is now widely used to assess how transcriptional control is orchestrated on individual promoters in a whole genome context. The general transcription factor TFIIA is reviewed in chapter 2. This chapter represents, in fact, an outline of the results, conclusions and perspectives presented in this thesis. In chapter 3, the identification of the contact residues between TBP and TFIIA in TAC is described. The results show that the helix 2 of TBP is essential for

30

GENERAL INTRODUCTION

formation of TAC and that residues in the stirrup region contribute to its stability. To begin investigating the role of TFIIA cleavage we identified the N-terminus of TFIIAβ, as described in chapter 4. Mutational analysis of the adjacent region was undertaken to assess the cleavage region, and this revealed the cleavage recognition sequence (CRS), a string of four residues that are essential for cleavage. Whereas no functional difference in transcriptional competence could be detected between wildtype TFIIA and the uncleavable CRS mutants, cleavage of TFIIA was found to affect its stability. The uncleavable forms of TFIIA were considerably more stable than wild-type TFIIA. The data described in this chapter suggest that TFIIA is a substrate for proteasomal degradation subsequent to cleavage through the N-end rule. The identification of the protease responsible for TFIIA cleavage is described in chapter 5. The TFIIA CRS was identical to the recently identified cleavage site of MLL, which is cleaved by Taspase1. Subsequently, Taspase1 was found to cleave TFIIA, revealing an exciting connection between two until then unrelated proteins. The hypothesis that TFIIA is a substrate for degradation through the N-end rule is elaborated upon in chapter 6. Over-expressing ATE1 leads to increased degradation of TFIIA, and consistently, the half-life of TFIIA is prolonged in ATE1-/- cells. This study indicates that TFIIA degradation follows the N-end rule, making TFIIA the first physiological substrate of ATE1 through an aspartic acid. Chapter 7 summarises the data presented in this thesis and discusses its implications.

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2 TFIIA Before and After Torill Høiby, Dimitra J. Mitsiou, Salvatore Spicuglia, Huiqing Zhou and Hendrik G. Stunnenberg

Manuscript in preparation

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CHAPTER 2

Abstract TFIIA was originally identified as one of the general transcription factors, and activates transcription by stabilizing TBP binding to DNA and by working as a coactivator and anti-repressor. Newly obtained data show that TFIIA instead of being a general transcriptional regulator is involved in transcriptional activation at a subset of promoters. In addition, recent studies concerning the proteolytical cleavage of TFIIA reveal unexpected layers of complexity in TFIIA regulation. This review will focus on functional characteristics of TFIIA and discuss novel insights in the role and regulation of this revived basal transcription factor.

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TFIIA BEFORE AND AFTER

Introduction In one of the groundbreaking discoveries in eukaryotic transcription, nuclear RNA polymerase I, II and III were identified and found to transcribe large ribosomal genes, protein coding and some small nuclear RNA genes and most small structural RNA genes, respectively1-3. The complexity and variability of the general transcription machinery in Eukarya was further extended through the discovery of specific accessory factors4-7. The inability of RNA polymerases to initiate transcription by themselves provided the basis for characterizing the basal transcription machinery, i.e. the general transcription factors (GTFs) that reconstitute efficient and selective transcription initiation. For RNA pol II, these GTFs were identified by fractionation of cellular extracts and include TFIIA, TFIID, TFIIE, TFIIF and TFIIH8-11. Despite the complexity conferred by the composition of the basal transcription machinery, subsequent research has unveiled an unexpected multitude of activators, repressors, multi-protein complexes mediating communication to the basal transcription machinery, and last but not least variation within the basal transcription machinery itself. It has also lead to the understanding that the highly compact nucleoprotein or chromatin structure in which the DNA is organised plays a central role in orchestrating transcription regulation12,13. One of the general transcription factors whose precise role in transcription has remained elusive is TFIIA. The TFIIA-containing fraction was originally found necessary to reconstitute basal transcription in vitro14. TFIIA has since been counted amongst the general transcription factors, though this classification has been debated, largely because of the contradicting results as to how general it actually is for basal transcription. Recent data suggest that promoters vary widely in their requirement for TFIIA for transcriptional activation, and it appears that TFIIA may not be generally

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involved in transcription, but rather have a role in transcription of a subset of genes. An enigmatic feature of TFIIA is its proteolytic cleavage. Recent observations that TFIIA cleavage appears to be a regulated event together with the discovery that the MLL protease Taspase1 is also responsible for TFIIA cleavage have revealed unexpected aspects of TFIIA biology.

The role of TFIIA in general transcription Role in vitro The HeLa-derived Phosphocellulose (PC) A fraction, or Trancription Factor Pol II in PC A (TFIIA) has been shown to stimulate transcription and TBP binding to the TATA box15 but does not stimulate TBP-mediated transcription from a core promoter using highly purified factors16-19. In assays using partially purified factors, however, TFIIA stimulates transcription by reversing the inhibitory effects of negative co-factors like NC1, Dr1/NC2, Dr2/Topo1, HMG1 and DSP1, as well as by counteracting the effects of BTAF1 and TAF1 present in the extracts16,20-27. TFIIA is crucial for basal and activated transcription from TATA-less promoters in vitro, suggesting it has a core promoter-specific role beyond the binding of TBP to DNA28. TFIIA functions as a co-activator for several activators (AP-1, Gal4-AH, Zta, VP16, CTF, NTF, Sp1)16,17,29-31 and promotes co-activators like PC4 and HMG218,32. In addition, TFIIA has been reported to regulate TBP or TFIID dimerization and thus accelerate DNA binding33. Some observations have lead to the implication that TFIIA is also involved in RNA pol III transcription, for example on the 5S and U6 RNA promoters34,35, whereas this has been contradicted by other results36-38.

48

TFIIA BEFORE AND AFTER

The initial classification of TFIIA as a general transcription factor has been debated because of contradictory observations9,16,17,19,39, and the inconsistent categorisation of TFIIA as essential40,41, stimulatory8 and dispensable42,43 for transcription are generally believed to originate from variations in the transcription factor composition employed in the respective studies.

Role of TFIIA in vivo TFIIA can activate RNA Pol II transcription by a variety of mechanisms, but one question that remains open is to what extent TFIIA is generally involved in RNA Pol II transcription. Various approaches have been employed to study its function in transcription by disrupting the TBP-TFIIA-DNA interaction. By employing a yeast TBP mutant unable to interact with TFIIA, Stargell and Struhl demonstrated a specific impairment in the response to acidic activators, but no effect on RNA Pol II transcription in general44 and by diminishing the interactions between TFIIA and TBP using yeast TOA2 mutants, Ozer et al. observed selective transcriptional effects and a partial inhibition of cell-cycle progression45. Ten times depletion of TFIIA leads to a modest decrease in RNA Pol II transcription from both TATA-containing and TATAless promoters37 and a variety of genes is affected when TFIIA is reduced to less than 1% of the wild-type level36. The effects on individual genes can be seen as minor (2-3 fold), but the fact that cells arrest specifically at G2/M argues that TFIIA has an important role in controlling genes related to cell cycle progression. This moderate effect contrasts with depletion of other general transcription factors like TBP, TFIIB, TFIIH subunits and RNA Pol II subunits which in all cases leads to a total elimination of RNA Pol II transcription in yeast36. Hitherto, the data support the notion that TFIIA

49

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acts as a co-activator more than a general factor and that promoters vary considerably in the extent to which they require TFIIA. Based on its co-activating features and association with TBP, TFIIA has been proposed to be a TBP-associated factor (TAF) that dissociates from the TFIID complex more readily than the other TAFs46. This classification is, however, not fully supported; recent in vivo studies in yeast have shown that the TBP/TAF ratio varies significantly amongst different promoters in contrast to the TBP/TFIIA ratio47. This suggests that some genes are TFIIA-dependent but TAF-independent, whereas other genes are both TFIIA- and TAF-dependent. Geisberg and Struhl recently proposed a novel mechanism based on yeast studies addressing the occupancy of Mot1 on promoters before and after stress48. These data suggested that in stressed cells Mot1 functionally replaces TFIIA in preinitiation complexes in vivo. The authors argued that this may explain the moderate effects on transcription upon TFIIA depletion. It has indeed been shown that Mot1 can assist in recruiting TBP to promoters during gene activation, much in the same way TFIIA is thought to do49,50. A large number of factors have been reported to genetically or physically interact with TFIIA (summarised in table 1). TFIIA has been shown to interact with Gcn5, Swi2 and Nhp6 suggesting that histone acetylation, chromatin regulation and architectural gene modulation contribute to the formation of a TBP-TFIIA-DNA complex61, but the overall characteristics of these factors support the notion that TFIIA modulates the activities of the TFIID- or SAGA-components, communicates between activators and the basal transcription machinery and counterbalances the effects of repressors.

50

TFIIA BEFORE AND AFTER

Table 1: TFIIA-interacting proteins Factor Function AP-1 Zta VP16 CTF NTF SP1 GAL4 PC4 HMG2 scTAF11 dmTAF4 TBP TRF2 CREM GCN5 SWI2 NHP6 TAF1 BTAF1 NC2 HMGB1 RBP SPT3 SPT8

Reference

Activator of transcription Activator of transcription Activator of transcription Activator of transcription Activator of transcription Activator of transcription Activator of transcription Co-activator of transcription Co-activator of transcription TBP-Associated Factor TBP-Associated Factor TATA-Binding Protein TBP-Related Factor 2 Activator of transcription in germ cells Histone acetylase, component of SAGA Chromatin remodeling Archetectural gene modulation TBP-Associated Factor Repressor of transcription Repressor of transcription Repressor of transcription Repressor of transcription Component of SAGA Component of SAGA

51 17,51 51,52 51 29,46 29 53 54 32 55 46,56 55,57,58 59 60 61 61 61 23,25,27 48,62 63,64 65 56 62 56,66

The architecture of the TFIIA subunits Yeast TFIIA was originally purified as two polypeptides with molecular masses of 32 and 13.5 kDa67. The subsequent cloning of the polypeptides identified two genes, TOA1 and TOA2, that are both essential in yeast40. In contrast to yeast, TFIIA was found to be composed of three polypeptides, α, β and γ, in H. sapiens and D. melanogaster16,46,68. Cloning of TFIIA revealed that the two larger subunits, α and β, are encoded by a single gene and post-translationally processed. Toa1 and Toa2 are the respective homologues of the TFIIAαβ and TFIIAγ subunits of higher eukaryotes16,46,68 (figure 1). he high sequence- and function conservation of TFIIA from yeast to human underlines the importance of TFIIA in fundamental aspects of eukaryotic transcription. Besides the highly conserved and essential N- and Cterminal domains of TOA1, a significant portion of the non-conserved middle region can be removed without loss of viability37, leading to the suggestion that this domain

51

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functions as a non-specific spacer region. Introducing the human acidic region or the very C-terminal region of TFIIAαβ has severe effects on yeast growth, arguing that, despite their conservation, some protein functions are not conserved across species69.

Acidic

Spacer

1 FHB I

II

1 54% I

CRS II

β-barrel

286 TOA1

IV

III

72% III

IV

III

IV

376 TFIIAαβ 478

1 I

II

β-barrel

1 FHB I

Figure 1. Sequence comparison of yeast and human TFIIA and human ALF. The four-helix barrel (FHB) domain and the βbarrel domain identified in the crystal studies are depicted, as are the spacer domain, the acidic fomain the the CRS.

122 TOA2

II

1

109 I

II

ALF

TFIIAγ

58%

The general transcription machinery The general transcription factors were long believed to be unique and essential for transcription of all RNA Pol II promoters. This view has been challenged by the discovery of TBP-free TAF-containing complexes like hTFTC, hPCAF, hSTAGA and ySAGA and the isolation of cell-and tissue specific paralogues of several basal transcription factors. TBP was originally defined as a universal transcription factor70, but is in fact supplemented with at least three paralogues in metozoans; the insectspecific TRF171-73, TLF/TRF2 which is found in all metozoan genomes examined74-78 and the vertebrate TBP279,80. Subsequent to the identification of the TBP paralogues, the TFIIA-Like Factor (ALF) was isolated81,82. ALF is homologous to TFIIAαβ (figure 1), and like TFIIAαβ, it is able to interact with the small TFIIA subunit (γ) to form a heterodimeric complex that stabilizes binding of TBP to promoter DNA. ALF undergoes proteolytical cleavage like TFIIAαβ83-85. The expression of ALF is

52

TFIIA BEFORE AND AFTER

restricted to testis (X. laevis and M. musculus), and oocytes (X. laevis), where it probably replaces TFIIAαβ during meiosis. Hitherto, only subunits of TFIID and TFIIA are complemented with cell-specific paralogues whereas RNA Pol II, TFIIB, TFIIE TFIIF and TFIIH are all present as a single gene in the D. melanogaster, mammalian and C. elegans genomes. An archeal homologue of TFIIA is missing, and similarly, the protozoa P. falciparum expresses no apparent subunits of TFIIA and no TAFs86. One way to interpret this is that TFIIA has evolved in organisms where activated transcription is more widely employed and requires more complex regulation.

Regulation of TFIIA by cleavage The TFIIAαβ precursor could originally not be detected in SDS-treated cell extracts from HeLa cells, leading to the assumption that cleavage of TFIIA is not regulated and occurs simultaneously with or directly after or during translation, and implying that functional cellular TFIIA is cleaved17,68. This common view was reconsidered years later when studies in P19 embryonal carcinoma (EC) cells demonstrated that these cells contain considerable levels of the uncleaved form of TFIIAαβ. In P19 EC cells, the uncleaved form of TFIIA interacts strongly with TBP in a TAF-free complex termed TAC that supports transcription87. Furthermore, overexpression of the co-activator p300 facilitates formation of TAC also in other cell lines88. These observations provided the first hint that cleavage of TFIIAαβ is a regulated event that may be linked to the differentiation state of cells. This observation motivated new efforts to identify the TFIIA cleavage site and the responsible protease(s). The mapping and characterisation of the TFIIA cleavage site lead to the discovery of the cleavage recognition sequence (CRS), a string of four residues (272-275) that are essential for TFIIA cleavage85. The importance of the CRS 53

CHAPTER 2

is underscored by its strict conservation between higher eukaryotes and S. pombe (figure 2). Inhibition of cleavage through a single mutation in the CRS significantly prolonged the half-life of TFIIA85. These and other results suggested that cleavage of TFIIA is linked to its degradation; cleaved TFIIAα and -β are substrates for proteasomal degradation, but uncleaved TFIIAαβ appears not to be, indicating that the level of TFIIA is regulated by cleavage. Several reports conclude that cellular TFIIA levels fluctuate; it has for example been shown that TFIIA expression declines dramatically upon HSV virus infection, suggesting that regulation of TFIIA levels is part of a cellular program enabling the transition from early to late viral gene transcription during infection, consistent with a requirement for TFIIA in transcription of early but not late genes89. Furthermore, inactivation of TFIIA during terminal differentiation of avian erythroid cells contributes to a general repression of gene activity in these cells90, and TFIIAγ expression is up- regulated during Ras-mediated photoreceptor induction in D. melanogaster91. Collectively, these results suggest that regulation of TFIIA levels contributes to the regulation of gene expression concomitant with cell differentiation and transformation. Regulating TFIIA stability through cleavage seems to be one way to achieve this.

Figure 2. Alignment of the CRS and the cleavage site of TFIIA and MLL from different organisms, human (h), mouse (m), Xenopus (x), pufferfish (p) and Drosophila (d). The conserved CRS is boxed. Cleavage of TFIIA and MLL by Taspase1 is at D/G, indicated with an arrow. D278 marked with ◊ is the identified N-terminal end of the β subunit of TFIIA purified from mammalian cells. The acidic stretch (residues in blue and purple) is relatively conserved in TFIIA and MLL.

54

TFIIA BEFORE AND AFTER

The identification of the protease responsible for TFIIA cleavage was facilitated by the observation that the CRS of TFIIA is identical to the protease cleavage site of the proto-oncogene Mixed-Lineage Leukemia (MLL)92, and consistent with that, MLL and TFIIA were shown to be cleaved by the same protease, Taspase193 (Zhou et al., manuscript in preparation). Whereas the N-terminus of the TFIIAβ-subunit purified from stable FM3A cells was identified as D27885, N-terminal sequencing of in vitro Taspase1-cleaved TFIIA revealed that its cleavage site was between D274/G275, identical to the cleavage site determined for MLL (Zhou and Stunnenberg, unpublished observations). The inconsistencies in the cleavage sites as determined in the two studies may be a result of a secondary cleavage event in vivo by either an endo- or an exopeptidase activity. The strict conservation of D278 in TFIIA across species and in the TFIIA homolog ALF together with the fact that mutations of D278 strongly impair cleavage argues that it is important for cleavage. D278 represents a potential N-terminal degron in TFIIA, and one of the implications of TFIIA cleavage could be the generation of a destabilising N-terminus that activates the destruction pathway and thus regulates the level and transcriptional activity of TFIIA in the cell. Transcriptional regulation in higher eukaryotes displays a much higher degree of complexity than in unicellular Eukarya and this is exemplified by the existence of cell- and tissue-specific paralogues of various general transcription factors. The occurrence of TFIIA (and ALF) cleavage adds additional complexity to the collection of transcription factors. From an evolutionary point of view, the cleavage process appears to have evolved subsequent to the divergence of S. cerevisiae which does not contain cleaved TFIIA, consistent with the absence of a CRS in its TFIIA and the absence of a Taspase1 like L-asparaginase. In contrast, the TFIIA of fission yeast (S.

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pombe), A. thaliana and C. elegans all contain a CRS and their genomes encode a Taspase1 like factor. It is therefore likely that TFIIA undergoes cleavage in these organisms.

Developmental role of TFIIA cleavage During X. laevis development, TFIIA cleavage is tightly regulated and correlates closely with Taspase1 activity (Spicuglia and Stunnenberg, unpublished observations), suggesting that in X. laevis, TFIIA cleavage is largely regulated by governing Taspase1 activity. In P19 EC cells, the higher ratio of uncleaved to cleaved TFIIAαβ does not correlate with reduced cellular protein levels of Taspase1 (Zhou et al., manuscript in preparation). Thus in these cells, regulation of TFIIA cleavage may occur either by regulating the Taspase1 activity itself or by post-translational modification of TFIIA that interfere with cleavage. In line with the latter, a fraction of TFIIAαβ/γ present in HeLa nuclear extracts appears uncleavable even when exposed to high levels of Taspase1 suggesting that human TFIIA may indeed be modified to prevent cleavage (Zhou and Stunnenberg, unpublished observations). With this in mind, it is interesting to note that mimicking phosphorylation by the mutation T276D located in a putative phosphorylation site adjacent to the CRS renders TFIIA uncleavable, whereas T276A behaves like wild-type TFIIA (Høiby and Stunnenberg, unpublished observations). Furthermore, putative phosphorylation sites in the cleavage region seem to regulate TFIIA stability85. The sites are potential GSK-3 (T276) or casein kinase II (CKII) (T276, T279, S280, S281) sites, and notably, CKII has been implicated in promoter selection and in the regulation of a number of transcription factors, including IκBα, c-Jun, IRF-1, RNA pol II and the phosphatase FCP1 which is involved in recycling of RNA Pol II for transcription elongation94-96.

56

TFIIA BEFORE AND AFTER

Additionally, phosphorylation of TFIIA by TAF1 has been reported to stimulate TBPTFIIA-DNA interaction and to contribute to transcriptional activation in yeast and human97,98. In addition to phosphorylation, TFIIA may be acetylated, because the role of p300 in TAC formation is dependent on the presence of its HAT domain, and consistently, preferentially the uncleaved form of TFIIAαβ is acetylated88. The exact role of p300 in regulation of TFIIA cleavage can only be speculated upon at present, but it is possible that it affects the efficacy of cleavage, either directly on the substrate TFIIA or by modulating the Taspase1 activity. TFIIA appears to be subjected to a relatively complex array of regulation steps involving several factors, including well-known co-activators like p300 as well as recently identified factors like Taspase1.

Perspectives The basal transcription machinery, once viewed as conserved and stoical, varies extensively in a promoter- and cell-type specific fashion. The classical TBPcontaining complex TFIID is complemented by tissue- and cell-specific TAFs, TBPlike factors and an array of TBP-free complexes. This diversity in the general transcription machinery extends to TFIIA; the TFIIA-like factor ALF is expressed in a cell type specific manner in higher eukaryotes and probably takes over the role of TFIIA during meiosis. Evolution of the CRS together with Taspase1-like proteases, suggests that cleavage of TFIIA occurs in most eukaryotic systems and may have evolved subsequent to the divergence of S. cerevisiae, likely resulting in additional complexity of transcriptional control. The assembly of the uncleaved form of TFIIAαβ and TBP into TAC suggests that it may be required for expression of a

57

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specific set of genes that are not receptive to cleaved TFIIA-containing complexes. In line with this notion are the observations that TFIIA cleavage is tightly regulated throughout development of X. laevis, and it seems that the uncleaved TFIIAαβ exists predominantly during early embryonal stages. A comprehensive analysis of the function of cleaved and uncleaved TFIIA will require genome-wide localization studies using organisms where wild-type TFIIA is replaced by an uncleavable form of TFIIA and antibodies that specifically recognize cleaved or uncleaved forms of TFIIA. The unexpected finding that TFIIA and MLL are substrates of the same protease, Taspase1, raises intriguing questions concerning a link between these proteins. Is cleavage of TFIIA and MLL a functionally co-regulated process during development or is their interconnectedness limited to sharing a protease? Insights into the role of p300, E1A and CKII in MLL processing could shed light on this issue. The facts that the two substrates for Taspase1 identified so far (TFIIA and MLL) are transcription factors and that Taspase1 associates with chromatin may argue that Taspase1 mediated cleavage is linked to the transcription process. Over the years, TFIIA has transformed from a basal transcription factor involved in general transcription to a co-activator affecting only a specific subset of genes. The role of its most notable feature, the posttranslational processing, remains a complicated matter. TFIIA cleavage seems to be tightly regulated during development suggestive of a specific role for uncleaved TFIIA on developmentally regulated genes. However, cleavage does not seem to affect the transcriptional competence of TFIIA and the only apparent distinction is the difference in half-life. The role for cleavage in determining promoter recognition specificity and/or attenuating the turnover of TFIIA will require a multifaceted mechanistic analysis involving

58

TFIIA BEFORE AND AFTER

Figure 3. Model of function and regulation of TFIIA. Uncleaved TFIIAαβ/γ can assemble with TBP into TAC and this is facilitated, directly or indirectly, by p300. TAC is responsible for the transcriptional activation of hitherto unknown embryo-specific promoters. TFIIAαβ/γ can be cleaved by Taspase 1 into TFIIAα/β/γ that can assemble with TFIID and activate general transcription. Ultimately, TFIIAα/β/γ is a substrate for proteasomal degradation, possibly through the N-end rule

transcriptional co-activators such as p300 and CKII, Taspase1 and the co-factors involved in TFIIA degradation. In an age of high-throughput functional screening, it is remarkable that the biology of a single basal transcription factor continues to unveil unexpected layers of complexity decades after its identification.

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Olave, I., Reinberg, D. & Vales, L. D. The mammalian transcriptional repressor RBP (CBF1) targets TFIID and TFIIA to prevent activated transcription. Genes and development 12, 1621-37 (1998). Ozer, J., Lezina, L. E., Ewing, J., Audi, S. & Lieberman, P. M. Association of transcription factor IIA with TATA binding protein is required for transcriptional activation of a subset of promoters and cell cycle progression in Saccharomyces cerevisiae. Molecular and cellular biology 18, 2559-70 (1998). Bryant, G. O., Martel, L. S., Burley, S. K. & Berk, A. J. Radical mutations reveal TATA-box binding protein surfaces required for activated transcription in vivo. Genes and development 10, 2491-504 (1996). Teichmann, M. et al. Human TATA-binding protein-related factor-2 (hTRF2) stably associates with hTFIIA in HeLa cells. Proceedings of the National Academy of Sciences of the United States of America 96, 13720-5 (1999). De-Cesare, D., Fimia, G. M., Brancorsini, S., Parvinen, M. & Sassone-Corsi, P. Transcriptional control in male germ cells: general factor TFIIA participates in CREM-dependent gene activation. Molecular endocrinology Baltimore, Md. 17, 2554-65 (2003). Biswas, D., Imbalzano, A. N., Eriksson, P., Yu, Y. & Stillman, D. J. Role for Nhp6, Gcn5, and the Swi/Snf complex in stimulating formation of the TATAbinding protein-TFIIA-DNA complex. Molecular and cellular biology 24, 8312-21 (2004). Madison, J. M. & Winston, F. Evidence that Spt3 functionally interacts with Mot1, TFIIA, and TATA-binding protein to confer promoter-specific transcriptional control in Saccharomyces cerevisiae. Molecular and cellular biology 17, 287-95 (1997). Kim, T. K., Zhao, Y., Ge, H., Bernstein, R. & Roeder, R. G. TATA-binding protein residues implicated in a functional interplay between negative cofactor NC2 (Dr1) and general factors TFIIA and TFIIB. Journal of biological chemistry, The 270, 10976-81 (1995). Xie, J., Collart, M., Lemaire, M., Stelzer, G. & Meisterernst, M. A single point mutation in TFIIA suppresses NC2 requirement in vivo. EMBO journal, The 19, 672-82 (2000). Dasgupta, A. & Scovell, W. M. TFIIA abrogates the effects of inhibition by HMGB1 but not E1A during the early stages of assembly of the transcriptional preinitiation complex. Biochimica et biophysica acta 1627, 101-10 (2003). Warfield, L., Ranish, J. A. & Hahn, S. Positive and negative functions of the SAGA complex mediated through interaction of Spt8 with TBP and the Nterminal domain of TFIIA. Genes and development 18, 1022-34 (2004). Ranish, J. A. & Hahn, S. The yeast general transcription factor TFIIA is composed of two polypeptide subunits. Journal of biological chemistry, The 266, 19320-7 (1991). DeJong, J. & Roeder, R. G. A single cDNA, hTFIIA/alpha, encodes both the p35 and p19 subunits of human TFIIA. Genes and development 7, 2220-34 (1993). Upadhyaya, A. B. & DeJong, J. Expression of human TFIIA subunits in Saccharomyces cerevisiae identifies regions with conserved and speciesspecific functions. Biochimica et biophysica acta 1625, 88-97 (2003). Hernandez, N. TBP, a universal eukaryotic transcription factor? Genes and development 7, 1291-308 (1993).

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77.

78. 79.

80.

81.

82.

83.

84.

85. 86.

Hansen, S. K., Takada, S., Jacobson, R. H., Lis, J. T. & Tjian, R. Transcription properties of a cell type-specific TATA-binding protein, TRF. Cell 91, 71-83 (1997). Takada, S., Lis, J. T., Zhou, S. & Tjian, R. A TRF1:BRF complex directs Drosophila RNA polymerase III transcription. Cell 101, 459-69 (2000). Holmes, M. C. & Tjian, R. Promoter-selective properties of the TBP-related factor TRF1. Science 288, 867-70 (2000). Martianov, I. et al. Distinct functions of TBP and TLF/TRF2 during spermatogenesis: requirement of TLF for heterochromatic chromocenter formation in haploid round spermatids. Development Cambridge, England 129, 945-55 (2002). Zhang, D., Penttila, T. L., Morris, P. L., Teichmann, M. & Roeder, R. G. Spermiogenesis deficiency in mice lacking the Trf2 gene. Science 292, 1153-5 (2001). Dantonel, J. C., Quintin, S., Lakatos, L., Labouesse, M. & Tora, L. TBP-like factor is required for embryonic RNA polymerase II transcription in C. elegans. Molecular cell 6, 715-22 (2000). Veenstra, G. J., Weeks, D. L. & Wolffe, A. P. Distinct roles for TBP and TBPlike factor in early embryonic gene transcription in Xenopus. Science 290, 2312-5 (2000). Martianov, I. et al. Late arrest of spermiogenesis and germ cell apoptosis in mice lacking the TBP-like TLF/TRF2 gene. Molecular cell 7, 509-15 (2001). Bartfai, R. et al. TBP2, a vertebrate-specific member of the TBP family, is required in embryonic development of zebrafish. Current biology CB 14, 5938 (2004). Jallow, Z., Jacobi, U. G., Weeks, D. L., Dawid, I. B. & Veenstra, G. J. Specialized and redundant roles of TBP and a vertebrate-specific TBP paralog in embryonic gene regulation in Xenopus. Proceedings of the National Academy of Sciences of the United States of America 101, 13525-30 (2004). Upadhyaya, A. B., Lee, S. H. & DeJong, J. Identification of a general transcription factor TFIIAalpha/beta homolog selectively expressed in testis. Journal of biological chemistry, The 274, 18040-8 (1999). Ozer, J., Moore, P. A. & Lieberman, P. M. A testis-specific transcription factor IIA (TFIIAtau) stimulates TATA-binding protein-DNA binding and transcription activation. Journal of biological chemistry, The 275, 122-8 (2000). Upadhyaya, A. B. et al. The germ cell-specific transcription factor ALF. Structural properties and stabilization of the TATA-binding protein (TBP)DNA complex. Journal of biological chemistry, The 277, 34208-16 (2002). Han, S. Y. et al. TFIIAalpha/beta-like factor is encoded by a germ cell-specific gene whose expression is up-regulated with other general transcription factors during spermatogenesis in the mouse. Biology of reproduction 64, 507-17 (2001). Hoiby, T. et al. Cleavage and proteasome-mediated degradation of the basal transcription factor TFIIA. EMBO journal, The 23, 3083-91 (2004). Coulson, R. M., Hall, N. & Ouzounis, C. A. Comparative genomics of transcriptional control in the human malaria parasite Plasmodium falciparum. Genome research 14, 1548-54 (2004).

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87.

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95.

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Mitsiou, D. J. & Stunnenberg, H. G. TAC, a TBP-sans-TAFs complex containing the unprocessed TFIIAalphabeta precursor and the TFIIAgamma subunit. Molecular cell 6, 527-37 (2000). Mitsiou, D. J. & Stunnenberg, H. G. p300 is involved in formation of the TBPTFIIA-containing basal transcription complex, TAC. EMBO journal, The 22, 4501-11 (2003). Zabierowski, S. & DeLuca, N. A. Differential cellular requirements for activation of herpes simplex virus type 1 early (tk) and late (gC) promoters by ICP4. Journal of virology 78, 6162-70 (2004). Bungert, J., Waldschmidt, R., Kober, I. & Seifart, K. H. Transcription factor IIA is inactivated during terminal differentiation of avian erythroid cells. Proceedings of the National Academy of Sciences of the United States of America 89, 11678-82 (1992). Zeidler, M. P., Yokomori, K., Tjian, R. & Mlodzik, M. Drosophila TFIIA-S is up-regulated and required during Ras-mediated photoreceptor determination. Genes and development 10, 50-9 (1996). Hsieh, J. J., Ernst, P., Erdjument-Bromage, H., Tempst, P. & Korsmeyer, S. J. Proteolytic Cleavage of MLL Generates a Complex of N- and C-Terminal Fragments That Confers Protein Stability and Subnuclear Localization. Molecular and cellular biology 23, 186-94 (2003). Hsieh, J. J., Cheng, E. H. & Korsmeyer, S. J. Taspase1: a threonine aspartase required for cleavage of MLL and proper HOX gene expression. Cell 115, 293-303 (2003). Lin, R., Beauparlant, P., Makris, C., Meloche, S. & Hiscott, J. Phosphorylation of IkappaBalpha in the C-terminal PEST domain by casein kinase II affects intrinsic protein stability. Molecular and cellular biology 16, 1401-9 (1996). Rossignol, M., Keriel, A., Staub, A. & Egly, J. M. Kinase activity and phosphorylation of the largest subunit of TFIIF transcription factor. Journal of biological chemistry, The 274, 22387-92 (1999). Friedl, E. M., Lane, W. S., Erdjument-Bromage, H., Tempst, P. & Reinberg, D. The C-terminal domain phosphatase and transcription elongation activities of FCP1 are regulated by phosphorylation. Proceedings of the National Academy of Sciences of the United States of America 100, 2328-33 (2003). Solow, S. P., Lezina, L. & Lieberman, P. M. Phosphorylation of TFIIA stimulates TATA binding protein-TATA interaction and contributes to maximal transcription and viability in yeast. Molecular and cellular biology 19, 2846-52 (1999). Solow, S., Salunek, M., Ryan, R. & Lieberman, P. M. Taf(II) 250 phosphorylates human transcription factor IIA on serine residues important for TBP binding and transcription activity. Journal of biological chemistry, The 276, 15886-92 (2001).

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3 THE CONVEX SURFACE OF TBP IS ESSENTIAL FOR TAC FORMATION Torill Høiby, Dimitra J. Mitsiou and Hendrik G. Stunnenberg

Manuscript in preparation

Abstract

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The general transcription factor TFIIA facilitates assembly of the preinitiation complex (PIC) by stabilizing TBP binding to the DNA, by operating as a co-activator and by counteracting negative effects from factors like NC2 and BTAF1. TFIIA consists of three subunits, α, β and γ, of which α and β are transcribed from one gene and post-translationally cleaved. The processed form of TFIIA associates with the TFIID complex, whereas the unprocessed form of TFIIA can associate with TBP in a complex (TAC) devoid of classical TAFs. To learn more about the TAC-specific interactions, we tested the ability of a large number of TBP mutants for their ability to form TAC. Similarly, we studied the binding affinity of these respective mutants to TAF1. Our data demonstrates that the convex surface of TBP is essential for TAC formation, whereas residues in the stirrup region contribute to its stability. Consequently, the TBP binding sites of TAF1 and TFIIA largely overlap, indicating that formation of TAC is incompatible with TBPTAF1 interaction. Since TAF1 is suggested to function as a scaffold for the other TAFs in TFIID, it could also explain why no TAFs have been found in TAC.

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Introduction Classically, the general transcription factors necessary for the assembly of the preinitiation complex (PIC) on an RNA pol II promoter ending in transcription initiation include TFIIA, -B, -D, (containing TATA-binding protein (TBP) and a number of TBP-associated factors (TAFs)), -E, -F and –H1. TBP has affinity for the TATA element, and has long been held to be crucial for transcription because of its involvement in the function of RNA pol I, RNA pol II and RNA pol III2. This dogma was challenged by the identification of the TBP-free complexes, like hTFTC (TBPfree-TAF-containing complex) that supports in vitro transcription from TATAcontaining promoters as well as TATA-less promoters3. Genome-wide analyses in yeast do in fact confirm that most of the genes are TFIID-dominated (90%) and these genes have a strong tendency towards being house-keeping and non-regulated. In contrast, the ySAGA complex, a TBP-free protein complex that share some TFIID subunits, generally control genes that are stress-induced and highly regulated4, though a marginal overlap between these two complexes exists. The classification of TFIIA as a general transcription factor is already long debated. In general, TFIIA stimulates and stabilises association of TBP to the promoter5,6, and it can operate as an anti-repressor as well as a co-activator7-10. Most probably, the contradicting effects of TFIIA in transcription are due to differences in the experimental setup. TFIIA consists of the subunits TOA1 (yeast) or TFIIAαβ (higher eukaryotes) and TOA2 (yeast) or TFIIAγ (higher eukaryotes)11-14. In contrast to yeast TOA1, TFIIAαβ in higher eukaryotes is post-translationally cleaved to yield the two subunits α and β that are highly homologous to TOA1 N- and C–terminus, respectively10,15,16, whereas TFIIAγ and TOA2 are conserved throughout the protein. The major interactions between TFIIA and TBP are between the TOA2 and the N-

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terminal stirrup region of TBP, as demonstrated by crystallographic17-19 and genetic studies20-22. In addition, genetic studies have indicated that the N-terminal helix 2 region of TBP is important for TFIIA interaction, probably through a strongly acidic domain of TFIIAβ. The largest subunit of the TFIID complex, TAF1, is suggested to serve as a binding scaffold for the other TAFs23. TAF1 has two bromo domains24, two kinase domains reported to phosphorylate Rap74 (TFIIF)25,26 and TFIIA26, a histone acetyl transferase domain that acetylate histones H3, H4 and H2A27 and a Ubiquitinactivating/conjugating activity shown to ubiquitylate histone H128. TAF1 has been associated with regulation of the cell cycle, cell differentiation, cell proliferation and cell survival29-31. Although TAF1 in general is seen as a co-activator, it negatively regulates transcription by sequestering TBP away from DNA, and by competing with TFIIA binding to TBP32-35. The N-terminal domain of dmTAF1 has been dissected into two main TBP-interaction domains, dmTANDI and dmTANDII (TAND for TAF1 N-terminal Domain), binding to the concave and the convex side of TBP, respectively32,36,37. In contrast to yeast TBP, which is largely monomeric, mammalian TBP does not exist as a free molecule2. A protein complex consisting of TBP and the uncleaved form of TFIIAαβ together with TFIIAγ (named TAC), originally identified in embryonal carcinoma (EC) cells, has been hypothesized to supplement complexes like hSTAGA, hPCAF or hTFTC with TBP38,39. TAC mediates transcription in P19 EC cells and its cell-specific assembly suggests a link between the proteolytical state of TFIIAαβ and cell differentiation. Whereas the TFIIA-TFIID interaction can be disrupted under low-stringent conditions12, TAC is remarkably stable and can be purified from cell extracts under high stringency conditions. This suggests that

70

TBP SURFACE AND TAC FORMATION

interactions between TFIIA and TBP in TAC are fundamentally different from interactions between TFIIA and TBP in TFIID. Recent studies have found that whereas the TBP/TAF ratio varies significantly between different promoters, the TBP/TFIIA ratio is constant40. This implies that some promoters are TFIIA-dependent but TAF-independent. We set out to study the biochemical characteristics of the TAC complex, and the properties that distinguish it from a TFIID-TFIIA complex; furthermore, to investigate how TBP is distributed between various protein complexes in cells. By employing a large set of TBP surface mutants, we have shown that residues found to be essential for TFIID-TFIIA interaction and activation of transcription play a less important role in the formation of TAC. This suggests that the ‘classical’ TFIIDTFIIA interactions are supplemented by additional interactions in TAC, consistent with its remarkable stability under high-stringency conditions. Explicitly, our data demonstrate that the helix 2 domain of TBP is essential for TAC formation whereas residues in the stirrup region contribute to its stability.

Results Competition between TFIIA and TAF1 for TBP binding A number of studies has demonstrated that TFIIA and TAF1 can bind competitively to overlapping surfaces of TBP but that TBP in the context of TFIID accommodates binding of both TAF1 and TFIIA33,41. The absence of TAF1 in TAC could imply that binding of TAF1 to TBP interferes with TAC formation. To address this question we set up a competitive assay by co-expressing TAF1, TBP and TFIIA in P19 EC cells. Figure 1B shows the expression of TBP, TFIIAαβ and increasing amounts of transfected TAF1. Both TFIIAαβ and TAF1 are detected through an HA-

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epitope to enable direct comparison in a Western blotting analysis. However, the results showed that obtaining sufficiently high levels of expressed TAF1 was difficult (compare the levels of TFIIAαβ and TAF1 in lanes 5, figure 1B). To facilitate this, two deletion mutants of TAF1 were generated; TAF1∆1 contains the two TBPinteracting domains TANDI and TANDII (residues 1-156), whereas TAF1∆2 in addition contains the N-terminal protein kinase domain of TAF1 and most of the histone acetylase domain (residues 1-894) (see figure 1A for a schematic depiction of TAF1 and its mutants). TAF1∆2 was readily expressed in P19 EC cells and TBP is stabilised upon co-expression of TAF1∆2 (Figure 1C, compare lane 2 with lane 5), confirming that the interaction between the two proteins is intact. Co-expression of TFIIA did not affect the overall level of TBP or TAF1∆2 in these experiments (compare lanes 4 and 5 with lane 6). Co-expression of TBP and TFIIA resulted in pronounced TBP stabilisation (fig. 1D, compare lane 1 with lane 4) indicating TAC

Figure 1 Co-expression of TAF1, TFIIA and TBP (A) Schematic picture of TAF1 and the deletion mutants TAF1∆1 and TAF1∆2. The known protein domains are depicted in the figure (B) Extracts of P19 EC cells transfected with TBP, TFIIA and increasing amounts of TAF1 were subjected to Western blot analysis with the HA-antobody (C) Extracts from P19 EC cells transfected with TBP, TFIIA and the TAF1 deletion mutant TAF1∆2 were analysed as in (B) (D) Extracts from P19 EC cells transfected with TBP, TFIIA and the deletion mutant TAF1∆1 were immunopricipitated using the anti-TBP antibody SL39. Peptide elution with the SL39 synthetic peptide was followed by analysis as in (B). Lanes 1-7 represent whole cell extracts and lanes 8-14 represent the eluted proteins.

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complex formation, consistent with previous results38. The shortest deletion mutant TAF1∆1 containing only the two TBP interaction domains TANDI and TANDII was expressed at high levels in P19 EC cells (Figure 1D, lane 3). The level of TBP increased dramatically upon co-transfection of TAF1∆1, similar to what occurs when TFIIA and TBP are co-expressed (compare lane 4 with lane 5). This suggests that the TBP interaction site is contained within the smallest mutant of TAF1 (Figure 1B). To extend the above data and to test whether TFIIA and TAF1 compete for the same interaction domain on TBP, extracts from P19 EC cells co-transfected with TFIIA, TAF1∆1 and TBP were used for immunoprecipitations with antibodies against TBP. TFIIAαβ and TFIIAγ were readily co-precipitated with TBP (Figure 1D, lane 11 and data not shown) confirming the formation of a genuine TAC complex. Similarly, the presence of TAF1∆1 in the precipitate established that a complex was formed between TAF1∆1 and TBP (Figure 1D lane 12).

Some TAF1∆1 was

immunoprecipitated from the cells where TBP was not transfected (Figure 1D, lane 13), most likely due to interaction between TAF1∆1 and endogenous TBP. In a competitive setting when all three proteins (TFIIA, TAF1∆1 and TBP) were coexpressed, both TFIIA and TAF1∆1 were co-immunoprecipitated by TBP (Figure 1D, lane 14). The amount of TFIIA in the precipitate was moderately less than in the absence of TAF1∆1 (Figure 1D, compare lane 11 with lane 14). Although these results do not prove that complexes are formed with either TFIIA or TAF1 and not both, they suggest that the interaction between TBP and TAF1∆1 is affecting the formation of TAC.

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Helix 2 in TBP is important for TAC formation To systematically study the potentially overlapping interaction sites of TAF1 and TFIIA on TBP, we employed a large number of TBP surface mutants in a set of experiments designed to identify single residues involved in the formation of the respective complexes. These mutants have previously been used to map TBP epitopes important for TFIIA-TBP-DNA complex formation (DA complex) and tested for their response to activated and basal transcription21. Bryant and colleagues showed that the main interaction lies in the so-called stirrup region of TBP, specifically residues A184, N189, A190, E191, R203 and R205, consistent with the crystallographic data of yeast TBP17,18,21, and these residues are also important for activated transcription in vivo. Fourty-seven TBP surface mutants were chosen for their structural proximity to the TFIIA interaction domain and used in this study. The TBP mutants were analysed by co-transfection with TFIIA in U2-OS and P19 EC cells, followed by Western blotting analysis. The extent to which TAC was formed was monitored by the observed stabilization of TBP upon co-transfecting TFIIA (Figure 2A and 2B). Of the fourty-seven mutants, five (R188E, Y192E, I201D, G223R, F253E) were expressed at very low level, or not at all. These mutants were probably unable to fold properly and were not considered further in the analysis. The results showed that mutants A190E, N193R, E227A, L244E, G245E, F246E, K249E, F250E, L251E and K254E modestly affected the formation of TAC, whereas the most severe effect was observed with the triple mutant R231E/R235E/R239E (named H2 for helix2) that was not able to form TAC at all (Figure 2B and 2C). This is consistent with previous data, except for mutants N193R, G245E, K249E, F250E and L251E that have been reported to behave like wild-type TBP in DA complex formation21. In converse, a

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TBP SURFACE AND TAC FORMATION

Figure 2 Effect of single mutations on TBP for formation of TAC and TAF1∆1-TBP (A, B) Extracts from P19 EC cells transfected with TBP wild-type or mutants, as depicted, (A) alone or (B) together with TFIIA were subjected to Western blot analysis using the HA antibody. (C) Extracts from P19 EC cells transfected with TFIIA and TBP wild-type or mutants as depicted, were subjected to immunoprecipitation using the Myc antibody against Myc-tagged TFIIAαβ. Peptide elution with the Myc synthetic peptide was followed by Western blot analysis using antibodies Myc (for TFIIAαβ and TFIIAα) and SL39 (for TBP). (D) Extracts from P19 EC cells transfected with TBP wild-type or mutants as depicted and the TAF1 deletion mutant TAF1∆1 (see figure 1A) were analysed as in 2A.

number of mutants (A184E, E191R, R203E, R205E, S216R) that have previously been shown to severely undermine DA complex formation, behaved as TBP wild-type in the TAC assay (Figure 2B and Table 1). This suggests that the critical interactions involved in ‘classical’ TFIIA-TFIID formation are complemented by additional interactions in TAC. However, the relatively modest effects obtained in these experiments needed further verification. To confirm the data, extracts from P19 EC cells transfected with TFIIA and TBP (wildtype or a subset of the mutants; A184E, E191R and the triple helix 2 mutant H2) were used for immunoprecipitations. A184E and E191R are in the stirrup region of TBP

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and were chosen because they fail to support transcription in vivo21. Figure 2C shows that TBP mutants A184E and E191R are able to form exogenous TAC, though to a lesser extent than wild-type TBP. However, TAC cannot be formed with the mutant TBP H2. Taken together, the above results demonstrate that helix 2 is essential for TAC formation. Amino acids in the stirrup region contribute to the stability of TAC but appear to be less important in this complex, whereas they are essential for formation of a DA complex and to support activated transcription. In addition, TAC appears to employ an additional contact surface including N193, G245, K249, F250 and L251 as compared to TFIIA-TFIID. Table 1: The effect of single TBP mutations on formation of TAC and interaction with TAF1∆1 Mutation S159E I161R V162R G175R C176R K177E K179R K181E A184E L185E R186E A190E E191R N193R P194E K195E R203E R205E E206R R208E S215E S216R E227R E228A R231E L232E R235E K236Q R239S V240Q L244E G245E F246E K249E F250E L251E D252R K254E S261E R231A R235A R239A

EMSA with TFIIA

TAC formation

TAF1∆1 interaction

+ +/+ + + + + +/+ + + + + + + + +/+ + + + + + + +/+ +/+ + + + + + -

+ + + + + + + + + + +/+ +/+ + + + + + + + + +/+ + + + + + + +/+/+/+ +/+/+ +/+ -

+ + + + + + + + + + + +/+/+ + + +/+ + + + + + + + + + + + +/+ + + + +/+/+ + + -

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TBP SURFACE AND TAC FORMATION

The binding surface between TBP and TAF1 To determine which surface of TBP is involved in TAF1 interaction, TBP and the mutant TAF1∆1 were co-transfected into P19 EC cells or U2-OS cells, followed by Western blotting analysis. The extent to which the proteins interact was monitored by the stabilization of the proteins; both TBP and TAF1∆1 were stabilized upon coexpression (Figure 1D, compare lanes 1, 3 and 5). Of the fourty-seven mutants tested, 6 mutations affected the stability of TBP-TAF1∆1, namely A190E, E191R, R203E, V240Q, F250E and L251E (Figure 2D and Table 1). As previously mentioned, R188E, Y192E, I201, G223R and F253E were probably unable to fold properly, and were excluded from the analysis. Our results are partly consistent with the study of the Berk group42 that reported TBP residues A184, N189, N193, R205, R231, R235, K236, R239, V240, Q242, K243, L244, F250, L251 and K254 were critically involved in TAF1 interaction. Mutation of either one of these residues leads to a binding efficacy reduced with 50% or more. Whereas the study of Berk did not test the mutant R203E, this work did not include mutants of residues N189, Q242 and K243. Nonetheless, seven mutants, namely A184E, N193R, R231E, R235E, K236Q, R239E and L244E gave conflicting results in the two studies (see discussion). Taken together, our results show that there are several overlaps in the binding of TFIIAαβ/γ (TAC-specific) and TAF1 to TBP. The triple mutant H2 completely prohibits formation of TAC, and has also been shown to reduce the interaction between TAF1 and TBP with 98% compared to the wild-type TBP42. Thus, these three residues are critically involved in both formation of TAC and TBP-TAF1. Furthermore, residues L244, F250 and L251, which all seats at the top of the saddle structure of TBP, near helix 2 (see figure 3), contribute to the stability of TAC as well as TAF1-TBP.

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A

B Figure 3. The TBP binding surface for TFIIA and TAF1∆1 overlap (A) Crystal structure of TBP binding to DNA. Residues involved in formation of TAC are depicted in dark. (B) As in (A), but residues in dark are involved in interactions with TAF1∆1

Discussion In this study, we have mapped the TBP residues that are crucial for formation of TAC by employing fourty-seven TBP surface mutants and testing them for interaction with TFIIAαβ/γ (into TAC) in vivo in P19 EC cells. The mutants cover most of the N-terminal region of TBP, including the domains that have been shown to be important for the formation of a TFIIA-TFIID-DNA complex in vitro and for activated transcription in vivo

21,42

. It has earlier been shown that TFIIA can interact

with TBP in the context of TFIID, but unlike the TAFs, TFIIA disassociates from TFIID at low-stringent conditions12. This is in strong contrast to the stability of TAC that survives high-salt conditions. We have found that ten single mutations in TBP reduced TAC formation (∼two-fold), and most of these residues are essential for optimal activated transcription by TFIID-TFIIA21. No TBP residues were found to be critically and exclusively involved in TAC formation, although four mutants that behaved like wild-type TBP in earlier studies, modestly affected TAC formation. On the other side, a number of the mutants that were critically impaired in their ability to form a TFIID-TFIIA complex and activate transcription readily formed TAC. An explanation for this is that interactions that are essential for ‘classical’ TFIIA-TFIID are complemented by additional interactions in the TAC complex. This could explain why TAC survives high-stringent conditions whereas TFIID-TFIIA dissociates under low-salt conditions. It also offers an explanation for why no single residues are critically and specifically important for TAC formation; individual/single mutations in 78

TBP SURFACE AND TAC FORMATION

TBP have less effect on TAC formation because more residues are involved in the binding. However, the strategy using single TBP mutations only is probably not sufficient to identify all the residues that are involved in TAC formation. The most severe effects on TAC formation were obtained with a triple mutation, R231E/R235E/R239E in the helix 2 sitting on top of TBP and residues clustered close to it (L244E, K249, F250E, L251E, V240Q). This domain has been hypothesised to be involved in TFIIA binding based on several arguments; its basicity complements the acidic stretch of TFIIA (residues 280-300 in humans)17,18, in addition, genetic studies have suggested that this domain is involved in TFIIA binding20,22. The acidic region of TFIIA did not crystallise and its function is not clear. In this context, it is interesting to note that the cleavage-exposed N-terminus of TFIIAβ has been identified as D278, immediately N-terminal of the acidic stretch43. Cleavage of TFIIA may have consequences for the structural flexibility of this region. Therefore, cleavage may change, directly or indirectly, the nature of the interaction between TFIIAαβ and TBP in TAC. It is likely that the uncleaved TFIIAαβ present in TAC makes additional contacts with TBP as compared to the interactions made between cleaved TFIIA and TFIID. Given that TFIIAαβ has been shown to be acetylated in TAC38,39, it is tempting to speculate that modified TFIIAαβ has different affinity for TBP than non-modified, cleaved TFIIA. Berk et al have shown that important contributions to TAF1-TBP interaction in vitro are made by the residues A184, R231, R235, K236 and R23942; however these results could not be confirmed in our studies. The inconsistencies in the two studies are likely due to the different experimental setups; the former study was performed using in vitro GST pull-downs and a protein is likely to bind differently in the context of a full set of transcription factors in vivo. Another explanation may be that the small

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TAF1 fragment (∼30kD) generated in this study lacks domains that contribute to the TBP-TAF1 interaction. Consistent with the latter, Martel et al suggest an interaction between the first TBP repeat and residues 1172-1344 (the HMG box) of TAF1 and this region is not present in the deletion mutant TAF1∆1. Taken together, this study shows that there are overlaps in the binding sites of TAF1 and TFIIA to TBP, consistent with previous reports. These overlaps indicate that formation of TAC is incompatible with the assembly of a TAF1-TBP complex, and may offer an explanation for why none of the classical TAFs have been observed in TAC. The co-existence of TAF-dependent and TAF-independent forms of transcriptionally active TBP are well-known in yeast40,44, and the occupancy of TFIIA correlates with TBP, suggesting that some promoters bind TBP and TFIIA but no TAFs. Whether the TAC complex present in mammalian cells represents the TAFindependent form of TBP in higher eukaryotes or whether TAC complements TBPfree complexes like PCAF or STAGA remains to be seen.

Materials and Methods Plasmids, mutagenesis and antibodies, protein extracts, SDS-PAGE, immunoprecipitation, immunoblotting Expression plasmids encoding hTBP (pSG5-hTBP), Myc-tagged hTFIIAαβ (pSG5-Myc-hTFIIAαβ) and hTFIIAγ (pSG5-hTFIIAγ) have been described38. HATAF1 was cloned by excising the BglII-BglII fragment (4241bp) fragment from pSVCMVTAF1 and ligate it into the BamHI site of pSG5-new (step1). In step 2, the PstIBamHI fragment from pSV-CMVTAF1 was excised and ligated into the PstI-BglII site of pSG5-new-TAF1 from step 1. TAF1∆1 was generated by placing the BglIISmaI fragment from pSV-CMVTAF1 into the SmaI site of pSG5-new. TAF1∆2 was

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generated by excising the BglII-PstI fragment from pSV-CMVTAF1 and clone it into the BglII (klenow)-BamHI site of pSG5-new. The monoclonal antibodies Myc, HA and SL39, preparation of cell extracts, SDS-PAGE, immunoblotting and immunoprecipiation have been described38.

Cell culture and transient transfections U2-OS cells were maintained in DMEM supplemented with 10% FCS, P19 EC cells were maintained as described38.

Transient transfections were performed as

previously38.

Acknowledgements We are grateful to A. Berk and M. Timmers for the generous gift of plasmids.

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4.

5.

6.

7.

8.

Roeder, R. G. The role of general initiation factors in transcription by RNA polymerase II. Trends in biochemical sciences 21, 327-35 (1996). Hernandez, N. TBP, a universal eukaryotic transcription factor? Genes and development 7, 1291-308 (1993). Wieczorek, E., Brand, M., Jacq, X. & Tora, L. Function of TAF(II)-containing complex without TBP in transcription by RNA polymerase II. Nature 393, 187-91 (1998). Huisinga, K. L. & Pugh, B. F. A genome-wide housekeeping role for TFIID and a highly regulated stress-related role for SAGA in Saccharomyces cerevisiae. Molecular cell 13, 573-85 (2004). Lagrange, T. et al. High-resolution mapping of nucleoprotein complexes by site-specific protein-DNA photocrosslinking: organization of the human TBPTFIIA-TFIIB-DNA quaternary complex. Proceedings of the National Academy of Sciences of the United States of America 93, 10620-5 (1996). Oelgeschlager, T., Chiang, C. M. & Roeder, R. G. Topology and reorganization of a human TFIID-promoter complex. Nature 382, 735-8 (1996). Auble, D. T. et al. Mot1, a global repressor of RNA polymerase II transcription, inhibits TBP binding to DNA by an ATP-dependent mechanism. Genes and development 8, 1920-34 (1994). Inostroza, J. A., Mermelstein, F. H., Ha, I., Lane, W. S. & Reinberg, D. Dr1, a TATA-binding protein-associated phosphoprotein and inhibitor of class II gene transcription. Cell 70, 477-89 (1992).

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TBP binding and transcription activity. Journal of biological chemistry, The 276, 15886-92 (2001). Mizzen, C. A. et al. The TAF(II)250 subunit of TFIID has histone acetyltransferase activity. Cell 87, 1261-70 (1996). Pham, A. D. & Sauer, F. Ubiquitin-activating/conjugating activity of TAFII250, a mediator of activation of gene expression in Drosophila. Science 289, 2357-60 (2000). Ruppert, S., Wang, E. H. & Tjian, R. Cloning and expression of human TAFII250: a TBP-associated factor implicated in cell-cycle regulation. Nature 362, 175-9 (1993). Wang, E. H., Zou, S. & Tjian, R. TAFII250-dependent transcription of cyclin A is directed by ATF activator proteins. Genes and development 11, 2658-69 (1997). Wassarman, D. A., Aoyagi, N., Pile, L. A. & Schlag, E. M. TAF250 is required for multiple developmental events in Drosophila. Proceedings of the National Academy of Sciences of the United States of America 97, 1154-9 (2000). Kokubo, T., Yamashita, S., Horikoshi, M., Roeder, R. G. & Nakatani, Y. Interaction between the N-terminal domain of the 230-kDa subunit and the TATA box-binding subunit of TFIID negatively regulates TATA-box binding. Proceedings of the National Academy of Sciences of the United States of America 91, 3520-4 (1994). Kokubo, T., Swanson, M. J., Nishikawa, J. I., Hinnebusch, A. G. & Nakatani, Y. The yeast TAF145 inhibitory domain and TFIIA competitively bind to TATA-binding protein. Molecular and cellular biology 18, 1003-12 (1998). Verrijzer, C. P., Chen, J. L., Yokomori, K. & Tjian, R. Binding of TAFs to core elements directs promoter selectivity by RNA polymerase II. Cell 81, 1115-25 (1995). Burley, S. K. & Roeder, R. G. Biochemistry and structural biology of transcription factor IID (TFIID). Annual review of biochemistry 65, 769-99 (1996). Liu, D. et al. Solution structure of a TBP-TAF(II)230 complex: protein mimicry of the minor groove surface of the TATA box unwound by TBP. Cell 94, 573-83 (1998). Nishikawa, J., Kokubo, T., Horikoshi, M., Roeder, R. G. & Nakatani, Y. Drosophila TAF(II)230 and the transcriptional activator VP16 bind competitively to the TATA box-binding domain of the TATA box-binding protein. Proceedings of the National Academy of Sciences of the United States of America 94, 85-90 (1997). Mitsiou, D. J. & Stunnenberg, H. G. TAC, a TBP-sans-TAFs complex containing the unprocessed TFIIAalphabeta precursor and the TFIIAgamma subunit. Molecular cell 6, 527-37 (2000). Mitsiou, D. J. & Stunnenberg, H. G. p300 is involved in formation of the TBPTFIIA-containing basal transcription complex, TAC. EMBO journal, The 22, 4501-11 (2003). Kuras, L., Kosa, P., Mencia, M. & Struhl, K. TAF-Containing and TAFindependent forms of transcriptionally active TBP in vivo. Science 288, 12448 (2000). Bagby, S. et al. TFIIA-TAF regulatory interplay: NMR evidence for overlapping binding sites on TBP. FEBS letters 468, 149-54 (2000).

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Martel, L. S., Brown, H. J. & Berk, A. J. Evidence that TAF-TATA boxbinding protein interactions are required for activated transcription in mammalian cells. Molecular and cellular biology 22, 2788-98 (2002). Hoiby, T. et al. Cleavage and proteasome-mediated degradation of the basal transcription factor TFIIA. EMBO journal, The 23, 3083-91 (2004). Li, X. Y., Bhaumik, S. R. & Green, M. R. Distinct classes of yeast promoters revealed by differential TAF recruitment. Science 288, 1242-4 (2000).

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CLEAVAGE AND PROTEASOME-MEDIATED DEGRADATION OF THE BASAL TRANSCRIPTION FACTOR TFIIA Torill Høiby, Dimitra J. Mitsiou, Huiqing Zhou, Hediye Erdjument-Bromage, Paul Tempst and Hendrik G. Stunnenberg

The EMBO journal 23, 3083-91 (2004).

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Abstract The transcription factor TFIIA is encoded by two genes, TFIIAαβ and TFIIAγ. In higher eukaryotes, the TFIIAαβ is translated as a precursor and undergoes proteolytic cleavage; the regulation and biological implications of the cleavage have remained elusive. We determined by Edman degradation that the TFIIAβ subunit starts at Asp 278. We found that a Cleavage Recognition Site (CRS), a string of amino acids QVDG at position -6 to -3 from Asp 278, is essential for cleavage. Mutations in the CRS that prevent cleavage significantly prolong the half-life of TFIIA. Consistently, the cleaved TFIIA is a substrate for the ubiquitin pathway and proteasome-mediated degradation. We show that mutations in the putative phosphorylation sites of TFIIAβ greatly affect degradation of the β-subunit. We propose that cleavage and subsequent degradation fine-tunes the amount of TFIIA in the cell and consequently the level of transcription.

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Introduction A critical step in transcription is the recruitment and assembly of the preinitiation complex on the promoter1,2. RNA polymerase II and the basal transcription factors are necessary and sufficient to support basal transcription in vitro 3,4

. The basal transcription factor TFIIA has been shown to enhance transcription by

interacting with TBP and stabilising its binding to DNA, thereby accelerating a ratelimiting step5. In addition, TFIIA possesses activator activity and can counteract negative co-factors like NC2/Dr1 and Dr2/PC36-10. Basal transcription factors were originally defined as such because they were thought to be universally required for transcription. The identification of cell type-specific and gene-specific basal transcription factors such as the TBP paralogs, TLFs or the TFIIA paralog, ALF11-17, implies a higher degree of complexity than previously assumed. TFIIA is encoded by two genes; the small subunits are referred to as TOA2 in budding yeast and TFIIAγ in higher eukaryotes and show a high degree of overall homology. Whereas the large subunits, called TOA1 in yeast and TFIIAαβ or TFIIAL in human, show extensive homology in both N- and C-terminus, the central part is less conserved and structured18. A remarkable phenomenon in higher eukaryotes is the proteolytic cleavage of TFIIAαβ into TFIIAα and TFIIAβ subunits19-21. The site and amino acid requirements for cleavage have not been described until now, and it is also unclear whether cleavage is a specific and regulated process. Originally, only the cleavage products of native TFIIAαβ were detected in cell extracts in association with TFIIAγ. However, we recently identified a novel transcription complex, TAC, in embryonal carcinoma (EC) cells that contains TFIIAαβ along with TFIIAγ in a complex with TBP22,23. This observation and data presented in this study suggest that

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cleavage of TFIIAαβ precursor is a regulated process and that the cleaved (α+β+γ) and uncleaved (αβ+γ) TFIIA may have distinct cellular functions. Key

transcriptional

regulatory

mechanisms

include

post-translational

modifications like acetylation, phosphorylation and ubiquitylation. Classically, ubiquitylation has been viewed as a protein disposal pathway, but cells also appear to use this system as a means of fine-tuning transcriptional regulation that involves nonproteolytic pathways24. It has recently been demonstrated that ubiquitin-proteasome function is required for the transcriptional activity of ERα and VP16 activator25-27. Ubiquitylation of transcription factors can therefore affect their activity, accumulation and localisation. In this study, we determine the N-terminal amino acid of TFIIAβ and elucidate the mechanism and biological consequences of the cleavage process. Edman degradation and mutational analysis of the region surrounding the cleavage site revealed the Cleavage Recognition Site (CRS), a string of four amino acids adjacent to the cleavage site that is essential for cleavage. We provide evidence that cleavage triggers degradation of TFIIA via the ubiquitin-proteasome pathway and determines its half-life. Our results further suggest that degradation of TFIIAβ is likely regulated. In conclusion, cleavage and degradation of TFIIA appear to control the level of this basal transcription factor to meet the demands of the cell to rapidly adapt transcription processes.

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Results Identification of the N-terminus of TFIIAβ To facilitate rapid purification of TFIIA we employed classical biochemical and immuno-affinity purification methods using a retrovirally transduced FM3A mouse cell line that stably expresses human TFIIAαβ with Myc-and HA-epitopes at the Nand C-termini, respectively. The transduced human TFIIAαβ protein is partially cleaved into TFIIAα and TFIIAβ subunits of the expected sizes (Figure 1A). Interestingly, over-expression of TFIIAαβ results in elevated levels of endogenous TFIIAγ (compare lane 1 with lane 2), likely resulting from stabilization of endogenous TFIIAγ due to interaction with transduced human TFIIAαβ. Thus, the transduced human TFIIAαβ is able to form a complex with mouse TFIIAγ. Whole cell extracts prepared from a pool of transduced FM3A cells were used for purification of TFIIA as outlined in Figure 1B. Fractions from the Superose 6 column were analysed by SDS-PAGE and silver staining (Figure 1C) and by immunoblotting

Fig. 1. Expression and purification of TFIIA. (A) hTFIIAαβ stably expressed in transduced FM3A cells is cleaved to yield TFIIAα and TFIIAβ subunits. Extracts from non-transduced (lane 1) and stably transduced cells expressing Myc-HAtagged hTFIIAαβ (lane 2) were analysed by SDSPAGE and immunoblotting using antibodies against TFIIAα, TFIIAβ and TFIIAγ. The band marked with an asterisk is a minor TFIIAα product. (B) TFIIA purification scheme using whole cell extracts from FM3A cells stably expressing hTFIIAαβ. (C) Superose 6 fractions were subjected to SDS-PAGE and silver staining. Fractions 12 and 13 contain TFIIA subunits (α, β and γ), as depicted by arrows. (D) TFIIAcontaining fraction 12 from Superose 6 column (lane 1) or extract from non-transduced FM3A cells (lane 2) were subjected to immunoprecipitation using the 12 CA5 (HA) antibody against HA-tagged-hTFIIAβ. The immunoprecipitates were eluted with an excess of the HA synthetic peptide and analysed by SDSPAGE and silver staining. endTFIIA=endogenous TFIIA.

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(data not shown). Fraction 12 containing the highest concentration of TFIIA was subjected to immunoprecipitation using the 12 CA5 monoclonal antibody against the HA-epitope on the C-terminus of the β subunit (Figure 1D). The immunoprecipitate contained, amongst others, polypeptides with molecular weights of about 40, 20 and 12 kDa, the expected sizes of the TFIIA subunits. Western blotting indeed identified these as the α, β and γ subunits of TFIIA, respectively (data not shown). The 40-kDa protein as well as other slower migrating polypeptides (Figure 2D) were excised and analysed by mass spectrometry. A tryptic fragment of 11 amino acids conserved between mouse and human identified the 40-kDa polypeptide as the TFIIAα subunit. The identity of the slower migrating polypeptides indicated that they were most likely non-specific contaminants (data not shown). The 20-kDa polypeptide corresponding to TFIIAβ was subjected to N-terminal sequence analysis (Edman degradation) that revealed Asp278 as the most N-terminal amino acid. The sequence of amino acids is presented in Figure 2A.

Analysis of the cleavage site To elucidate the molecular determinants of the cleavage, we performed a mutational analysis (alanine scan) of the region surrounding the cleavage site. Wildtype and mutant TFIIAαβ were co-transfected along with TFIIAγ into U2-OS cells and analysed by immunoblotting (Figure 2B). Interestingly, mutation of the amino acids at positions -6 to -3 with respect to the cleavage site (Q272A, V273A, D274A and G275A) either abolished cleavage or yielded only trace amounts of cleavage products. Mutation of the amino acid at +1, i.e. the cleavage site (D278A), caused a significant reduction in the levels of the cleavage products TFIIAα and TFIIAβ as well as increased accumulation of TFIIAαβ. In contrast, mutation of the amino acids -

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CLEAVAGE AND DEGRADATION OF TFIIA

2 and -1 (T276A and G277A, respectively) did not significantly affect cleavage. Mutations at -8, +3 and +5 (V270A, S280A and E282A) slightly affected cleavage and yielded elevated levels of the precursor. S280A affected the efficacy of the cleavage as well as yielded a doublet at the position of TFIIAβ. Whether this doublet is due to alternative cleavage or a post-translational modification is currently under investigation.

Fig. 2. Mutational analysis of the TFIIAαβ cleavage region. (A) Sequence alignment indicates conservation of the GTG/DT TFIIAαβ cleavage site (marked by arrow) among Human, Mouse, Xenopus, Drosophila and S. pombe. The twelve residues following the arrow were from cycle 1-12 in the Edman degradation. The amino acids of the Cleavage Recognition Site (CRS) that are essential for cleavage are boxed. (B) Extracts from U2-OS cells transfected with plasmids expressing Myc-tagged hTFIIAαβ (wt or the Ala mutants of the indicated residues) and hTFIIAγ were analysed by SDS-PAGE and immunoblotting using antibodies against TFIIAα (upper panel), TFIIAβ and TFIIAγ (lower panel). The asterisk marks the position of residue A267 that was not included in the analysis. (C) U2-OS cell extracts from panel B, were subjected to immunoprecipitation using an antibody against TFIIAγ. The immuniprecipitates were analysed by SDSPAGE and immunoblotting as in B. (D) Whole cell extracts (WCE) from U2OS cells transfected with plasmids expressing hTBP, Myc-tagged hTFIIAαβ (wt or the indicated mutants) and hTFIIAγ, as indicated, were used for electrophoretic mobility shift assay with a synthetic oligonucleotide comprising the adenovirus 2 major late TATA box (lanes 4-13). Recombinant TBP and TFIIA were used giving rise to the DA (TBP-TFIIA-DNA) complex (lane 3).

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To test whether the different TFIIAαβ mutants and in particular the uncleaved mutant forms were assembled into functional complexes, co-immunoprecipitation experiments were performed under high-stringency conditions using an antibody against the TFIIAγ subunit (Figure 2C). Cleaved and the uncleaved TFIIAαβ were recovered with equal efficacy as judged from their relative abundance in the input and in the immunoprecipitates. Interestingly, both TFIIAβ polypeptides obtained with mutant S280A were efficiently co-immunoprecipitated with TFIIAγ, demonstrating that both peptides are part of a TFIIA complex. Bandshift assays performed with crude extracts from transfected U2-OS cells revealed that the mutated, uncleaved TFIIA facilitates a so-called DA-mobility shift in the presence of TBP similar to that observed with wild type TFIIA (Figure 2D, lanes 3, 5 and 8-13). Similarly, the uncleaved mutants of TFIIA raised activation of transcription from a tk-promoter

22

to levels similar to that observed with the wild-

type protein (data not shown). Sub-cellular localization experiments also showed that the uncleavable TFIIA was distributed similarly to the wild-type protein (data not shown). Taken together, our data show that uncleavable and cleavable TFIIA equally participate in TFIIA complex formation and support TBP-TFIIA binding to DNA in vitro and transcriptional activation in vivo.

The cleavage site is highly conserved The mutational analysis revealed a Cleavage Recognition Site (CRS), a string of four residues in the TFIIAα subunit essential for cleavage. Alignment of the region surrounding the cleavage site revealed a high degree of homology with TFIIAαβ from other organisms except budding yeast (Figure 2A). First, the Asp at position +1 is

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CLEAVAGE AND DEGRADATION OF TFIIA

conserved in all species for which the sequence of TFIIAαβ is known. Second, the CRS is fully conserved in vertebrates and highly similar in D. melanogaster, C. elegans and S. pombe. Furthermore, the spacing between the cleavage site and the CRS is conserved with the exception of C. elegans and S. pombe. The cleavage site is followed by several potential CKI and CKII phosphorylation sites (TSSEED)28. The high homology at and preceding the cleavage site is even more striking considering that the CRS is embedded in a part of TFIIAαβ that is of low sequence complexity and overall poorly conserved. Interestingly, a CRS is absent in S. cerevisiae and consistently TFIIA is not cleaved in this organism. Therefore, the high conservation of the CRS in addition to its critical role in the cleavage process strongly suggest that the CRS determines the position of the cleavage and furthermore predicts that cleavage of TFIIA also occurs in C. elegans and S. pombe.

Human ALF is cleaved As the CRS is also highly conserved in the germ cell-specific TFIIA-like Factor (ALF), a TFIIA variant (Figure 3A), we examined whether human ALF is cleaved as well. Extracts from U2-OS cells transfected with Myc-tagged ALF alone or together with TFIIAγ were analysed by immunoblotting using the Myc-antibody (Figure 3B). Western blotting identified a polypeptide that migrates at the position of full-length, recombinant ALF as well as a second ALF-derived polypeptide migrating with the relative mobility expected for the N-terminal moiety of ALF (left panels, lane 2). This is in accordance with a recent report that Xenopus laevis ALF is subject to cleavage in Xenopus oocytes29. Co-immunoprecipitation experiments further showed that both forms of ALF indeed form a complex with TFIIAγ (Figure 3B, right panels, lane 4). ALF could not be detected in the absence of TFIIAγ, suggesting that ALF,

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like TFIIAαβ, is unstable and formation of a complex between ALF and TFIIAγ is a prerequisite for its accumulation. Given the fact that a TFIIAγ paralog has not been found in the genome, the recent study in Xenopus29 and our data in mammalian cells strongly suggest that TFIIAγ is the natural partner of TFIIAαβ as well as ALF.

Fig. 3. The TFIIA-like Factor ALF is cleaved. (A) Sequence alignment among hTFIIAαβ and human and mouse ALF. The CRS is marked in the figure. (B) Extracts from U2-OS cells transfected with plasmids expressing hTFIIAγ together with Myc-tagged hTFIIAαβ (lanes 1, 3) or Myc-tagged hALF (lanes 2, 4) were subjected to immunoprecipitation using an antibody against TFIIAγ. The immunoprecipitates were analysed by SDS-PAGE and immunoblotting using the Myc antibody (upper panels) and the TFIIAγ specific antibody (lower panels).

Cleavage of TFIIA affects its stability Our results show that the uncleavable forms of TFIIAαβ such as G275A accumulated to higher levels than the wild type protein (Figure 2B). Accordingly, TFIIAγ accumulated to higher levels when co-transfected with the uncleavable TFIIAαβ mutants than with wild-type TFIIAαβ. To investigate whether these mutations affected stability of the different forms of TFIIA, we performed pulse-chase experiments to assess their half-life in vivo. Figure 4A shows that significant cleavage of TFIIAαβ occurred already during the pulse period, i.e. within 1 hour. Yet, cleavage takes several hours to complete, suggesting that there is a limiting factor that is required for cleavage. Furthermore, the uncleavable mutant appeared to be more stable than wild type TFIIA that undergoes cleavage (compare lanes 7-11 with lanes 2-6). Quantitation analysis revealed that equal amounts of wild type products

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CLEAVAGE AND DEGRADATION OF TFIIA

Fig. 4. Uncleavable TFIIA is more stable than cleavable TFIIA. (A) U2-OS cells transfected with plasmids expressing hTFIIAγ together with Myc-tagged hTFIIAαβ wt (lanes 2-6) or mutant G275A (lanes 7-11) were labelled for 1 hour with 35 STrans followed by a chase for 1, 2, 4, 8 and 24 hours. Extracts from these cells were subjected to immunoprecipitation using the Myc antibody followed by SDS-PAGE and fluorography. (B) Quantitation of labelled proteins from (A) was performed by Phosphoimager. The results represent the average of three independent experiments. (C) Extracts from U2-OS cells transfected with plasmids expressing hTFIIAγ and Myc-tagged hTFIIAαβ wild-type or mutant G275A and treated with CHX were analysed by SDS-PAGE and immunoblotting using antibodies against TFIIAα and GFP. Plasmid expressing GFP was cotransfected as the internal control. (D) Extracts from U2-OS cells transfected with plasmids expressing hTFIIAγ and Myctagged hTFIIAαβ wild-type or mutants as indicated and hTFIIAγ and treated with CHX, were analysed by SDS-PAGE and immunoblotting using antibodies against TFIIAα. (E) Quantitation of (D) was done by Phosphoimager. The result represents the average of three independent experiments.

(TFIIAαβ+α+β) and uncleavable mutant TFIIA G275A were detected immediately after the pulse (Figure 4B), showing that both forms are produced at about equal rate. However, clear differences in accumulation can be observed after 8 and 24 hours of chase, at which point uncleavable TFIIA G275A is roughly four times more abundant than wild type protein. These data suggest that cleavage causes destabilisation of TFIIA. Experiments performed in the presence of cycloheximide to prevent de novo protein synthesis demonstrated similar differences in accumulation of the wild-type and mutant forms of TFIIA (Figure 4C and data not shown). To extend our analysis, a number of mutants that displayed impaired cleavage were tested in identical settings

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(Figure 4D and E). Similarly, these results show that CRS mutants are stabilised up to 4 fold compared to the wild-type protein.

Cleaved TFIIA is a substrate for the 26S proteasome Having established that cleavage of TFIIA reduces its stability, we tested whether TFIIA is a substrate for the ubiquitin-proteasome pathway. Extracts from U2OS cells co-transfected with TFIIAαβ and TFIIAγ and treated with the proteasome inhibitor MG132 were analysed by immunoblotting. Figure 5A shows that the TFIIAα and TFIIAβ subunits are stabilised upon treatment with MG132 (compare lane 2 with lane 1), indicating that these subunits are degraded by the 26S proteasome. In striking contrast, the uncleaved form of the protein is unchanged upon MG132 treatment, demonstrating that TFIIAαβ is not targeted for proteasome-mediated degradation. Stabilisation of endogenous p53 in the presence of MG132 served as an internal control

30

. Thus, the cleaved TFIIAα and TFIIAβ, but not TFIIAαβ are

degraded via the proteasome which supports the notion that cleavage of TFIIA is a prerequisite for degradation and is in agreement with the half-life studies. Given that most substrates for the proteasome are ubiquitylated, we next investigated whether cleaved TFIIA is ubiquitylated. Extracts from U2-OS cells co-transfected with a HA-tagged form of ubiquitin along with TFIIA were used for immunoprecipitation with an antibody against TFIIAβ under high stringency conditions. Western blot analysis using the HA antibody revealed a characteristic laddering of ubiquitylated proteins only when TFIIA and HA-ubiquitin were co-expressed (Figure 5B, compare lanes 5-7 with lane 8). A weak laddering was observed when HA-ubiquitin was transfected alone, which may be due to ubiquitylation of endogenous TFIIA (lane 6). The smallest ubiquitylated polypeptide migrates at the position of ~29 kDa (asterisk),

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CLEAVAGE AND DEGRADATION OF TFIIA

consistent with the predicted size of mono-ubiquitylated TFIIAβ. Importantly, ubiquitylated TFIIA subunits could not be detected following immunoprecipitation using antibodies against TFIIAα and TFIIAγ (data not shown). Collectively, the above data suggest that ubiquitylation of TFIIAβ destabilises or even disrupts the TFIIA complex.

Fig. 5. Cleaved TFIIA is a substrate for proteasome-mediated degradation. (A) Inhibition of proteasome activity results in stabilization of hTFIIAα and hTFIIAβ subunits. Extracts from U2-OS cells transfected with plasmids expressing hTFIIAαβ and hTFIIAγ and treated (lane 2) or not (lane 1) with MG132 were analysed by SDS-PAGE and immunoblotting using antibodies against TFIIAα (upper panel) TFIIAβ (middle panel) and p53 (lower panel). (B) Extracts from U2-OS cells transfected with plasmids expressing hTFIIAγ, Myc-tagged hTFIIAαβ, and HA-tagged Ubiquitin, as indicated, were subjected to immunoprecipitation under high stringency conditions using an antibody against TFIIAβ. Extracts (lanes 1-4) and immunoprecipitates (lanes 5-8) were analysed by SDS-PAGE and immunblotting using antibodies against TFIIAα (lanes 1-4, upper panel), TFIIAβ and TFIIAγ (lanes 1-4, lower panel) and HA (lanes 5-8). (C) Extracts from U2-OS cells transfected with plasmids expressing hTFIIAγ and Myc-tagged hTFIIAαβ wild-type or mutants as indicated were analysed by SDS-PAGE and immunoblotting using antibodies against TFIIAα, TFIIAβ and TFIIAγ. (D) Extracts from U2-OS cells transfected with plasmids expressing hTFIIAγ and Myc-tagged hTFIIAαβ wild-type or mutant as indicated and treated with CHX, were analysed by SDS-PAGE and immunoblotting using antibodies against TFIIAα and TFIIAβ (E) Quantitation of (D) was done by Phosphoimager. The results represents the average of three independent experiments (F) Extracts from U2-OS cells transfected with plasmids expressing hTFIIAγ and Myc-tagged hTFIIAαβ and treated with CHX in We have demonstrated that inhibiting TFIIA cleavage through mutations in the the presence or the absence of α-amanitin or ActD, as indicated, were analysed by SDS-PAGE and immunoblotting using antibodies against TFIIAα and GFP (upper panel), TFIIAβ (middle panel) and TFIIAγ (lower panel). Plasmid expressing GFP was cotransfected as the internal control. CRS increases the stability of the protein, linking processing of TFIIA to its

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degradation. Furthermore, we have shown that treating cells with proteasome inhibitor stabilises the cleaved forms of TFIIA but not the uncleaved form, suggesting that TFIIAα and TFIIAβ are substrates for proteasomal degradation. Interestingly, cleavage of TFIIA creates an N-terminal aspartate, a secondary destabilising residue according to the N-end rule31. To study the significance of the N-terminus of TFIIAβ for its stability, we generated two mutants; D278M, yielding a stabilising N-terminal residue, and D278R, yielding a primary destabilising N-terminus. Extracts from U2-OS cells transfected with TFIIAγ and wild-type or mutant TFIIAαβ (D278A, D278M and D278R, respectively) were analysed by western blotting using antibodies against different TFIIA subunits (Figure 5C). In agreement with data presented in Figure 2, all D278 mutants showed reduced cleavage efficacy, indicating that residue D278 is important but not essential for cleavage, in contrast to residues within the CRS. Interestingly, while the steady-state levels of TFIIAγ remained the same, significant differences were observed with respect to TFIIAβ levels. TFIIAβ accumulated to a higher level when Asp was mutated to Met compared to Arg, supporting the hypothesis that TFIIAβ is a substrate for the N-end rule (Figure 5C, compare lane 4 with 5). D278A behaved similar to D278R, consistent with alanine being an unstable N-end residue. The effect was observed to a lesser extent for TFIIAα, suggesting that destabilisation of TFIIAβ also affects the stability of other subunits. Quantitation of the endogenous TFIIA subunits revealed that TFIIAβ is under-represented as compared to TFIIAα in extracts from U2-OS cells (data not shown), in agreement with the above observations. To extend and corroborate these observations, we compared the turn-over of TFIIAαβ, TFIIAα and TFIIAβ using extracts from U2-OS cells transfected with wildtype or mutant TFIIAαβ (D278M and D278R) in the presence of cycloheximide. 98

CLEAVAGE AND DEGRADATION OF TFIIA

Figure 5D and 5E show that the stability of TFIIA is dependent on the identity of residue 278 and that both D278M and D278R mutants of TFIIAαβ exhibit reduced cleavage as compared to wild-type protein. Interestingly, mutant D278M stabilises the TFIIAβ subunit whereas D278R increases its degradation (Figure 5D and E). The above results support our notion that cleavage of TFIIAαβ is linked to turn-over of the protein and that TFIIAβ is a substrate for N-end rule degradation. Our observation that TFIIAβ is likely degraded via the proteasome combined with recent publications showing that transcription is required for the proteasomemediated degradation of liganded hERα and SREBP27,32 led us to assess whether degradation of TFIIA subunits is linked to transcription. Therefore, TFIIA was expressed in U2-OS cells and the effect of transcription inhibitors was tested in the presence of cycloheximide. Figure 5F shows that treatment with α-amanitin or actinomycin D (ActD) neither affects cleavage nor degradation of TFIIA subunits. Similar results were also obtained with pulse-chase experiments in the presence of transcription inhibitors (data not shown). Taken together, our data indicate that degradation of TFIIAβ via the proteasome, probably via the N-end rule, occurs independently of its transcriptional activity.

TFIIAβ stability is regulated by residues C-terminal of the cleavage site Our results demonstrate that the cleaved TFIIA is degraded by the proteasome and indicate that TFIIAβ is ubiquitylated. As previously mentioned, the TFIIAβ subunit contains three conserved, potential phosphorylation sites directly C-terminal of the cleavage site (TSS). The single mutations T279A and S280A displayed no apparent effect on cleavage or stability, whereas S281A reduced the steady-state levels of TFIIAαβ (Figure 2 and 6A, lanes 2-4) suggesting that S281A is unstable or 99

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cleaved at a higher rate than wild type TFIIAαβ. To remove all phosphorylation sites simultanously, we mutated T279/S280/S281 to alanines (TSS-A). This had a dramatic effect on the stability of TFIIAα and particularly the TFIIAβ, which was only detectable after long exposure (Figure 6A, compare lanes 1 and 5). Similarly, mutating the recognition sites of casein kinase I and II by the triple mutation E282A/E283A/D284A dramatically destabilized the β-subunit (data not shown). The β-specific antibody is raised against the most C-terminal 76 amino acids

Fig. 6. Residues adjacent to the cleavage site are important for stability of TFIIAβ (A) Extracts from U2-OS cells transfected with plasmids expressing hTFIIAγ and Myc-tagged hTFIIAαβ wild-type or mutants as indicated and labelled with 32 PO4 were subjected to Ni-NTA purification and immunoprecipitation with the Myc antibody followed by analysis with SDS-PAGE and immunoblotting using antibodies against TFIIAα, TFIIAβ and TFIIAγ or fluorography. (B) Extracts from U2-OS cells transfected with plasmids expressing hTFIIAγ and hTFIIAαβ wild-type or mutants as indicated were analysed by SDS-PAGE and immunoblotting using antibodies against TFIIAα, TFIIAβ and TFIIAγ.

of the precursor and recognition by the antibody of the respective TFIIAαβ mutants is unaffected, arguing against reduced epitope recognition. In addition, identical results were obtained using antibodies against the C-terminal HA-tag of the β-subunit. To assess directly whether residues 279-281 are phosphorylated, we performed in vivo labelling experiments using TFIIA wild-type, the mutants T279A, S280A and S281A, and the triple mutant TSS-A. Figure 6A shows that TFIIAαβ and TFIIAβ but not TFIIAα and TFIIAγ, are phosphorylated (lane 6). None of the mutations significantly affected the overall

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32

P incorporation into TFIIA and the

CLEAVAGE AND DEGRADATION OF TFIIA

phosphorylation level roughly corresponded to the protein level in all cases (Figure 6B, compare lane 1 to 6, 2 to 7,3 to 8, 4 to 9 and 5 to 10). Mimicking constitutive phosphorylation by mutating T279/S280/S281 to Asp or Glu yielded ambiguous results. Whereas T279D and S280D destabilized the β-subunit, T279E, S280E, S281E and S281D behaved similar to wild type (Figure 6B). In conclusion, we have found that the residues immediately C-terminal to the newly created N-terminus are critical for the stability of TFIIAβ and consequently to TFIIA.

Discussion It has been known for quite some time that TFIIA undergoes co- or posttranslational cleavage19-21 but neither the cleavage site nor the biological implications of the cleavage have been described. In the present study, we characterised the cleavage site of TFIIAαβ and shed light on the function of the cleavage. We identified the CRS, a string of four residues N-terminal of the cleavage site that is essential for the cleavage process and showed that the identity of the amino acid at the cleavage site is less critical. The CRS partially overlaps with a region in TFIIAαβ that has the propensity to form a β-sheet and is directly followed by a highly acidic, probably unstructured part. Given the high conservation of the charged region following the conserved CRS, it is likely that the cleavage site and the CRS are surface-exposed. The amino acid sequence of the cleavage site and CRS do not match the recognition site of a known protease. The only protease that is known to cleave Nterminal to an Asp is the endopeptidase N-Asp from Pseudomonas fragi, which does not appear to have a homologue in eukaryotes. Recent studies addressing the

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processing of MLL, a human homologue of the Drosophila trithorax protein, revealed a cleavage recognition site -QV/LDG- which is virtually identical to the CRS in TFIIAαβ33,34. Furthermore, in MLL the CRS also precedes a highly acidic stretch containing multiple potential phosphorylation sites. MLL is cleaved in the CRS, between D and G, while we identified the cleavage site of TFIIA more C-terminal of the CRS. Currently, we cannot exclude the possibility that cleavage of TFIIA occurs within the CRS and that the N-terminal D278 is generated by a secondary cleavage or exopeptidase activity. Identification and characterisation of the CRS-specific TFIIAαβ protease will shed more light on the regulatory role of the cleavage and the possible link between TFIIA and MLL processing. In our experiments, the uncleaved and cleaved forms of TFIIA behave similarly with respect to TBP-DNA binding and transcriptional activity. While these assays are generally used to assess the ‘classical’ characteristics of TFIIA like stabilising TBP-DNA interactions, their in vivo value is rather limited, as they do not provide insight into potential promoter-specific differences. Although it seems likely that cleaved and uncleaved TFIIA may have distinct roles as could be inferred from the presence of the uncleaved TFIIA in the TAC complex, appropriate assays are currently under investigation. However, the present study provides evidence that cleavage has an important consequence for the half-life of TFIIA. Inhibition of cleavage as obtained with the mutation in the CRS impair or prevent protein degradation, leading to accumulation of the protein which is consistent with the observed increased half-life of the uncleaved TFIIA. Our results demonstrate that the cleaved, but not the uncleaved TFIIA complex is a substrate of the 26S proteasome. Several pieces of evidence suggest that ubiquitylation occurs on TFIIAβ followed by its degradation, and subsequently the

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TFIIAα and TFIIAγ subunits are degraded. First, the stabilising effect of the proteasome inhibitor MG132 is seen with the α- and β-subunits but not with the TFIIAαβ precursor. Second, the smallest ubiquitylated polypeptide is approximately 29 kDa, consistent with mono-ubiquitylated TFIIAβ. Third, under high-stringency conditions the poly-ubiquitylated forms of TFIIA can only be immunoprecipitated with antibodies raised against TFIIAβ but not TFIIAα or TFIIAγ. Last, the instability of the β-subunit is markedly enhanced by mutating sites adjacent to the cleavage site, indicating an important role for these residues in TFIIA degradation and suggesting that degradation of different subunits can occur partly independent on each other. The precise role of the putative phosphorylation sites can only be speculated upon at present. It is likely that phosphorylation of residues T279-S281 protects the β-subunit against proteasomal degradation, and that dephosphorylation accelerates degradation of TFIIAβ as well as the other subunits of TFIIA, similar to what has been reported for c-fos and NF-κB 35,36. Cleavage of TFIIA creates an N-terminal aspartate, which is a secondary destabilizing residue according to the N-end rule pathway31. This N-terminal aspartate, though highly conserved in TFIIA and ALF of all higher eukaryotes, is important but not essential for cleavage. It is therefore likely that the aspartate is conserved mostly to render the cleavage product instable. Recent studies have shown that caspase-mediated cleavage of DIAP1 (Drosophila IAP1) converts the more stable full-length protein into a highly unstable Asn-bearing N-degron and that its subsequent degradation by the N-end rule pathway is essential for regulation of apoptosis37,38. It is therefore conceivable that TFIIAαβ represents a stable pro-Ndegron and that its cleavage results in production of the unstable Asp-bearing N-

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degron (TFIIAβ). Whether TFIIA indeed represents another physiological N-end rule metazoan substrate remains to be investigated. We hypothesize that the biological significance and implications of cleavage is to generate a destabilizing N-terminus that triggers destruction of the protein to finetune the level of this basal transcription factor and consequently the transcriptional activity of the cell. The level of expression of the TFIIA protease and its activity are likely to have consequences for transcriptional initiation and re-initiation. Regulation of cleavage of TFIIA and subsequent degradation of the protein may provide directionality to the transcription process and prevent recycling of the basal factor.

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Materials and Methods Plasmids, mutagenesis and antibodies Expression plasmids encoding hTBP (pSG5-hTBP), Myc-tagged hTFIIAαβ (pSG5-Myc-hTFIIAαβ) and hTFIIAγ (pSG5-hTFIIAγ) have been described earlier22. Expression

plasmid

encoding

Myc-HA-tagged

hTFIIAαβ

(pSG5-Myc-HA-

hTFIIAαβ) was constructed by insertion of a double-stranded oligonucleotide encoding for the HA epitope at the 3’-end of hTFIIAαβ in pSG5-Myc-hTFIIAαβ. pSRα-tk-neo-Myc-HA-hTFIIAαβ was constructed by subcloning the appropriate fragment from pSG5-Myc-HA-hTFIIAαβ into the EcoRI site of pSRα-tk-neo. Plasmid expressing Myc-tagged hALF (pSG5-Myc-ALF) was constructed by subcloning the appropriate fragment from pRSET-ALF (provided by J. DeJong) into pSG5-Myc plasmid22. Plasmid pSG5-Myc-hTFIIAαβ was used for mutagenesis according to the manufacturer’s instructions (Quick Site-directed Mutagenesis, Stratagene). Plasmid expressing enhanced green fluorescence protein (pEGFP-N1) was from Clontech. The monoclonal antibodies Myc, HA and SL39 have been previously described22. Polyclonal antibodies against hTFIIA were as follows: αΝ-specific (against the N-terminus, amino acids1-63), β-specific (against amino acids 301-376) and γ-specific against the TFIIΑγ subunit. Polyclonal antibody against GFP was purchased from Clontech.

Cell culture, retroviral transduction, transient transfections, treatment with cycloheximide (CHX), α-amanitin, actinomycin D (ActD) and the proteasome inhibitor MG132, pulse-chase labelling and in vivo phosphate labelling

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U2OS, HeLa, COS7 and Bosc23 cells were maintained in DMEM supplemented with 10% FCS. P19 EC cells were maintented as described39. FM3A cells were maintained in RPMI medium supplemented with 5% FCS. FM3A cells stably expressing hTFIIAαβ were generated by retroviral transduction. Supernatant from Bosc23 packaging cells transfected with plasmid pSRα-tk-neo-TFIIAαβ was used to transduce FM3A cells at a density of 100,000 cells/ml, followed by selection with G418 (800 µg/ml)40,41. Transient transfections were performed as previously described22. Transfected cells were washed with PBS, incubated with fresh medium for 24 hours and then treated or not with CHX (20 µg/ml) alone or together with α-amanitin (2.5 µg/ml) or ActD (50 ng/ml), for the indicated periods of time. For treatment with the proteasome inhibitor MG132, transfected cells were incubated with 20 µM MG132 for 5 hours at 37 oC. For pulse-chase labelling, 48 hours after transfection, cells were incubated in methionine- and cysteine-free DMEM supplemented with 10% dialyzed FBS (against 0.15 M NaCl, cut-off 10 kDa), for 15 minutes at 37 oC. After starvation, cells were labelled in DMEM supplemented with 10 % FBS and 20 µCi/ml Tran35S Label (ICN) for 1 hour, followed by different chase periods in fresh medium. For in vivo phosphate labelling, 36 hours after transfection, cells were incubated in phosphate-free DMEM supplemented with 0.5 mCi/ml 32P PO4 (ICN) for 4 hours.

Ni2+-agarose affinity chromatography, ion exchange and gel filtration chromatography, protein extracts, SDS-PAGE, immunoblotting, treatment with

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bacterial alkaline phosphatase (BAP), immunoprecipitation, fluorography and electrophoretic mobility shift assay (EMSA) Purification of TFIIA from FM3A extracts by Ni2+-NTA-agarose affinity chromatography and MonoQ column were carried out as described19. Fractions containing TFIIA were loaded on a Superose 6 column in 20 mM Tris (pH 7.3), 100 mM KCl, 20 % glycerol, 0.2 mM EDTA, 5 mM DTT and 0.5 mM PMSF. Fractions were collected and assayed by immunoblotting. Preparation of cell extracts, SDSPAGE and immunoblotting have been described39. For quantitative immunoblotting, proteins were detected by ECL plus (Amersham) and analysed by PhosphorImager (Molecular Dynamics). For treatment with bacterial alkaline phosphatate (BAP), cell extracts were incubated with 0.05 unit of BAP/µl of extract, in the presence or the absence of 0.1 M NaH2PO4, for 1 hr at 30 oC. EMSA, immunoprecipitation and fluorography were as previously described22.

Chemical sequencing and Edman degradation analysis Chemical sequencing was done using a Procise 494 instrument from Applied Biosystems (AB) as described42.

Step-wise liberated PTH-amino acids were

identified using an "on-line" HPLC system (AB) equipped with a PTH C18 (2.1x220 mm; 5 micron particle size) column (AB). Gel-resolved proteins were digested with trypsin, partially fractionated, and the resulting peptide mixtures analyzed by matrix-assisted laser-desorption / ionization reflectron time-of-flight (MALDI-reTOF) mass spectrometry (MS) (Reflex III; BRUKER Daltonics, Bremen, Germany), as described43; and also using an electrospray ionization (ESI) triple quadrupole MS/MS instrument (API300; ABI/MDS SCIEX, Thornhill, Canada) modified with an ultra-fine ionization source44.

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Selected precursor or fragment ion masses from the MALDI-TOF MS or NanoESMS/MS spectra were taken to search the human segment of a protein non-redundant database, as described

45

. MS/MS spectra also were inspected for y" ion series to

compare with the computer generated fragment ion series of the predicted tryptic peptides.

Acknowledgements We thank members of the Stunnenberg laboratory and Susanne Mandrup for discussions and critical reading of the manuscript. We thank D. Reinberg, R.G. Roeder, J. DeJong, O. Witte and P.M. Lieberman for providing plasmids and antibodies. T.H. was partly financed by the Norwegian Research Council, project number 137372/300. P.T was supported by NCI Cancer Center Support Grant P30 CA08748.

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5 TFIIA IS A SUBSTRATE FOR TASPASE1, A CHROMATIN-ASSOCIATED PROTEASE Huiqing Zhou, James J.-D. Hsieh, Dimitra J. Mitsiou, Torill Høiby, Xavier Le Guezennec, Stanley J. Korsmeyer, and Hendrik G. Stunnenberg

Submitted for publication

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Abstract In higher eukaryotes, the large subunit of the general transcription factor TFIIA is encoded by a single gene TFIIAαβ and post-translationally cleaved into an α- and β-subunit. The molecular mechanisms and biological significance of this proteolytic process have remained obscure. Here, we show that TFIIA is a substrate of Taspase1 as reported for the trithorax group protein, MLL. We demonstrate that recombinant Taspase1 cleaves TFIIA in vitro. Transfected Taspase1 enhances cleavage of TFIIA and RNAi knock-down of endogenous Taspase1 diminishes cleavage of TFIIA in vivo. Taspase1 is localized in the nucleus and associated with chromatin suggesting that cleavage by Taspase1 might occur on chromatin. We propose that cleavage by Taspase1 regulates the levels of TFIIA, MLL and other substrates in the cell and plays a critical role in transcription and development.

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Introduction In eukaryotes, initiation of RNA polymerase II (pol II) transcription requires the assembly of a preinitiation complex (PIC). Specific binding of TBP to promoters is a key step in formation of PIC, which is followed by recruitment of general transcription factors and pol II. The basal transcription factor TFIIA interacts with TBP and stabilizes its binding to DNA1,2. TFIIA is also shown to interact with several activators3,4, and is required for transcriptional activation of certain genes5-8. In higher eukaryotes, purified TFIIA is composed of three subunits, α, β and γ. TFIIA α and β are encoded by one single gene, and cleaved post-translationally to α and β subunits. The γ subunit is conserved among different species, whereas sequence similarity in TFIIAαβ is limited mostly to the N-terminal region of the α subunit and the C-terminus covering most of the β subunit9. Recently, the cleavage recognition site (CRS) that is essential for TFIIA cleavage has been identified as -QVDG- (aa 272-275), and the N-terminus of the β subunit was determined to be at D278 located three amino acids downstream of the CRS (Fig. 1B)10. The CRS shares remarkable similarity in different evolutionarily distinct species and is embedded in an otherwise non-conserved and probably unstructured region11-13. The germ cell-specific TFIIAlike factor (ALF), a TFIIA variant that contains the CRS, was also shown to be cleaved10,14. The biological significance of cleavage has however remained elusive. Both uncleaved αβ and cleaved α and β subunits can be found in association with the TFIIAγ subunit in vivo15,16, and both forms interact with TBP on DNA and support transcription to a similar extent in vitro and in reporter assays10,17. TFIIA is mainly found in the cleaved form (α+β+γ) in differentiated cells. In embryonal carcinoma P19 cells, a substantial amount of the uncleaved TFIIA (αβ+γ) is detected and stably interacts with TBP in the TAC complex to mediate transcription15,16. Therefore, 115

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uncleaved and cleaved forms of TFIIA may have distinct gene regulatory functions in differentiation. The observation that cleavage is the prerequisite for proteasomemediated degradation of TFIIA10 indicates that cleavage regulates protein levels of TFIIA, and may play a role in transcription. Elucidation of the biological function of TFIIA cleavage is hampered because the protease(s) that specifically cleaves TFIIA has not been identified. The recently identified cleavage site in TFIIAαβ, G277/D27810, did not match known consensus sequences of proteases. We did notice however that the CRS QVDG- of TFIIA is virtually identical to the cleavage sites of MLL, Mixed-Lineage Leukemia protein, -QVD/G- (aa 2664-2667) and -QLD/G- (aa 2716-2719) (Fig. 1B)18,19. MLL is a 500 kDa nuclear protein of the trithorax (Trx) group proteins and required for maintenance of proper HOX gene expression. Chromosomal translocation results in different MLL fusion proteins that are involved in various leukemias20. MLL is proteolytically cleaved at two adjacent cleavage sites by a single protease, Taspase1, an endopeptidase with an asparaginase_2 homology domain21. Moreover, there is an acidic stretch downstream of the cleavage site in both MLL and TFIIA. These high similarities strongly indicate a molecular and/or functional link between TFIIA and MLL processing and prompted us to assess whether TFIIA is a substrate of Taspase1. Here, we show that TFIIA is a genuine substrate of Taspase1. Taspase1 cleaves TFIIA efficiently in vitro and in vivo, and RNAi knock-down of Taspase1 reduces cleavage of TFIIA. TFIIA interacts with Taspase1, and Taspase1 is localized in the nucleus and associated with chromatin. These results suggest that cleavage occurs on chromatin and may be a regulating step in various transcription processes mediated by TFIIA, MLL and other substrates. Moreover, identification of Taspase1

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as the protease for TFIIA provides a new tool to further our understanding of the role of TFIIA cleavage and function of TFIIA.

Results Specific protease activity for TFIIA cleavage in HeLa cell nuclear extracts To identify the protease for TFIIA, we set up an in vitro cleavage assay using purified recombinant TFIIA comprised of uncleaved αβ and γ subunits as the substrate to test for a cleavage activity for TFIIA in HeLa cell extracts. HeLa nuclear extracts (Fig. 1A, lanes 1, 2) showed reasonable levels of cleavage activity for recombinant wild-type TFIIA, yielding bands with apparent correct sizes for the cleaved α and β subunits. Fractionation of nuclear extracts on a P11 column showed that the cleavage activity was eluted at 500 mM KCl (PC-C fraction) (Fig. 1A, lanes 7, 8). The PC-C fraction was further fractionated on a Mono S column, and the cleavage activity was recovered at approximately 300 mM KCl in fractions 17, 18 and 19 (Fig. 1A, lanes 11-13). To assess whether the observed cleavage activity is specific and displays the same amino acid sequence requirements as in vivo, we tested in our in vitro assay a TFIIA cleavage site mutant G275A that was shown uncleavable in vivo10. As shown in Fig. 1A, the protease activity in the Mono S peak fraction 18 was able to cleave the wild-type TFIIA but not the G275A mutant (lanes 14 and 15), showing that the cleavage activity for TFIIA in HeLa nuclear extracts is specific. Having identified the cleavage recognition site (CRS) for TFIIA10, we noticed that the CRS in TFIIA, -QVDG- (aa 272-275), is identical or similar to the cleavage sites in MLL, -QVDG- (aa 2664-2667) and -QLDG- (aa 2716-2719) that are both cleaved at D/G by Taspase1 (Fig. 1B). This similarity indicated that TFIIA and MLL might be cleaved by the same protease. To test whether HeLa nuclear fractions enriched for TFIIA cleavage activity contain Taspase1, western blotting analysis was

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Fig. 1 Taspase1 activity in HeLa nuclear extract. A) A Protease assay was performed to detect TFIIA cleavage activity in HeLa nuclear extract. Highly purified recombinant TFIIA was used as the substrate as indicated, and western blotting analysis was used to detect the cleavage products. The protease activity was further fractionated on a P11 column (lanes 3-10), followed by a Mono S column (lanes 11-13). The specificity of the protease activity was tested by incubation of purified wild-type TFIIA or the cleavage site mutant G275A with the Mono S fraction 18 (lanes 14, 15). Nonspecfic bands are indicated with *. B) Alignment of the CRS and the cleavage site of TFIIA and MLL from different organisms, human (h), mouse (m), Xenopus (x), pufferfish (p) and Drosophila (d). The conserved CRS is boxed. Cleavage of TFIIA and MLL by Taspase1 is at D/G, indicated with a red arrow. D278 marked with ◊ is the identified N-terminal end of the β subunit of TFIIA purified from mammalian cells. The acidic stretch (residues in blue and purple) is relatively conserved in TFIIA and MLL. C) Endogenous Taspase1 was detected by a Taspase1 specific antibody in all fractions containing the protease activity.

performed using an anti-Taspase1 antibody21. Figure 1C shows that auto-cleaved Taspase1 is present in fractions with cleavage activity for TFIIA including the nuclear extracts (lane 1), the PC-C fraction (lane 4) and the Mono S fractions 17, 18 and 19 peaking in fraction 18 that has the highest cleavage activity (lanes 7-9), but not in fractions without cleavage activity. The full-length Taspase1 could not be observed in whole cell extracts 21 or nuclear extracts (lane 1) but was detectable in PC-C fraction and was further enriched in Mono S fractions 17-19. These data strongly suggest that Taspase1 is the protease for TFIIA cleavage.

Cleavage of TFIIA by Taspase1 in vitro To directly assess whether TFIIA is a substrate of Taspase1, we first tested cleavage in vitro using recombinant TFIIA and Taspase1. Recombinant wild-type Taspase1 cleaved TFIIA efficiently (Fig. 2A, lanes 1-5), while the active site mutant of Taspase1, T234A, did not cleave wild-type TFIIA (Fig. 2A, lanes 6-10). Although

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the in vitro assays showed that Taspase1 cleaves TFIIA, the determined cleavage site of MLL is different from that of TFIIA purified from cell extracts and analyzed by Edman degradation. Edman sequencing showed that cleavage in MLL occurs at D/G within the conserved CRS, -QVDG- or -QLDG-18, whereas in TFIIA, the most Nterminal amino acid of the β subunit was determined to be D278, three amino acids downstream of the CRS (Fig. 1B) 10. To resolve this ambiguity, the N-terminus of the TFIIAβ generated in vitro by recombinant Taspase1 (Fig. 2A, the marked bands in the left panel) was subjected to Edman degradation. The analysis yielded an amino acid sequence GTGDTSSE, showing that cleavage of TFIIA by Taspase1 occurred at D274/G275 (Fig. 2A). This cleavage site is within the conserved CRS that is essential for TFIIA cleavage, and it is consistent with cleavage sites of MLL by Taspase121. Having shown that TFIIA is cleaved by Taspase1 in vitro and the cleavage site is identical to that of MLL, we tested whether cleavage of TFIIA by Taspase1 has the same amino acid requirement as cleavage of TFIIA in vivo as shown previously10. A panel of mutants covering the CRS which was tested previously in vivo and wild-type TFIIA were expressed in E. coli and purified with Ni-NTA resin, and subsequently, the Ni-NTA eluates were analyzed in the in vitro assay with recombinant Taspase1. In this assay, wild-type TFIIA (Fig. 2B, lanes 1, 2) and mutants outside the CRS (data not shown) were readily cleaved by Taspase1, whereas cleavage of the CRS mutants was either completely blocked (D274A and G275A) or occurred weakly (Q272A and V273A) (Fig. 2B, lanes 7-10 and 3-6, respectively). Thus, the in vitro data match the in vivo cleavage profile (Fig. 3B,10), showing that cleavage by Taspase1 requires the CRS, and that Taspase1 is the primary protease for TFIIA and it cleaves TFIIA at D274/G275.

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Fig. 2 Cleavage of TFIIA by Taspase1 in vitro. A) Coomassie staining was performed to detect cleavage of recombinant TFIIA complex by recombinant Taspase1. wtTFIIA was incubated with different amounts of wtTaspase1 (lanes1-5) or mtTaspase1 (T234A) (lanes 6-10) as indicated. The β-subunit of TFIIA marked with ◄ was cut out from the gel and subjected to Edman analysis. Edman analysis showed that G275 is the N-terminal end of the β subunit. B) Western blot analysis was performed to test cleavage of TFIIA mutants covering the CRS. These mutants were expressed in complex with the γ subunit in E. coli, and one-step Ni-NTA purification was applied to obtain semi-purified proteins. Nonspecific bands are indicated with *.

Cleavage of TFIIA by Taspase1 in vivo To corroborate and extend our in vitro observations, we tested whether TFIIA is cleaved by Taspase1 in vivo in transient transfection assays. Expression of wildtype Taspase1 followed by western blot analysis with Taspase1 antibody against the N-terminal region of Taspase1 21 revealed two polypeptides of approximately 50 kDa and 28 kDa in size (Fig. 3A, lanes 3, 5) corresponding to the full-length Taspase1 (Taspase1-FL) and the auto-cleaved N-terminus (Taspase1-N28)18. Co-expression of TFIIA and Taspase1 led to complete cleavage of TFIIAαβ (lanes 5), while expression of mutant Taspase1 T234A that cannot undergo auto-cleavage did not change the ratio of uncleaved and cleaved TFIIA (compare lanes 6 and 2), showing that TFIIA is cleaved specifically by Taspase1 in vivo. Interestingly, we did not observe a clear increase of TFIIA α and β subunits upon complete cleavage of TFIIAαβ (compare lanes 5 and 6), suggesting that in vivo the levels of cleaved TFIIA are measured and

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maintained in cells. To assess whether the CRS is essential for Taspase1 cleavage in vivo, we again utilized the alanine scanning nning mutants covering the CRS. Without overexpression of Taspase1, mutations in the CRS either completely abolished cleavage of TFIIA (Q272A, D274A, G275A) (Fig. 3B, upper panel, lanes 4, 6, 7) or yielded only small amount of the cleaved products (V273A) (lane 5), as observed previously

10

. Co-expression of Taspase1 with wild-type TFIIA and mutants outside

the CRS resulted in significant reduction of the uncleaved αβ subunits (Fig. 3B, lower panel, lanes 2, 3 and 8-11). Mutants Q272A and V273A showed elevated cleavage in the presence of overexpressed Taspase1, but the cleaved products remained at low levels (lanes 4, 5). Importantly, mutants at the cleavage site D274A and G275A cannot be cleaved even upon overexpression of Taspase1 (lanes 6, 7), demonstrating

Fig. 3 Cleavage of TFIIA by Taspase1 in vivo. A) wild-type (wt) TFIIA was transfected either alone or together with either wild-type (wt) or mutant (mt) Taspase1 (T234A) in U2OS cells, and cleavage was analyzed by western blotting. B) TFIIA mutants covering the CRS were tested either alone or together with Taspase1 for their cleavage in U2OS. Green Fluorescent Protein (GFP) was cotransfected as the internal control. Nonspecific bands detected by Taspase1 antibody are indicated with *. C) Endogenous Taspase1 was knockeddown by RNAi duplex oligos. Control oligos (C) and Taspase1 oligos (T) were used in this experiment. To test the effect on transiently transfected TFIIA, oligos were transfected for 48 hours and removed, followed by transfection of TFIIA constructs. To test the effect on endogenous TFIIA, U2OS cells were treated with oligos for 3 and 4 days as indicated. Nonspecific bands detected by TFIIAα-specific antibody are marked with *.

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that the amino acid requirement for TFIIA cleavage in vivo is identical to that of Taspase1 and supporting the notion that Taspase1 is the primary protease for TFIIA. The role of endogenous Taspase1 in TFIIA cleavage was further tested using an RNAi approach. In an experiment with transfected TFIIA, Taspase1 RNAi knockdown led to a clear accumulation of the uncleaved TFIIAαβ subunit and a small decrease of the cleaved products (Fig. 3C, compare lanes 2 and 4). To investigate the effect on endogenous TFIIA, a prolonged treatment of Taspase1 RNAi oligos was applied, and this treatment gave rise to a clear decrease of the endogenous Taspase1 level, and concomitantly, an accumulation of the uncleaved form of endogenous TFIIAαβ and a slight decrease of the cleaved α and β subunits (compare lanes 5 and 6, lanes 7 and 8). In summary, our in vivo results show that TFIIA is a genuine substrate for Taspase1.

Regulation of Taspase1 cleavage in different cell lines We noticed previously that TFIIA is cleaved to different extents in different cell lines

15

. After Ni-NTA purification of cell extracts, a considerable amount of

Fig. 4 Regulation of Taspase1 cleavage in different cell lines. A) Endogenous TFIIA from different cell lines was purified with Ni-NTA resin and detected using a TFIIAα-specific antibody. Endogenous Tapase1 from corresponding cell lines was also visualized by western blotting and αtubulin served as the loading control. B) The ratios of TFIIAαβ to α and Taspase1 to α-tubulin were quantitated.

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uncleaved TFIIA was observed in embryonal carcinoma P19 and HeLa cells extracts, while in COS7 and U2OS extracts, the cleaved TFIIA appeared to be the major form (Fig. 4A). Quantitating the ratios of uncleaved αβ subunit to the cleaved α subunit revealed that P19 cells contained the highest amount of uncleaved TFIIA (Fig. 4B). Next, we assessed whether the difference in TFIIA cleavage is due to different expression levels of Taspase1 in the various cell types. Therefore, endogenous Taspase1 from different cell lines was monitored by western blotting (Fig. 4A) and quantitated using α-tubulin as the reference (Fig. 4B). Quantitation showed that the Taspase1 level is higher in P19 cells than in U2OS cells, which does not correlate with the presence of higher uncleaved TFIIA in P19 cells as compared to U2OS cells. These data indicate that the observed difference in efficiency of TFIIA cleavage is not controlled by different expression levels of Taspase1, suggesting that regulation of TFIIA cleavage maybe exerted by the Taspase1 activity or post-translational modification of the TFIIA cleavage site.

Fig. 5 Nuclear localization and chromatin association of overexpressed Taspase1. A) Immunoprecipitation was performed to detect interaction of TFIIA and Taspase1. pSG5-TFIIA with a myc epitope at the N-terminus was transfected either alone or together with Taspase1. IP was performed with anti-myc antibody. Input (1/5 of total), supernatant (sup, 1/10 of total) and precipitate (IP) were analyzed by western blotting. B) U2OS cells were transfected with TFIIA or protein C-tagged Taspase1 and subjected to immunostaining using TFIIAα-specific antibody and protein C epitope antibody. DAPI was used to stain the nuclei. C) Chromatin association of Taspase1 was tested using transiently transfected proteins. U2OS cells transfected with wt Taspase1 and mutant T234A were crosslinked with formaldehyde, followed by CsCl purification. The nuclear extracts and the chromatin-bound fractions were de-crosslinked and analyzed by western blotting.

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Nuclear localization and chromatin association of Taspase1 Having shown that TFIIA is a substrate of Taspase1, we tested whether Taspase1 and its substrate TFIIA can form a stable complex using transient transfection assays. Following immunoprecipitation of TFIIA, the cleaved form of Taspase1 was readily detectable in the precipitated fraction (Fig. 5A, lanes 9-12) even under stringent RIPA conditions. Since the uncleaved Taspase1 was masked by the IgG heavy chain migrating at a similar position in the precipitated fraction, the supernatant was tested. The amount of Taspase1 co-expressed with TFIIA was reduced significantly in the supernatant for both the uncleaved and cleaved forms (lane 8) compared to that of Taspase1 alone (lane 7), showing that both forms of Taspase1 interact with TFIIA. The observed interactions reinforce the notion that TFIIA is a substrate for Taspase1. Since TFIIA is a nuclear protein involved in transcription and it interacts with Taspase1, we raised the question whether TFIIA and Taspase1 are localized to the same subcellular compartment. The presence of Taspase1 activity in HeLa nuclear extracts also appeared to be at odds with our previous observations in which Taspase1 was detected mainly in the light membrane fraction of HEK 293T cells 21. To assess the subcellular localization of Taspase1 and TFIIA, we carried out immunostaining in U2OS cells. Interestingly, overexpressed Taspase1 was detected predominantly in the nucleus (Fig. 5B). The nuclear localization is consistent with the presence of a nuclear localization

signal

predicted

in

Taspase1

at

residues

200-220,

KRKLELAERVDTDFMQLKKRR (NLSdb, CUBIC). Overexpressed TFIIA was present mainly in the nucleus and weakly in the cytoplasm (Fig. 5B).

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The nuclear localization of Taspase1 combined with the fact that TFIIA and MLL are regulators of transcription prompted us to test whether Taspase1 associates with chromatin. Formaldehyde cross-linked chromatin was isolated from U2OS cells transfected with Taspase1, and purified on CsCl density gradients and analyzed for its associating proteins. Western blot analysis showed that both precursor and autocleaved forms of Taspase1 were associated with chromatin (Fig. 5C). Similarly, the overexpressed Taspase1 mutant T234A was also associated with chromatin. To confirm whether Taspase1 expressed at physiological level is associated with chromatin, we established a Taspase1 stable HeLa cell line (HT) which expressed a protein A-TEV (Tobacco Etch Virus protease) cleavage sites-myc-tagged Taspase1 (A-myc-Taspase1) at near physiological levels. To quantitate the expression level of the tagged Taspase1, we used recombinant Taspase1 as the reference (Fig. 6A, lanes 4-6), and compared expression of the tagged Taspase1 relative to that of the endogenous Taspase1 (lanes 1-3) using western blotting analysis. After removal of the protein A domain of the tagged Taspase1 by TEV protease, the level of the myctagged Taspase1 appeared to be about 2 to 3-fold higher than that of the endogenous Taspase1 (Fig. 6A, compare lanes 3 and 1), showing that it is expressed at a physiological level in HT cells, and the higher signal detected from A-myc-Taspase1 is due to cross-reaction of its protein A domain to any IgG. In this cell line, the endogenous TFIIA could be cleaved to a further extent as compared to in the wildtype HeLa cells (Fig. 6A, lanes 7, 8), showing that the tagged Taspase1 is functional. Having established that the expression level of the A-myc-tagged Taspase1 is comparable to that of the endogenous protease, we tested whether the tagged Taspase1 is associated with chromatin. Shown in Fig. 6B, formaldehyde cross-linking of HT cells for 10 or 30 minutes was sufficient to detect association of Taspase1 with

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chromatin (Fig. 6B, upper panel, lanes 7, 8). Cross-linking of Taspase1 was at least as efficient as compared to that of the TATA-box binding protein (TBP), whereas

-

tubulin was barely detectable in the chromatin fraction (Fig. 6B, lower panel, lanes 58). Furthermore, the endogenous Taspase1 was also detected in the CsCl density purified fraction (Fig. 6B, middle panel, lane 5-8). Collectively, these data show that Taspase1 is able to associate with chromatin, suggesting that cleavage of TFIIA (and maybe MLL) by Taspase1 may occur on chromatin and be a part of the transcription regulatory process. Fig. 6 Chromatin association of Taspase1 expressed at physiological level. A) A stable Taspase1 cell line, HT, was established in HeLa cells that expressed a protein A domain-2xTEV cleavage sites-myc-tagged Taspases1, and its Nterminal part (A-myc-N28) was visualized by western blotting using the Taspase1 specific antibody. To estimate the expression level of Taspase1 in this cell line, equal amount of cell extracts from wild-type HeLa cells (wt) and HT were analyzed by western blotting (lanes 1, 2), and similarly, cell extracts from HT was also analyzed following TEV protease treatment to physically separate the protein A domain from the myc-tagged Taspase1 (myc-N28) and prevent overestimation of the expression level of Taspase1 caused by the tagged protein A domain (lane 3). Recombinant Taspase1 was detected in the same western blot and used as the reference (lanes 4-6). Cleavage of the endogenous TFIIA in HT was also shown in lanes 7, 8. B) Chromatin association of A-myctagged Taspase1 was tested by CsCl purification. Wild-type HeLa (wt) and stable HT cells (HT) were cross-linked (CL) with formaldehyde for either 10 (+) or 30 (++) minutes, and nuclear extracts were subjected to CsCl purification and analyzed by western blotting. Endogenous TBP was used as the positive control, and α-tubulin in the nuclear extract preparation served as the negative control for chromatin association.

Discussion In this study, we have provided several lines of evidence that Taspase1 is the protease for TFIIA. Firstly, Taspase1 cleaves TFIIA efficiently in vitro and in vivo, whereas the TFIIA cleavage site mutants D274A and G275A cannot be cleaved by Taspase1. Secondly, knock-down of endogenous Taspase1 reduces cleavage of overexpressed and endogenous TFIIA. Thirdly, TFIIA and Taspase1 interact upon co-

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transfection and immunoprecipitation. Finally, Taspase1 is localized in the nucleus and is associated with chromatin. We showed that Taspase1 cleaves TFIIA at D274/G275 that is within the highly conserved CRS and identical to MLL21.

Both our in vitro and in vivo

experiments showed that cleavage of TFIIA by Taspase1 has the same amino acid requirement in the CRS as TFIIA cleavage by the endogenous protease10, strongly arguing that Taspase1 is the primary protease for TFIIA. The conclusion that G275 rather than D278 is the primary N-terminal residue for the β subunit is also supported by the observation that mutations of D274 and G275 abolished cleavage completely whereas mutation of D278 reduced but did not abolish cleavage (Fig. 3B)10. The Nterminal residue D278 in the TFIIAβ subunit reported previously is probably generated by a secondary protease with either an endo- or an exopeptidase activity that removes three more amino acids to generate D278 as the N-terminus. We hypothesize that TFIIA undergoes two consecutive cleavage events for TFIIA in vivo, a primary cleavage by Taspase1 and a secondary cleavage by an unknown protease. Cleavage by this second protease is dependent on the primary cleavage by Taspase1 and ultimately yields the N-terminal D278 that prone to degradation as proposed previously. Alternatively, the N-terminus at D278 might be due to proteolytic degradation during protein purification. Whether cleavage of MLL is linked to degradation as observed for TFIIA to regulate its transcriptional activity is an interesting question. In our transient transfection experiments, we did not observe a clear increase in the levels of the cleaved α and β subunits upon complete cleavage of TFIIAαβ. Moreover, in the RNAi experiments, we only observed a slight decrease of the levels of the cleaved subunits while a clear increase of uncleaved TFIIAαβ was

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detected (Fig. 3). These in vivo observations suggest that the levels of the cleaved TFIIA is measured and maintained at in cells. One possibility is that the excessive amount of the cleaved subunits is degraded through the proteosome-dependent pathway. However, upon proteosome inhibitor treatments in which proteosomedependent degradation was blocked, we still could not observe an increase of the cleaved α and β subunits when the uncleaved αβ form was completely cleaved by overexpressed Taspase1 (data not shown), suggesting that apart from the proteosomedependent pathway, there may be other mechanisms involved in maintaining the cleaved protein levels. The CRS of TFIIA is evolutionarily conserved between different species (Fig. 2B), with the exception of the large subunit of yeast TFIIA, TOA1, that does not contain a CRS and is not cleaved22. In addition to the CRS, a downstream acidic stretch is also conserved in TFIIA in different species as well as in Trx-group proteins (Fig. 2B). Apart from the CRS and acidic stretch, there is little homology in surrounding regions in different TFIIA proteins, and no overall homology between TFIIA and MLL. These findings suggest that the CRS together with acidic stretch is necessary and probably sufficient for cleavage by Taspase1. The acidic stretch may play a role in cleavage recognition or facilitate docking or positioning of the active site of Taspase1 on the CRS. TFIIA is the second identified substrate for Taspase1 so far. Searching for the CRS sequence QV/LDG in the Swissprot database revealed about 150 proteins that contain the QV/LDG sequence, and about 1/10 of these proteins contain acidic stretches (data not shown). It will be of interest to test whether they are also substrates of Taspase1. Proteolysis often leads to cleaved products that have functions distinct from those of the uncleaved precursor, such as the membrane-bound transcription factor

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SREBP that is activated by protease cleavage23. In case of TFIIA, we have previously shown that the uncleaved TFIIA interacts with TBP to form a distinct TAC complex in embryonal carcinoma P19 cells15,16. Moreover, the ratio of uncleaved (αβ+γ) to cleaved (α+β+γ) TFIIA is higher in P19 EC cells than in other differentiated cells analyzed thus far, and this difference does not appear to be due to different expression levels of Taspase1 (Fig. 4), suggesting that cleavage of TFIIA is regulated by Taspase1 activity or modifications on the cleavage site of TFIIA in different cell types or developmental stages. Therefore, cleavage of TFIIA is not likely to simply function as a step to activate the uncleaved precursor. Our data suggest that, in addition to involvement in degradation, cleavage may also generate cleaved TFIIA products that have different roles and/or associate with different subsets of proteins during differentiation and development. We hypothesize that uncleaved TFIIA associated with TBP plays a transcriptional role in embryonal stage, and after differentiation, cleaved TFIIA associated in different complexes takes over the major role in transcription. In both stages, the level of TFIIA is regulated by cleavage and proteosome-dependent degradation. Reported studies on MLL also suggest that the role of MLL cleavage is not a simple activation of the precursor. It was shown that various Hox genes were differentially affected upon Taspase1 RNAi treatment21, suggesting that uncleaved and cleaved MLL have different specificities in controlling Hox gene expression. It was also reported that cleavage generated two subunits with opposite transcriptional activities19, reinforcing that the uncleaved and cleaved forms might have distinct roles in transcription. Whether the uncleaved or the cleaved, or both forms of TFIIA and MLL are transcriptionally active remains to be elucidated. Previously Taspase1 was purified from the light membrane fraction of the cell extracts of 293 cells21. In our extracts prepared from U2OS cells, a considerable

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amount of Taspase1 could also be detected in crude cytoplasmic fractions (data not shown). Immunostaining experiments showed however that overexpressed Taspase1 was predominantly localized in the nucleus. In accordance with this observation, Taspase1 has a predicted nuclear localization signal indicating that Taspase1 can be transported into the nucleus and may have a function in the nucleus. One intriguing explanation for the observed discrepancy is that subcellular localization of Taspase1 is a regulated process or cell-type specific. Importantly, in U2OS and HeLa cells, Taspase1 is in the nucleus and associated with chromatin, which suggests that cleavage of TFIIA, MLL and other substrates by Taspase1 might occur on chromatin. The fact that two substrates of Taspase1 identified so far, TFIIA and MLL, are transcription factors, supports our hypothesis that cleavage might be transcriptionally linked. Cleavage and subsequent degradation might control and regulate the levels of transcription factors that are substrates of Taspase1, and play a role in transcription regulation. By controlling cleavage and transcriptional activities of TFIIA, MLL and other substrates, Taspase1 is likely to be an important player in development. To understand the role of Taspase1 in transcription and development, the link between control of cleavage by Taspase1 and regulation of specific target genes of TFIIA and Trx-group proteins needs to be investigated in more detail.

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TFIIA IS CLEAVED BY TASPASE1

Material and Methods Plasmids and antibodies Mammalian expression vectors, myc-tagged TFIIAαβ (pSG5-myc-TFIIAαβ), its CRS mutants (alanine mutants from L271-T279), HA-tagged TFIIAγ (pSG5-HATFIIAγ), green fluorescent protein construct (pEGFP-N1)10, pcDNA-Taspase1 and its mutant T234A21 were described previously. TFIIAαβ and γ genes were subcloned from their mammalian expression vectors into a single polycistronic vector pST39

24

between the SacI and KpnI sites and XbaI and BamHI sites, respectively, to generate pST-IIAγαβ for expression in E. coli. The CRS mutants (Q272A, V273A, D274A and G275A) in pST vector were generated by excision of the MscI and NotI fragments containing the mutated sequences from the mammalian vectors, and replacement of the wild-type sequence in pST-IIAγαβ.

Oligonucleotides, pRAV-myc1f (5’-

AATTTAATGGAGCAGAAGCTTATCAGCGAGGAGGACCTGGGCGGGG-3’) and pRAV-myc1r (5’-AATTCCCCGCCCAGGTCCTCCTCGCTGATAAGCTTCTG CTCCATTA-3’), containing the myc epitope coding sequence and were annealed and cloned into pRAV-FLAG at the EcoRI site25 to generate pRAV-myc. Subsequently, the EcoRI and BamH1 fragment from pRAV-myc containing one Protein A domain, two TEV cleavage sites and a myc epitope was cloned into PZ-1-N (Cellzome) to generate PZXN. PZXN-Taspase1 construct for viral infection was generated by inserting Taspase1 gene into PZXN at the EcoRI site. Protein C antibody was purchased from Roche Molecular Biochemicals. Monoclonal myc antibody, polyclonal GFP antibody, polyclonal TFIIAα-specific, β-specific and γ-specific15 and Taspase121antibodies were previously described.

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Cell

culture,

transient

transfection,

RNAi,

protein

extraction,

immunoprecipitation, Immunostaining Maintenance and transfection of U2OS cells and extract preparation were performed as previously described10. Immunoprecipitation was performed with the anti-myc monoclonal antibody under RIPA wash conditions15. RNAi knock-down of Taspase1 was carried out using duplex RNAi oligos as described previously21. After 48-hour RNAi treatment of U2OS cells, RNAi oligos were removed and cells were transfected with TFIIA plasmids. To detect the effect of Taspase1 RNAi knock-down on endogenous TFIIA, U2OS cells were treated with RNAi oligos for 3 and 4 days. For immunostaining experiments, U2OS cells were seeded and transfected on cover slips. Forty hours after transfection, cells were fixed in 4% paraformaldehyde for 10 minutes, and then permeabilized in 0.2% TritonX-100. Following blocking in 0.5% BSA, cells were incubated in the anti-TFIIAα-specific antibody to detect TFIIA or an anti-protein C epitope antibody to detect the protein C-tagged Taspase1. Subsequently, cells were incubated in corresponding secondary antibodies conjugated with either FITC or Texas Red dyes, and visualized under fluorescence microscope.

Establishment of a protein A-myc-Taspase1 stable cell line in HeLa cells Phoenix amphotropic packaging cell line was transfected with the retroviral plasmid PZXN-Taspase1 using calcium phosphate method. Forty-eight hours after transfection, virus containing supernatant was filtered through a 0.22-µm filter, and 105 HeLa cells were transduced with 3ml filtered virus containing supernatant in the presence of 8µg/ml of polybrene for two infection rounds of 24 hours each. Cells were then recovered for 24 hours, and a polyclonal pool of cells was selected with

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1µg/ml of puromycin. Subsequently, individual clones were picked up and screened for physiological expression.

Protein expression and purification, Edman sequencing The polycistronic expression plasmid pST-IIAγαβ (and its CRS mutants) carrying both TFIIAαβ (and its mutants) and TFIIAγ genes was transformed into BL21(DE3)plysS cells, and induced with 0.2 mM IPTG. Overexpressed wild-type TFIIAαβ and TFIIAγ proteins were purified as a complex through Ni-NTA, Mono Q, Mono S columns to nearly homogeneity. The purified TFIIA complex was functionally assayed in EMSA after each purification step. TFIIAγαβ CRS mutants expressed from pST constructs were semi-purified with Ni-NTA resin and eluted with 250 mM imidazole before subjected to the in vitro protease assays. Endogenous TFIIA from P19, COS7, HeLa and U2OS cell extracts were semi-purified with NiNTA resin, eluted with 250mM imidazole and analyzed by western blotting. Quantitation of TFIIA subunits was performed by Phophorimager (Molecular Dynamics) using ECL plus (Amersham). To purify the protease activity for TFIIA cleavage, HeLa nuclear extracts were prepared in high salt buffer containing Hepes pH 7.8, KCl 50 mM, NaCl 300 mM, EDTA 0.1 mM, DTT 1 mM, glycerol 10%, PMSF 0.1 mM, 1x complete protease inhibitors (Roche Molecular Biochemicals), and fractionated on a P11 column, followed by step elutions with 100, 300, 500 and 1000 mM KCl. The PC-C fraction (500 mM fraction) containing the protease activity was further fractionated on a Mono S column. For the Mono S column, a gradient from 10-1000 mM KCl was applied, and the activity eluted at 300 mM KCl. Protease activity was monitored with the in vitro protease assay. To perform Edman N-terminal sequencing analysis, recombinant TFIIA was digested with recombinant Taspase1

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followed by SDS-PAGE stained with Coomassie. The band corresponding to the β subunit was excised and subjected to Edman N-terminal sequencing analysis as described before18

In vitro protease assay. The purified recombinant TFIIA complex and semi-purified TFIIA (and its mutants) were incubated at 37°C with the recombinant Tapase1 for 1 hour or with HeLa nuclear fractions for 12 hours. The reaction buffer contained 20 mM Tris, pH 8.0, 100 mM KCl, 0.2 mM EDTA, 2 mM DTT, 10% glycerol. The reaction mix was subsequently analyzed by western blotting, and probed for TFIIAαβ, α, β and γ subunits using respective antibodies. A-myc-Taspase1 in HeLa cells extracts was incubated with Tobacco Etch Virus protease (TEV) at 37°C for 1 hour to cleave the protein A domain. TEV protease was purchased from Invitrogen.

Chromatin cross-linking and preparation, CsCl purification U2OS cells transfected with pcDNA-Taspase1 and T234A mutant were crosslinked in 11% formaldehyde for 30 minutes after 40-hour transfection. Wild-type HeLa cells and the Taspase1 stable HeLa cell line, HT, were cross-linked in formaldehyde for either 10 or 30 minutes.

Chromatin preparation and CsCl

purification of chromatin-bound fractions were performed as previously described15

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Acknowledgements We thank Dr. Salvatore Spicuglia for critical reading and comments on the manuscript and we thank Michiel Vermeulen for providing reagents. This work is supported by grant 812.08.006 from NWO (ALW) in the Netherlands.

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Yokomori, K. et al. Drosophila TFIIA directs cooperative DNA binding with TBP and mediates transcriptional activation. Genes and development 8, 231323 (1994). Reinberg, D. & Roeder, R. G. Factors involved in specific transcription by mammalian RNA polymerase II. Transcription factor IIS stimulates elongation of RNA chains. Journal of biological chemistry, The 262, 3331-7 (1987). Ozer, J. et al. Molecular cloning of the small (gamma) subunit of human TFIIA reveals functions critical for activated transcription. Genes and development 8, 2324-35 (1994). Yokomori, K., Admon, A., Goodrich, J. A., Chen, J. L. & Tjian, R. Drosophila TFIIA-L is processed into two subunits that are associated with the TBP/TAF complex. Genes and development 7, 2235-45 (1993). Lieberman, P. M., Ozer, J. & Gursel, D. B. Requirement for transcription factor IIA (TFIIA)-TFIID recruitment by an activator depends on promoter structure and template competition. Molecular and cellular biology 17, 662432 (1997). Lieberman, P. M. & Berk, A. J. A mechanism for TAFs in transcriptional activation: activation domain enhancement of TFIID-TFIIA--promoter DNA complex formation. Genes and development 8, 995-1006 (1994). Stargell, L. A., Moqtaderi, Z., Dorris, D. R., Ogg, R. C. & Struhl, K. TFIIA has activator-dependent and core promoter functions in vivo. Journal of biological chemistry, The 275, 12374-80 (2000). Stargell, L. A. & Struhl, K. The TBP-TFIIA interaction in the response to acidic activators in vivo. Science 269, 75-8 (1995). Ranish, J. A., Yudkovsky, N. & Hahn, S. Intermediates in formation and activity of the RNA polymerase II preinitiation complex: holoenzyme recruitment and a postrecruitment role for the TATA box and TFIIB. Genes and development 13, 49-63 (1999). Hoiby, T. et al. Cleavage and proteasome-mediated degradation of the basal transcription factor TFIIA. EMBO journal, The 23, 3083-91 (2004). Bleichenbacher, M., Tan, S. & Richmond, T. J. Novel interactions between the components of human and yeast TFIIA/TBP/DNA complexes. Journal of molecular biology 332, 783-93 (2003). Geiger, J. H., Hahn, S., Lee, S. & Sigler, P. B. Crystal structure of the yeast TFIIA/TBP/DNA complex. Science 272, 830-6 (1996). Tan, S., Hunziker, Y., Sargent, D. F. & Richmond, T. J. Crystal structure of a yeast TFIIA/TBP/DNA complex. Nature 381, 127-51 (1996). Han, S. Y. et al. TFIIAalpha/beta-like factor is encoded by a germ cell-specific gene whose expression is up-regulated with other general transcription factors during spermatogenesis in the mouse. Biology of reproduction 64, 507-17 (2001).

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Mitsiou, D. J. & Stunnenberg, H. G. TAC, a TBP-sans-TAFs complex containing the unprocessed TFIIAalphabeta precursor and the TFIIAgamma subunit. Molecular cell 6, 527-37 (2000). Mitsiou, D. J. & Stunnenberg, H. G. p300 is involved in formation of the TBPTFIIA-containing basal transcription complex, TAC. EMBO journal, The 22, 4501-11 (2003). Sun, X., Ma, D., Sheldon, M., Yeung, K. & Reinberg, D. Reconstitution of human TFIIA activity from recombinant polypeptides: a role in TFIIDmediated transcription. Genes and development 8, 2336-48 (1994). Hsieh, J. J., Ernst, P., Erdjument-Bromage, H., Tempst, P. & Korsmeyer, S. J. Proteolytic Cleavage of MLL Generates a Complex of N- and C-Terminal Fragments That Confers Protein Stability and Subnuclear Localization. Molecular and cellular biology 23, 186-94 (2003). Yokoyama, A., Kitabayashi, I., Ayton, P. M., Cleary, M. L. & Ohki, M. Leukemia proto-oncoprotein MLL is proteolytically processed into 2 fragments with opposite transcriptional properties. Blood 100, 3710-8 (2002). Daser, A. & Rabbitts, T. H. Extending the repertoire of the mixed-lineage leukemia gene MLL in leukemogenesis. Genes and development 18, 965-74 (2004). Hsieh, J. J., Cheng, E. H. & Korsmeyer, S. J. Taspase1: a threonine aspartase required for cleavage of MLL and proper HOX gene expression. Cell 115, 293-303 (2003). Ranish, J. A., Lane, W. S. & Hahn, S. Isolation of two genes that encode subunits of the yeast transcription factor IIA. Science 255, 1127-9 (1992). Sundqvist, A. & Ericsson, J. Transcription-dependent degradation controls the stability of the SREBP family of transcription factors. Proceedings of the National Academy of Sciences of the United States of America 100, 13833-8 (2003). Tan, S. A modular polycistronic expression system for overexpressing protein complexes in Escherichia coli. Protein expression and purification 21, 224-34 (2001). Knuesel, M. et al. in Molecular and cellular proteomics MCP 1225-33 (2003).

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6 REGULATION OF TFIIA DEGRADATION BY THE ARGININE tRNA TRANSFERASE 1 Torill Høiby, Chris Brower, Alexander Varshavsky and Hendrik G. Stunnenberg

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Abstract The N-end rule states that the stability of a protein is dependent on its Nterminus, and the various amino acids can consequently be categorized as primary/secondary destabilizing or stabilizing residues. ATE1 recognises N-terminal D and E and converts them to substrates for proteasomal degradation. The proteolytical cleavage of the general transcription factor TFIIA has been shown to promote its proteasomal degradation, and the cleavage-generated N-terminus of TFIIAβ, D278, is a secondary destabilizing residue according to the N-end rule. We found that TFIIA was stabilized in an ATE1 knock-out cell line compared to a wildtype cell line. Co-expression of ATE1 and TFIIA lead to a faster degradation of TFIIA wild-type, but did not affect the stability of the uncleavable mutant TFIIA G275A. Collectively, our data suggest that TFIIA is a substrate for the N-end rule through cleavage and ATE1-mediated proteasomal degradation, and indicate that TFIIA may be the first physiological substrate for proteasomal degradation through an N-terminal Asp.

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Introduction The activity of most transcription factors is tightly regulated because cells need to swiftly respond to environmental changes by altering its gene expression profile. Several mechanisms contribute to supply cells with the necessary plasticity for progression through the cell cycle, commitment to a particular differentiated state or response to stress. Protein activity can be regulated through post-translational modification, exemplified by the interaction between cAMP-responsive element binding protein (CREB) and CREB-binding protein (CBP)1 and subcellular relocalisation which is important in the Smad signaling pathway2. Protein activity can also be controlled through proteasomal degradation of which important examples are the consecutive degradation of cyclins enabling progression through the cell cycle and the degradation of SCC1 facilitating chromatin segregation3,4. This form of regulation may be crucial to ensure irreversibility in processes where uni-directionality is critical. Various proteolytical processes have been implicated in destruction of transcription factors, for example the contribution of calpain-mediated proteolysis to degradation of p535 and the selective lysosomal proteolysis of IκB6. The most commonly used pathway for protein disposal, however, is poly-ubiquitin-mediated proteasomal degradation7. The proteasomal degradation system involves conjugation of Ub to the substrate through the sequential action of the ubiquitin-activating enzyme E1, the ubiquitin-conjugating enzyme E2 and the ubiquitin ligase E38,9. Several substrates have been reported to contain an element, a so-called degron, signaling ubiquitylation. Most degrons are poorly defined, but amongst the known ones are cyclin destruction boxes10, regions rich in proline, glutatamate, serine and threonine (PEST sequences)11 and N-end rule degrons12. In addition to the destabilizing N-

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terminal residue, the degradation signal in an N-end rule substrate consists of a lysine that serves as a determinant for (poly)ubiquitylation. The N-end rule has a hierarchal organization, where Asp is a secondary destabilizing residue that must undergo arginylation by arginine-tRNA transferase (ATE1) (see figure 1) to be recognized by the ubiquitin ligase13-15. It has been shown that the presence of ATE1 is required for cardiogenesis and angiogenic remodeling during mouse embryogenesis, but the mechanistic background for this is unknown.

Figure 1 The N-end rule pathway for the type 1 substrates Asp and Glu. N-terminal residues are denoted by single letter abbreviations for amino acids. The ovals represent the rest of a protein substrate. Primary destabilising residues are recognised by functionally overlapping E3s that 15 include UBR1 (E3α) and UBR2 .

The physiological significance of the N-end rule is unclear because few substrates have been found in nature16. The first bona fide substrate to be identified was the cohesion subunit SCC1 that undergoes cleavage and subsequent degradation through its exposed N-terminal Arg, and whose degradation is essential for chromatin stability3. Furthermore, the γ2 subunit of the G protein heterotrimer is targeted for degradation through cleavage and exposure of Arg17, and a recently identified N-end substrate, DIAP1, undergoes caspase-mediated cleavage and exposure of Asn, followed by degradation through deamidation and arginylation by ATE118. The human general transcription factor TFIIA consists of the three subunits α, β and γ, of which TFIIAαβ is expressed from one gene and post-translationally cleaved19,20. Recently, the protease responsible for TFIIA cleavage was identified (Zhou et al. manuscript in preparation), and found to be Taspase1, a protease known to cleave MLL. The function of cleavage has remained elusive because uncleaved and

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cleaved forms of TFIIAαβ are equally able to interact with TFIIAγ, bind DNA, support trans-activation and activate transcription in vivo21. It appears, though, that cleavage affects the half-life of TFIIA and is a pre-requisite for proteasome-mediated degradation. Because of the link between cleavage of TFIIA and its degradation, in addition to the cleavage-mediated exposure of an N-terminal Asp, we wished to assess the role of ATE1 in the regulation of TFIIA stability. We found that over-expressing ATE1 and TFIIA results in faster degradation of TFIIA in U2-OS cells, compared to when ATE1 is omitted. Importantly, ATE1 over-expression does not appear to affect the stability of the uncleavable TFIIA mutant G275A. Further, we find that in an ATE1 knock-out cell line, endogenous TFIIA subunits are stabilized compared to a wildtype MEF cell line. Our findings indicate that TFIIA is the first identified protein to undergo degradation through arginylation of an N-terminal Asp.

Results and Discussion ATE1 over-expression leads to increased degradation of TFIIAα, -β and -γ We have previously shown that TFIIA is degraded via the proteasome and that cleavage is a pre-requisite for this degradation21. Since Edman sequencing of the purified TFIIAβ in FM3A cells identified the N-terminus as Asp, which is a secondary destabilizing residue according to the N-end rule, we wished to study the role of the N-end rule pathway in TFIIA degradation. We therefore set out to test the effect of ATE1 on TFIIA levels by co-transfecting the proteins in U2-OS cells. Analysis by western blotting revealed that over-expressing ATE1 lead to a reduction in the steady-state levels of TFIIAα, TFIIAβ and TFIIAγ (Figure 2A, compare lane 2 and 3), whereas the levels of TFIIAαβ remained unchanged (Figure 2A and B). This

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is consistent with ATE1 having a role in TFIIA stability following cleavage. Green fluorescent protein (GFP), which was used as a control, was unaffected by ATE1, demonstrating that there was no global effect on transfection efficacy or protein expression. Levels of TFIIAα, β and γ subunits inversely correlated with the ATE1 levels and TFIIA stability was reduced upon increasing amounts of transfected ATE1 (Fig. 2B, compare lanes 5-8). At high levels of ATE1, also the level of TFIIAαβ was modestly affected, albeit clearly less than the cleaved subunits of TFIIA.

Figure 2 Effect of ATE1 on TFIIA stability (A) Extracts from U2-OS cells transfected with TFIIIA and ATE1 were analysed by western blotting. GFP was added as a control. (B) Extracts from U2-OS cells transfected with TFIIA and increasing concentrations of ATE1 were analysed by western blotting.

To investigate the physiological relevance of ATE1 for TFIIA, we compared TFIIA levels in MEF ATE1+/+ and MEF ATE1-/- cells. The MEF ATE-/- cells have previously been demonstrated to lack the N-end degradation pathway for proteins with secondary destabilizing N-termini, establishing the role of ATE1 for the degradation of these substrates15. We found that the levels of the endogenous, cleaved TFIIA subunits and particularly TFIIAβ were stabilized in the MEF ATE1-/- cells (Figure 3A, compare lanes 1 and 2), while the level of TFIIAαβ was largely unchanged. α-tubulin as a control confirmed that equal amounts of extracts were used. These results suggest that ATE1 affects the stability of endogenous TFIIA and further support the model that TFIIA is a substrate for the N-end rule. To further establish that the ATE1-induced reduction in TFIIA stability was a result of increased

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turn-over of the protein, the stability of transfected TFIIA was studied in the presence of cycloheximide to prevent de novo protein synthesis. The degradation rates of the various TFIIA subunits were compared in MEF ATE1-/- cells without and with restorage of ATE1 expression. At time=0 the levels of TFIIAαβ were similar, whether or not ATE1 was expressed (figure 3B compare lanes 1 and 5). In contrast to that, levels of TFIIAα, β and γ were considerably reduced when ATE1 was expressed (Figure 3B, compare lanes 1 and 5). At later time points, all TFIIA subunits were dramatically reduced upon ATE1 expression, and after 24 hours the protein was hardly detectable. Interestingly, the TFIIAαβ appeared to have a faster turn-over in the presence of ATE1 (Figure 3B, compare lanes 2, 3 and 4 to lanes 6, 7 and 8) in this setting, which could reflect increased cleavage as a response to accelerated depletion of the processed subunits.

1 2

3

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3

4

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8

Figure 3 Effects of ATE1 on -/TFIIA in MEF ATE1 and MEF +/+ ATE1 cells (A) Extracts from -/MEF ATE1 cells and MEF +/+ cells were analysed by ATE1 western blotting. (B) Extracts -/cells from MEF ATE1 transfected with TFIIA (lanes 1-4) and TFIIA + ATE1 (lanes 5-8) and treated with cycloheximide were analysed by western blotting. -/(C) Extracts from MEF ATE1 cells transfected with the TFIIA G275A mutant (lanes 1-3) and ATE1 (lanes 4-6) and treated with cycloheximide were analysed by western blotting.

Previously, we have found that inhibition of cleavage through a mutation in the CRS prolongs the half-life of TFIIA21. The increased stability of an uncleavable TFIIA mutant could be explained if the increase in TFIIA degradation upon ATE1 expression is due to arginylation of the exposed Asp on TFIIAβ. If this is true, ATE1 over-expression should not affect the level of uncleavable TFIIA. To this end, we employed the mutant TFIIAαβ G275A and compared it to TFIIA wild-type. In 143

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contrast to wild-type TFIIA, ATE1 expression has no effect on the stability of TFIIAαβ G275A (figure 3C, compare lanes 1, 2, 3 to lanes 4, 5, 6). Taken together, our data show that ATE1 expression leads to degradation of TFIIAα, -β and -γ. Importantly, ATE1 has no effect on stability of the uncleavable mutant TFIIAαβ G275A, supporting the hypothesis that cleavage of TFIIA generates a substrate for N-end coupled degradation of TFIIA through its exposed Asp. Surprisingly, the results showed that the cleavage efficacy or degradation rate of TFIIAαβ is increased upon ATE1 expression, suggesting a feedback loop that serves to generate more cleaved TFIIA as a result of increased degradation of the protein. To investigate whether the ATE1-induced increase in protein degradation depended on the amino acid identity of the N-end of TFIIA, we compared the stability of TFIIA mutants with different N-termini. According to the N-end rule, replacing Asp with Met should stabilize TFIIA, whereas Arg should have a destabilizing effect. To investigate the half-life of the respective TFIIA mutants, experiments were performed in the presence of cycloheximide to prevent de novo protein synthesis, and

B 120

% of initial TFIIA

100 80 60 40 20 0

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2

-A TE1/+ A TE1

4 3.5 3 2.5 2 1.5 1 0.5 0

D278M

8

Figure 4 Effect on TFIIA stability by mutating D278 (A) Extracts from U2-OS cells transfected with TFIIA or mutants as depicted and treated with cycloheximide were analysed by western blotting. (B) The results from (A) were quantified and represented graphically. (C) Extracts from U2-OS cells transfected with TFIIA or TFIIA+ATE1 and treated with cycloheximide were analysed by western blotting and the results were quantified and represented as concentration of TFIIA-ATE1/concentration of TFIIA+ATE1 (figure 4A and B from21)

4.5

WT

4

D278R

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the stability of the respective TFIIA subunits was monitored over time. Figure 4A demonstrates that a severe complication in the interpretation of these results is the effect on cleavage efficacy upon mutating the residue D278. The mutants are cleaved less efficiently than wild-type and because of the continuous generation of cleaved TFIIAα and β over time, it is not possible to accurately determine the half-life of the cleaved subunits (Fig. 4, compare lanes 1-4 with 5-8 and 9-12). Because of these problems, the data were interpreted by comparing the half-life of each single mutant by adding the total level of TFIIAαβ+TFIIAα+TFIIAβ. The level of wild-type TFIIA is reduced to 20% after 8 hour, whereas D278M remains roughly unchanged and D278R is halved (Fig. 4A, B). To investigate the effect of ATE1 on the respective mutants, ATE1 was co-expressed with TFIIA and the stability was quantified as described above. After 8 hours, over-expressing ATE1 lead to 4-5 fold increase in protein turn-over of wild-type TFIIA (Figure 4C). In contrast, restoring ATE1 expression did not significantly affect the levels of TFIIA D278M or TFIIA D278R (Figure 4B, C). Taken together, these results suggest that the ATE1-mediated effect on stability of TFIIA is dependent on the identity of residue 278. The mutant D278M is stabilized and D278R is destabilized compared to TFIIA wild-type, whereas neither D278M nor D278R are affected upon ATE1 expression, suggesting a role for D278 in substrate recognition. Our data suggest that ATE1 may play a role in TFIIA degradation and that TFIIA is a substrate for the N-end rule.

The CRS is conserved in ALF We have identified the cleavage re cognition site (CRS) of TFIIA, which is a stretch of four residues essential for cleavage21. The CRS is conserved in the proto-

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Figure 5. The CRS is essential for efficient cleavage of ALF. Extracts from U2-OS cells transfected with ALF and mutants as depicted were analysed by western blotting.

oncogene MLL and in the TFIIA homologue ALF. This strongly suggests that the CRS is essential also for cleavage of the ALF, and furthermore, that the CRS and the responsible protease may serve a more global role regulating other proteins with a conserved CRS. To test the role of the CRS for efficient cleavage of ALF, single residues within the CRS were mutated to alanine, and the mutants were analysed. Our results showed that any single mutation within the CRS virtually abolished cleavage (Figure 5), consistent with the results from TFIIA21. Importantly, the effect is dramatic when D354, analogous to TFIIA D278, was mutated to Ala, supporting the notion that this residue is crucial for cleavage of ALF.

Conclusions The intracellular levels of many short-lived transcription factors are largely determined by the rate of proteasomal degradation rather than de novo synthesis, as illustrated by p53, whose activity is mainly regulated through protein stability22. TFIIA was found to have an intermediate half-life (a few hours)21. Our results suggest that cleavage of TFIIA, rather than causing a rapid protein removal, places a ‘timer’ on it and marks it irreversibly for destruction. It has been argued by Reinberg et al. that the two forms of TFIIA (uncleaved and cleaved) may fulfill the same function, but that one may be more efficient than the other19,23. It is conceivable that the two

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forms have similar or largely overlapping functions in the cell, the main difference being that the cleaved form is primed for destruction. This would permit the protease activity to regulate the stability of the protein without significantly altering the function of it, and the cell could even regulate cleavage of TFIIA to keep a ‘pool’ of stable TFIIA (i.e. the TAC form of TFIIA). Our hypothesis does not exclude, however, that the two forms of TFIIA have promoter-specific functions. The discrepancy between the N-termini of TFIIAβ cleaved in vitro (Zhou et al., manuscript in preparation) and in vivo21 may be a result of a secondary cleavage event with either an endo- or an exopeptidase activity. The fact that only D278 was identified in the Edman analysis of the FM3A-derived material suggests that the second event is rapid after primary cleavage. However, the relatively long in vivo half-life of cleaved TFIIA may indicate that the N-terminal Asp is initially inaccessible for ATE1 until an unknown step exposes it. The high conservation of the CRS and the adjacent Asp in other proteins, including ALF and MLL, indicate that this manner of regulating protein levels/activities has a more global function and could involve other proteins as well.

Materials and methods Cell lines, plasmids, antibodies, protein extracts, SDS-PAGE, immunoblotting, mutagenesis U2-OS cells21 and MEF ATE-/- AND+/+ cells15 were cultivated as described earlier.

Transfections, protein extracts, SDS-PAGE and western blotting were

performed as described earlier24. PSG5-TFIIAαβ, pSG5-TFIIAγ, pSG5-myc-ALF21 and pCATE15 have all been described. Plasmid pSG5-Myc-hALF was used for

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mutagenesis according to the manufacturer’s instructions (Quick Site-directed Mutagenesis, Stratagene)..

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7 SUMMARY AND GENERAL DISCUSSION

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Why is TFIIAαβ cleaved? This question was the main motivation for initiating the work described in this thesis. Despite it being a fundamental aspect of TFIIA -TFIIA likely undergoes cleavage in all higher eukaryotes– this is a subject that hitherto has remained largely ignored and unexplained. Initially, most or all cellular TFIIA was found to be cleaved, suggestive of a lack of a physiological role of uncleaved TFIIA and regulation of cleavage, and these findings were not considered interesting enough to invest time and effort. Renewed interest into the topic was evoked by the identification of the cell-specific protein complex TAC, consisting of the uncleaved form of TFIIA together with TBP. Its presence in a subset of cell lines indicated that cleavage of TFIIA is regulated and that the regulation may be linked to the differentiation state of the cell. These findings strongly called for further studies addressing the characteristics of the cleavage process. We specifically asked ourselves the fundamental questions: What is the cleavage site of TFIIA and what are the requirements for cleavage? What is/are the protease(s) responsible for cleavage of TFIIA? What other factors regulate the timing and process of TFIIA cleavage? How does cleavage of TFIIA relate to general transcription? And ultimately, why is TFIIA cleaved and what are the functional differences between the two forms of TFIIA? The complexity of the gene-specific regulation in Eukarya that was unveiled by the discovery of multiple forms (I, II and III) of polymerase in addition to separate sets of general transcription factors, now represents only the very beginning of a yet on-going uncovering of the remarkable machinery that regulates eukaryotic gene transcription (Chapter 1). Eukaryotic gene regulation requires distinct multi-protein complexes to modulate chromatin structure, to imprint a transcriptional signature through covalent modifications (“histone/protein code”), to bind to DNA elements (enhancers, promoters), to communicate between the basal transcription machinery

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and activators as well as to generate accurate transcripts. Recent technical developments like chromatin immunoprecipitations (ChIP) and ChIP-on-chip allow studies as to how promoters stage the interplay between these factors. The ongoing research in the transcription field is by a large amount focused on mapping this using massive data collections and global target site analyses. TFIIA was identified as one of the general transcription factors in the initial screening of factors involved in assembly of the preinitiation complex and transcriptional activation, but later research has shown that depleting TFIIA gives but minor effects on general RNA pol II transcription, in contrast to depletion of any other general transcription factors (Chapter 2). Thus, TFIIA is probably not critical for transcription of all genes, and defining it as a general transcription factor may even be misleading. Nevertheless, TFIIA is essential in yeast and probably has an important role on a subset of promoters, for example on cell cycle regulating genes. The regulation of TFIIA levels has been shown to be involved in the cellular gene program change upon differentiation and viral infection, and cleavage of TFIIA, which has specifically evolved in higher eukaryotic organisms, may add another level of TFIIA regulation, evolved to fine-tune the transcriptional activity of the general transcription factor. The interaction of the uncleaved form of TFIIA with TBP in TAC is remarkably strong, compared to the interaction between TFIIA and TFIID. This suggests that interactions specific for TAC are formed and that the uncleaved form of TFIIA may have a different affinity for TBP than the cleaved form. This hypothesis was tested by studying a large number of TBP surface mutants for their ability to form TAC (Chapter 3), and the study reveals that helix 2 in TBP is critically involved in TAC formation whereas the stirrup region contributes to the overall stability of TAC.

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These conclusions are in contrast to earlier studies of the TFIIA-TFIID interactions, where the stirrup region was shown to be essential for formation of a DA complex and to support activated transcription in vivo1. In addition, single mutations in TBP had less severe effects on TAC assembly than on the previously assessed formation of a functional TFIIA-TFIID complex. An explanation for this is the formation of additional interactions between TBP and TFIIA in the TAC complex, and then specifically in the convex region of TBP. The uncleaved and cleaved forms of TFIIA could therefore associate in different complexes and may have separate roles in regulation of transcription. However, the cleavage of TFIIA had never been paid much attention; the cleavage site had not been identified and the responsible protease(s) remained unknown. To characterize the cleavage site, the N-terminus of the TFIIAβ-subunit was determined by Edman degradation to be D278 (Chapter 4). Mutational studies of the adjacent region revealed that the molecular determinants for the cleavage event are a string of four residues N-terminal of D278, named the cleavage recognition sequence (CRS). The CRS appears critical for efficient cleavage of TFIIA and any single mutant almost entirely inhibited cleavage. These mutants allowed the functional comparison of an uncleavable form of TFIIA with the wild-type TFIIA, but to our surprise the studies revealed no differences between the two with regard to binding TBP, stabilizing of TBP binding to DNA and activation of transcription in vivo. Despite the fact that uncleaved and cleaved TFIIA may be present in different transcriptionally potent complexes, there appears to be no significant differences in functional characteristics, at least as far as ‘classical’ TFIIA function is considered. However, the transcriptional variation between uncleaved and cleaved TFIIA could be promoter-specific, and this issue should be pursued in the future.

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Recent results from studying MLL cleavage, a human homologue of the Drosophila Trithorax protein, revealed a cleavage recognition site –Q[V/L]DG- which is virtually identical to the CRS in TFIIAαβ2,3. Taspase1 was identified as the protease responsible for MLL cleavage, and consistent with the conserved CRS, was successively found to cleave TFIIA as well (Chapter 5). Experiments performed both in vitro and in vivo demonstrated that Taspase1 cleaves TFIIA efficiently and specifically, and knock-down of Taspase1 in U2-OS cells reduces the efficacy of endogenous TFIIA cleavage, confirming that Taspase1 is the genuine protease responsible for TFIIA cleavage. Interestingly, Taspase1 is localized in the nucleus and associated with chromatin, implying that Taspase1 is active on DNA. Given that the substrates identified so far, MLL and TFIIA (and ALF) are transcription factors, it is tempting to speculate on a role for cleavage in transcriptional regulation. Studies have indeed shown that various Hox genes were affected differently upon Taspase1 RNAi treatment2, suggesting that uncleaved and cleaved MLL have different specificity in control of Hox gene expression. Cleavage might control and regulate the levels of transcription factors that are substrates of Taspase1, and in turn play a role in transcription regulation. N-terminal sequencing of Taspase1-cleaved, recombinant TFIIA revealed that the cleavage site was at D274/G275, consistent with the cleavage site of MLL, but contrasting previous data obtained from our in vivo experiments (Chapter 4). These discrepancies may be a result of a secondary cleavage event with either an endo- or an exopeptidase activity. The conservation of the N-terminal residue of TFIIA, Asp, throughout evolution suggests that this residue is important. Furthermore, mutation of this residue reduces cleavage of TFIIA and inhibits cleavage of ALF.

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One important difference between cleavable and uncleavable forms of TFIIA emerging from these studies was a difference in stability; prevention of cleavage by single mutations in the CRS increased the half-life by 3-4 fold, and cleavage of TFIIA appeared to be prerequisite for proteasome-dependent degradation (Chapter 4). Thus, these data support a link between cleavage and the turn-over of TFIIA. Cleavage of TFIIA creates an N-terminal Asp, which is a secondary destabilizing residue according to the N-end rule4. The N-terminal Asp, though highly conserved in TFIIA of all higher eukaryotes, is important but not essential for cleavage. It is therefore possible that the Asp is conserved mostly to render the cleavage product unstable and that TFIIAαβ represents a stable pro-N-degron, which upon cleavage by one or more protease activities exposes an unstable Asp-bearing N-degron (TFIIAβ). Secondary destabilizing residues are recognized by ATE1, which conjugates to it an Arg, turning it into a primary destabilizing residue and a substrate for an N-end rule E3 ligase. Upon testing the effect of ATE1 on TFIIA stability, we find that over-expression of ATE1 destabilises the cleaved subunits of TFIIA whereas the uncleavable form is non-affected, arguing that ATE1 affects the degradation of the TFIIA subunits subsequent to cleavage (Chapter 6). This was supported by studies in ATE1-knockout cells where endogenous TFIIA appears to be stabilized compared to that of wildtype cells. A similar scenario is the caspase-mediated cleavage of DIAP1 (Drosophila IAP1) that converts the more stable full-length protein into a highly unstable Asnbearing N-degron and its subsequent degradation by the N-end rule pathway is essential for regulation of apoptosis5,6. There are some important aspects to keep in mind; the Edman sequencing of TFIIAβ yielded an N-terminal Asp, not N-terminal Arg-Asp. Furthermore, the Aspbearing TFIIA fragment remained a part of the TFIIA complex on a Superose-6 size

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exclusion column. Thus, if the conjecture of the involvement of the N-end rule pathway is correct, one must assume that the Asp-bearing C-terminal fragment is sterically unavailable for arginylation (and degradation) in the complex until for example dissociation of the complex or (de-)modification, leads to the fragment becoming an actual substrate of ATE1. For the function of TFIIA cleavage, a number of scenarios can be imagined. There are several examples of proteins where cleavage represents an activating step. The well-known mammalian caspases can be activated through cleavage and they can themselves cleave and activate CAD, thereby initiating endonucleolytic chromosome degradation7. However, the fact that the uncleavable TFIIA performs similarly to the wild-type protein in all assays tested so far, makes this a rather unlikely possibility. The opposite setting where cleavage of TFIIA represents an inactivating step is possible but somewhat contra-intuitive, since most of the cellular TFIIA is in fact cleaved. Another protein that undergoes proteolytical cleavage is MLL; cleavage has been suggested to confer subnuclear localization8, furthermore inhibiting MLL cleavage interferes with proper HOX gene expression. Uncleavable TFIIA mutants however localized to similar cellular compartments as wild-type TFIIA (results not shown). Whether the two forms of TFIIA have separate promoter preferences remains an open issue to be pursued. At this point, however, the cleavage does not seem to affect the transcriptional competence of TFIIA and the only apparent distinction is the difference in half-life. The studies from chapter 4 suggest that the half-life of the cleaved subunits of TFIIA is not dramatically short; this is also consistent with the steady-state levels of TFIIA in most cell lines, in which the major fraction of TFIIA is cleaved. It seems a more likely explanation that rather than resulting in a rapid protein removal, cleavage

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of TFIIA places a ‘timer’ on TFIIA and labels it for destruction. Importantly, this would, in contrast to posttranslational (de)modifications like (de)phosphorylation, represent an irreversible step; upon cleavage, TFIIA has commenced its path to death and from this point has only a certain time to perform its role in the cell. It is possible that the two forms function indistinguishably, the main difference being that the cleaved form is primed for destruction. If this is true, regulation of the protease activity regulates the stability of the protein without significantly altering its function and the inhibition of TFIIA cleavage (by modification of TFIIA/the protease activity) could be pictured as a way to store a more stable form of TFIIA (i.e. TAC) (see figure 1). Interestingly, this manner of regulating TFIIA is conserved also in ALF (chapter 6) and possibly has a more global function.

Figure 1. Model of function and regulation of TFIIA. Uncleaved TFIIAαβ/γ can assemble with TBP into TAC and this is facilitated, directly or indirectly, by p300. TAC is responsible for the transcriptional activation of hitherto unknown embryospecific promoters. TFIIAαβ/γ can be cleaved by Taspase 1 into TFIIAα/β/γ that can assemble with TFIID and activate general transcription. Ultimately, TFIIAα/β/γ is a substrate for proteasomal degradation, possibly through the N-end rule

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Concluding remarks Returning to the initial question; why is TFIIAαβ cleaved? Frequently in science, simple questions do not have simple answers, and whereas this work has succeeded in filling some gaps in our knowledge about TFIIA, the most intriguing question is still a matter of speculation and of favoring one theory over another. The function of cleavage may be merely to destabilize TFIIA but the fact that Taspase1 associates with chromatin may reflect a role of cleavage more directly linked to the transcription process. Our data go against a direct requirement of transcriptional activity for cleavage to occur (chapter 4), but this cannot exclude the possibility that cleavage does occur at a certain transcription step, without changing the transcriptional competence of TFIIA. This regulation may be to ensure that TFIIA only undergoes a limited number of initiation rounds. Furthermore, the engagement of the cleaved and uncleaved TFIIA in two separate complexes, TFIID and TAC, may suggest that these two forms of TFIIA have distinct responsibilities in promoter regulation (see figure 1). Cleavage of TFIIA appears to have evolved from S. cerevisiae as an additional regulative step in higher eukaryotes. TFIIA cleavage is mediated through a highly conserved stretch of four amino acids, which is essential for efficient cleavage. TFIIA is cleaved by Taspase1, a chromatin associated protease that is also responsible for cleavage of MLL. The cleavage process is likely regulated, and factors that are potentially important are firstly p300; over-expression of p300 leads to accumulation of TAC (uncleaved TFIIA) in differentiated cell lines like Cos7. Whether this is a direct effect on TFIIA or an indirect effect on for example the protease remains to be seen. Secondly, our results suggest that phosphorylation sites adjacent to the cleavage site affect not cleavage but stability of TFIIA. These are potential CKII sites, and

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CKII has been implicated in the regulation of a number of transcription factors, including IκBα, c-Jun, IRF-1, RNA pol II and the phosphatase FCP1 which is involved in recycling of RNA Pol II for transcription elongation9-12. CKII has also recently been shown to be involved in promoter selection and transcription reinitiation13. How these factors interplay through development to adjust the levels of TFIIA accordingly should offer an interesting direction for future experimenting. Thus far, experiments with transcriptional assays in vivo have not succeeded in uncovering any functional differences in transcriptional competence between uncleavable and cleavable TFIIA; nonetheless, the strict conservation of the CRS within higher eukaryotes implies that whatever the role, it is essential for the organism. It follows that the work described here hopefully provides the necessary ground for identifying the function of TFIIA cleavage in higher eukaryotes.

1.

2.

3.

4.

5. 6. 7.

8.

Bryant, G. O., Martel, L. S., Burley, S. K. & Berk, A. J. Radical mutations reveal TATA-box binding protein surfaces required for activated transcription in vivo. Genes and development 10, 2491-504 (1996). Hsieh, J. J., Cheng, E. H. & Korsmeyer, S. J. Taspase1: a threonine aspartase required for cleavage of MLL and proper HOX gene expression. Cell 115, 293-303 (2003). Yokoyama, A., Kitabayashi, I., Ayton, P. M., Cleary, M. L. & Ohki, M. Leukemia proto-oncoprotein MLL is proteolytically processed into 2 fragments with opposite transcriptional properties. Blood 100, 3710-8 (2002). Varshavsky, A. The N-end rule: functions, mysteries, uses. Proceedings of the National Academy of Sciences of the United States of America 93, 12142-9 (1996). Varshavsky, A. The N-end rule and regulation of apoptosis. Nature cell biology 5, 373-6 (2003). Ditzel, M. et al. Degradation of DIAP1 by the N-end rule pathway is essential for regulating apoptosis. Nature cell biology 5, 467-73 (2003). Earnshaw, W. C., Martins, L. M. & Kaufmann, S. H. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annual review of biochemistry 68, 383-424 (1999). Hsieh, J. J., Ernst, P., Erdjument-Bromage, H., Tempst, P. & Korsmeyer, S. J. Proteolytic Cleavage of MLL Generates a Complex of N- and C-Terminal Fragments That Confers Protein Stability and Subnuclear Localization. Molecular and cellular biology 23, 186-94 (2003).

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9.

10.

11. 12.

13.

Lin, R., Beauparlant, P., Makris, C., Meloche, S. & Hiscott, J. Phosphorylation of IkappaBalpha in the C-terminal PEST domain by casein kinase II affects intrinsic protein stability. Molecular and cellular biology 16, 1401-9 (1996). Friedl, E. M., Lane, W. S., Erdjument-Bromage, H., Tempst, P. & Reinberg, D. The C-terminal domain phosphatase and transcription elongation activities of FCP1 are regulated by phosphorylation. Proceedings of the National Academy of Sciences of the United States of America 100, 2328-33 (2003). Liu, C. et al. Control of beta-catenin phosphorylation/degradation by a dualkinase mechanism. Cell 108, 837-47 (2002). Price, M. A. & Kalderon, D. Proteolysis of the Hedgehog signaling effector Cubitus interruptus requires phosphorylation by Glycogen Synthase Kinase 3 and Casein Kinase 1. Cell 108, 823-35 (2002). Lewis, B. A., Sims, R. J., 3rd, Lane, W. S. & Reinberg, D. Functional Characterization of Core Promoter Elements: DPE-Specific Transcription Requires the Protein Kinase CK2 and the PC4 Coactivator. Molecular cell 18, 471-81 (2005).

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SAMENVATTING Lange tijd werd het verschil in complexiteit tussen ‘lagere’ organismen en hoogst ontwikkelde en intelligente organismes zoals wij zelf, simpelweg veklaard door het gepostuleerde verschil in aantal genen. Volgens schattingen bevatte een redelijk ontwikkeld mens toch al gauw zo’n 100.000 verschillende genen in zijn of haar genoom, de veel simpelere fruitvlieg 15.000 en het genoom van de eencellige bakkersgist omvatte slechts 6.000 genen. De schok was dan ook groot toen de conclusies van het Human Genome Project inhielden dat de mens ‘slechts’ 20.00025.000 genen bevatte, net ietsje meer dat het fruitvliegje en maar 3 tot 4 keer de zoveel als gist. Het verschil in complexiteit diende dus op andere wijze te worden verkregen én verklaard; het huidige dogma stelt dan ook dat niet de quantiteit aan genen, maar de manier waarop deze genen gereguleerd worden veel complexer, dynamischer en subtieler is in de mens. Dit lijkt deels te danken aan een grotere variëteit aan zogenoemde transcriptiefactoren: alhoewel de meeste eukaryotische organismen dezelfde basis aan transcriptie regulerende functionaliteiten bevat (general transcription factors), bevatten hogere eukaryoten voor veel functies meerdere varianten, die elk hoogstwaarschijnlijk transcriptie net iets anders beinvloeden, eventueel op andere momenten, in andere celtypes, etc. Één zo’n general transcription factor is TFIIA, focus van deze thesis. Niet alleen bevat TFIIA een variant in hogere eukaryoten, ALF (TFIIA like factor), TFIIA zelf is in hogere eukaryoten (maar niet in bakkersgist) ook onderhevig aan posttranslationele proteolyse. Hierdoor bestaan 2 mogelijk cellulaire varianten van TFIIA, een ongekliefde (TFIIAαβ) vorm en een gekliefde vorm (TFIIAα/β), een vroege observatie die hardnekkig genegeerd werd door het wetenschappelijke veld.

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Experimenten waaruit geconcludeerd werd dat de aanwezigheid van gekliefd dan wel ongekliefd TFIIA afhankelijk is van de differentiatie status van de cel, zette het proces van klieving weer in de spotlight. De vraagstelling aan het begin van deze thesis was dan ook simpel: Waarom wordt TFIIA gekliefd? Om dit te onderzoeken zijn we begonnen om de exacte positie van klieving te bepalen. Edman degradatie op gezuiverd humaan TFIIA liet zien dat de N-terminus van TFIIAβ begint op aminozuur D278. Mutagenese van de regio rondom deze klievingssite toonde dat de identiteit van D278 weliswaar belangrijk was voor klieving, maar dat de identiteit van elk van vier opeenvolgende aminozuren Nterminaal van D278 essentieel was voor dit proces (vergelijkbare resultaten werden behaald met betrekking tot de TFIIA variant, ALF (alhoewel het ALF residu dat correspondeert met TFIIA D278, D354, volstrekt essentieel voor klieving is). Deze onkliefbare mutanten verschaften uitstekende reagenten om eventuele verschillen tussen

kliefbaar

TFIIA

en

onkliefbaar

TFIIA

te

bestuderen

in

cellen.

Verbazingwekkend genoeg bleek er geen duidelijk verschil tussen de 2 vormen te vinden met betrekking tot affiniteit voor de tweede subunit van TFIIA (TFIIAγ), transcriptionele activatie van een minimale promoter, binding aan TBP of stimulatie van TBP associatie met DNA. Het enige aanwijsbare verschil was dat de onkliefbare TFIIA mutanten accumuleerden tot hoge levels in de cel, wat suggereerde dat deze eiwitten een langer halfleven hadden dan kliefbaar TFIIA. Pulse-Chase experimenten lieten zien dat onkliefbaar TFIIA inderdaad een duidelijk langer halfleven heeft dan kliefbaar TFIIA. Inhibitie van proteasomale degradatie liet verder zien dat alleen geklieft TFIIA een substraat is voor proteasomale degradatie en dat een van de kleinere subunits geconjugeerd wordt aan ubiquitine, een kenmerk van proteasomale degradatie. Deze data suggereerden dat

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TFIIA klieving noodzakelijk is voor regulatie van TFIIA levels in de cel. Het feit dat de N-terminus van TFIIAβ (D278), maar niet van TFIIAαβ (M1) een secundair onstabiel residue is volgens the N-end rule ondersteunde deze notie. Volgens het proteosomale degradatie mechanisme van de N-end rule dient een secundair onstabiele N-terminus alvorens het gedegradeerd kan worden, omgezet te worden in een primair onstabiel residu door fusie van een arginine (R), een proces gemedieerd door ATE1 (arginine tRNA transferase 1). Als een doel van TFIIA klieving inderdaad het genereren van een potentieel N-end rule substraat is dan zou deregulatie van ATE1 het halfleven van TFIIA dienen te beinvloeden. Inderdaad, deletie van ATE1 leidde to accumulatie van zowel endogeen als exogeen TFIIA en herintroductie van ATE1 in deze cellen verkortte het halfleven van TFIIA. Vergelijkbaar, overexpressie van ATE1 in U2OS cellen verkortte het halfleven van kliefbaar TFIIA drastisch, maar niet het halfleven van onkliefbaar TFIIA. Mutatie van D278 tot een zeer stabiel Nterminaal residue, namelijk methionine (M) zou degradatie van geklieft TFIIA moeten inhiberen, en complementair, mutatie van D278 in het primair unstabiele R zou degradatie kinetiek onafhankelijk moeten maken van ATE1. Met behulp van transfectie experimenten konden beide postulaties bevestigd worden. Tesamen lijken al deze datasets er sterk op te wijzen dat een belangrijk (zo niet het enige) doel van TFIIA klieving de regulatie van het halfleven van TFIIA is. Een verdere open deur voor opvolgend onderzoek is de rol van fosforylatie is klieving en degradatie van TFIIA. Onze experimenten lieten zien dat de identiteit van 3 residuen direct C-terminaal van D278 (namelijk T279, S280, S281) belangrijk is voor regulatie van de levels van geklieft TFIIA. Deze residuen zijn bekend als fosforylatie targets en mutagenese van de herkenningssequentie (E282-D284) voor potentiële fosforylatie van T279-S281 door CKII (Casein kinase II) had hetzelfde

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destabiliserende effect als mutagenese van TSS naar Alanine. Tenslotte bleek de fosforylatie imiterende T276D mutatie, maar niet de T276A mutatie, volledig onkliefbaar. Dit suggereert dat de fosforylatie status van T276 (toevallig ook een potentiële CKII site) een rol speelt in TFIIA klieving. Één belangrijke speler in het leven van TFIIA bleef lang ongrijpbaar. Tot een publicatie de protease verantwoordelijk voor klieving van het trithorax eiwit MLL (Mixed lineage leukaemia) identificeerde, Taspase1. Deze protease kliefde midden in een sequentie die identiek van aan de sequentie van de CRS in TFIIA, de residuen essentieel voor TFIIA cleavage. Zowel in vitro als in vivo experimenten lieten zien dat Taspase1 in staat is TFIIA te klieven. Edman degradatie van in vitro, Taspase1 gemedieerd, geklieft TFIIA liet zien dat de N-terminus van TFIIAβ begon op dezelfde plek als in MLL, op G275 en dus niet begon op D278 zoals de in vivo resultaten stelden. Als een gevolg hiervan eindigt deze thesis zoals vaak met een aantal antwoorden, maar ook weer veel nieuwe vragen, hier een aantal ervan: Hoe zijn de verschillen tussen de in vivo en in vitro N-terminus van TFIIAβ te verklaren? Vindt er secundaire klieving leidend tot een instabiele N-terminus? Wat is de exacte physiologische rol van verschillende eiwitten waaronder Tapase1, ATE1, p300, E1A en CKII in regulatie van TFIIA klieving en halfleven? Op welke wijze speelt het transcriptieproces een rol in TFIIA klieving? p300, CKII en Taspase1 zijn chromatine geassocieerde eiwitten en alhoewel onze experimenten suggereren dat klieving niet noodzakelijk is voor transcriptie progressie en transcriptie progressie niet noodzakelijk is voor klieving, is het wel zeer mogelijk dat klieving en halfleven van TFIIA gerelateerd zijn aan het transcriptieproces (mogelijkerwijs door het plaatsen van een ‘timer’ op TFIIA vóór, tijdens of na transcriptie). Zijn er daadwerkelijk

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functionele verschillen tussen ongeklieft en geklieft TFIIA (bijv. promoter specificiteit, diferentiatie specifieke functies), of is klieving simpelweg een irreversibele stap in de degradatie van TFIIA?

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ACKNOWLEDGEMENTS And so it is time to set the final mark on my thesis and thank the people that have helped making it possible. Finishing a thesis while raising a child tends to make life a bit more crowded, so I would like to thank everyone in the lab for making it easier (and more pleasant). First I want to thank Henk as my supervisor for teaching me a lot over the last years, both professionally and personally. As you said yourself, the first half of my PhD was too long and the second half too short. Luckily that means it ended uphill. There are two people without whom this would never have been finished, and Dimitra, you are one of them. Thank you for being such a relaxed and nice person who was ever so patient through my first frustrating meeetings with the art of transfection. Thank you for, together with Yannis and Greta, presenting me with the not unsignificant greek hospitality and cooking. I hope to see you in Greece (or Bergen) soon again. I will also remember the way I was taken in under Gretas and Catelijns wings - as well as under their roof - when I first arrived in Nijmegen, with two suitcases and a blanket. Marion, you have been extraordinarily helpful and positive and your spirit has meant a lot to me and my choices, both scientifically and personally. I cannot thank you enough for your great positivism over the last years! Sergei, Salva and Jo made out the TFIIA group the majority of my time in Nijmegen and our TFIIA meetings were a source for progress and good ideas. In addition to being the closest to my project and my bench mate, Jo you also became a great friend. Thanks also to Josephine, Maria and Anita for helping with anything from cloning advice to housing and band-aid and to providing the essential warm mothering of the whole lab. My beginning was made a lot nicer by Paul and Colin: thanks for the great moments in the first year sharing not only office but also tomato plants with names

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and hazardous pizza events in the lab. Paul and Marjolein, it has been so nice to have your friendship through my good as well as rough times in the Netherlands…may we soon drink another overprized yet undertasting beer in Norway. Musketeers, our paths have divided! Jurgen, Xavy and Coen, despite our frustrations, it has been amazingly nice, whether is was our ridiculous working pattern, horse-back riding, skiing on the Norwegian mountains, crawling through tiny holes in Belgian caves, wind-surfing (wind-crawling more like it) on French beaches or simply one of our many evenings eating, drinking and talking. We were always each others most enjoyable distraction from whatever disturbing events going on scientifically. Michiel, my longest colleague amongst the Ph.Ds at the Department of Molecular Biology, over the years I have deeply come to enjoy your ability to utter the right comment at the wrong time (or was it the other way around?), and to add a certain absurdity to situations. Åshild and Ylva, you provided a Scandinavian room in Nijmegen where nationalism was nurtured shamelessly, never will Cornelis Vreeswijk be performed with more emotion and sincerity (and alcohol consumption…) Thanks to my room mates during the first three years: Noor, Janet, Robbie, Ruud and Ron, for introducing me to Dutchness such as skating, bicycling on the dikes or stamppot and for dragging me outside in the weekends. Finally, my most important thanks go to family and friends for their support and patience in my absence. Thanks for all the visits that brought a little of Norway to the Netherlands. Coen, you have been a never-ending source of energy that has pulled me through during my more gloomy moments as a Ph.D student, and without your support a finished thesis like this one would not have been lying here right now.

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CURRICULUM VITAE Torill Høiby was born on the 23rd of November 1972 in Kongsvinger, Norway. She graduated from Skarnes videregående skole in 1991 and moved to the west coast of Norway, to study Mathematics, Physics and Chemistry at the University in Bergen. Winter and Spring 1994 was spent travelling in Asia and Oceania, before she continued her studies in biochemistry and received her Master degree in 1998 studying the effect of vitamin A and D on breast cancer. She continued with this work as a technician till August 1999. From September 1999 until June 2004 she worked as an AiO at the Department of Molecular Biology at Radboud Universiteit Nijmegen, under the supervision of Prof. Dr. Hendrik G. Stunnenberg. In August 2005, she returned to the University of Bergen to work as a PostDoc in the Department of Biomedicine.

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