Characterisation of Ctbp

Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1089

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Characterisation of CtBP

A Co-Repressor of Transcription that Interacts with the Adenovirus E1A Protein

BY ANDERS SUNDQVIST

ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2001 Dissertation for the Degree of Doctor of Philosophy, Faculty of Medicine, in Medical Virology presented at Uppsala University in 2001

ABSTRACT Sundqvist, A. 2001. Characterisation of CtBP. A Co-Repressor of Transcription that Interacts with the Adenovirus E1A Protein. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1089. 50 pp. Uppsala. ISBN 91-554-5144-6.

In this study, adenovirus E1A has been used to target and analyse the transcriptional function of the cellular C-terminal Binding Protein (CtBP). Transcription is a complex biochemical process that represents a major regulatory step in gene expression. Formation of condensed chromatin by histone deacetylation and inhibition of efficient assembly of the transcription machinery are hallmarks of transcriptional repression. During a virus infection, an extensive modulation of the host cell gene expression in favour of viral gene expression can be observed. For example, the transcription regulatory E1A protein from adenovirus has been proven to be a valuable research-tool in characterising cellular proteins controlling eukaryotic gene expression. Expression of a CtBP-binding peptide, encoded by the second exon of E1A, de- repressed transcription from a broad range of promoters, suggesting that CtBP functioned as a repressor of transcription. Artificial promoter recruitment of CtBP, by using different Gal4- fusion proteins, confirmed that CtBP functioned as a repressor. Repression of transcription by Gal4E1A-recruited CtBP was efficiently prevented by a CtBP binding competent E1A peptide, indicating that E1A relieved CtBP mediated repression by displacing CtBP from the promoter. Furthermore, Gal4CtBP repressed both basal and activated transcription in a distance dependent manner, suggesting that CtBP might repress transcription by interfering with the assembly of the basal transcription machinery. Interestingly, CtBP was found to interact with histone deacetylase-1 (HDAC-1) both in vivo and in vitro and endogenous CtBP could also recruit histone deacetylase activity. This might indicate that histone deacetylation was involved in CtBP mediated repression. However, Gal4CtBP mediated repression was insensitive to inhibition of histone deacetylase activity, suggesting an alternative function of HDAC-binding in CtBP mediated repression. In conclusion, this work demonstrates that CtBP can act as a general repressor of activated and basal transcription. Furthermore, although CtBP was shown to recruit histone deacetylase activity the relevance of this binding remains unclear.

Key words: CtBP, HDAC, adenovirus, E1A, gene expression, repression.

Anders Sundqvist, Department of Medical Biochemistry and Microbiology, Biomedical Centre, Box 582, SE-751 23 Uppsala, Sweden

ISSN 0282-7476 ISBN 91-554-5144-6

Printed in Sweden by Eklundshofs Grafiska AB, Uppsala 2001 This thesis is based on the following articles, which in the text are referred to by their roman numerals:

1) Anders Sundqvist, Kerstin Sollerbrant and Catharina Svensson. 1998. The carboxy-terminal region of adenovirus E1A activates transcription through targetting of a C-terminal binding protein-histone deacetylase complex. FEBS Lett. 429: 183-188.

2) Anders Sundqvist, Edyta Bajak, Sindhulakshmi D. Karup, Kerstin Sollerbrant and Catharina Svensson. 2001. Functional knock-out of the co- repressor CtBP by the second exon of adenovirus E1A relives repression of transcription. Exp Cell Res. 268(2): 284-293.

3) Anders Sundqvist and Catharina Svensson. 2001. Molecular characterisation of transcriptional co-repressor CtBP. Manuscript.

Reprints were made with permission from the publishers: Elsevier Science B.V. and Academic Press. Back at the lab bench…at last! TABLE OF CONTENTS

ABBREVIATIONS AND DEFINITIONS...... 1

INTRODUCTION ...... 2

CHROMATIN ...... 2 Chromatin modifications ...... 3 Acetylation-deacetylation ...... 4 Phosphorylation...... 5 Methylation...... 5 Ubiquitylation...... 5 The "histone code"...... 5 Chromatin remode ...... 7

TRANSCRIPTION ...... 7 Promoter structure ...... 8 The basal transcription machinery ...... 9 RNA polymerase II...... 10 DNA sequence-specific transcription factors ...... 10 Co-factors ...... 11 TAFs...... 11 Mediator ...... 12 USA...... 12 Co-activators and co-repressors...... 12 Elongation...... 13 How transcription might be regulated...... 13 Regulation of transcription factor activity ...... 17 Protein stability...... 17 Cellular localisation ...... 18 DNA binding ...... 18 Phosphorylation and dephosphorylation ...... 18

THE ADENOVIRUS MODEL SYSTEM ...... 19 Adenovirus ...... 19 The adenovirus life cycle ...... 19 Early region 1A...... 20 Activation of transcription by E1A ...... 21 Repression of transcription by E1A ...... 23 The role of E1A in cellular transformation...... 23

CTBP, A CELLULAR CO-REPRESSOR OF TRANSCRIPTION ...... 24 CtBP interacting proteins ...... 25 Mechanisms of CtBP mediated repression ...... 26 The biological role of CtBP...... 26 Additional characteristics of CtBP...... 27

PRESENT INVESTIGATION ...... 28 Paper I: The carboxy-terminal region of adenovirus E1A activates transcription through targeting of a C-terminal binding protein-histone deacetylase complex...... 28 Paper II: Functional knock-out of the co-repressor, CtBP, by the second exon of adenovirus E1A relieves repression of transcription...... 30 Paper III: Molecular characterisation of the transcriptional co-repressor, CtBP...... 31 Conclusions and perspectives ...... 32

ACKNOWLEDGMENTS...... 35

REFERENCES ...... 36 ABBREVIATIONS AND DEFINITIONS

Activator DNA-sequence specific transcription factor that stimulates basal transcription Basal transcription a low steady state level of transcription driven by the general transcription factors and RNAP II Binding-site cis-regulatory promoter element, consisting of specific DNA- sequences to which activators and repressors bind Co-factor complexes (Mediator, TAFs) or factors (USA, co-activators and co-repressors) required for activated, but not for basal, transcription Core histone H2A, H2B, H3 and H4, which together form the octamer of the nucleosome Core promoter the core element (Inr, DPE and the TATA-box) of the promoter CTD the carboxy-terminal domain of the largest subunit of RNAP II DPE downstream promoter element Euchromatin comprises all of the genome in the interphase except for the heterochromatin GTF general transcription factor, termed: TFIIA, B, D, E, F and H HAT Histone acetyltransferase, adding acetyl-groups to the amino- Terminal tail of the core histones HDAC Histone deacetylase, an enzyme removing acetyl-groups from the amino-terminal tail of the core histones Heterochromatin region of chromosome that remains condensed and transcriptional inactive during interphase of the cell cycle Histone small, conserved DNA binding proteins Histone tail the amino-terminal of the core histones HMTase Histone methyl transferase Inr Initiator, core promoter element centred around the transcriptional start site Linker histone Histone H1, required for formation of the 30 nm fiber and heterochromatin Nucleosome the basic structural subunit of chromatin, consists of an octamer of the core histones around which approximately 146 base pairs of DNA are wrapped PIC Pre-initiation complex of transcription Repressor DNA-sequence specific transcription factor that inhibits transcription RNAP II RNA polymerase II, an enzyme that synthesize RNA using a DNA-template RNAP core enzyme RNAP II itself, consists of 12 subunits RNAP holoenzyme the yeast holoenzyme contains; RNAP II, a subset of GTFs and the Mediator complex TAFs TBP associated factors TBP TATA-box binding protein USA Upstream stimulatory activity

1 INTRODUCTION

The blueprint of life is stored in very long chains of DNA. The functional units of the DNA chains are called genes. The information stored in protein-encoding genes can be copied into messenger RNAs (mRNAs) by a process called transcription. These mRNAs are subsequently synthesised (translated) into proteins, which are the functional and structural macromolecules of the cell. Even if the majority of cell-types in higher organisms contain the same genetic information, they exhibit different characteristics. These distinct properties are determined by the expression of specific subsets of genes. According to this view, transcription plays the ultimate role in regulation and determination of the function and properties of a distinct cell type. Transcription is mainly controlled by a non-coding element - called a promoter - located in the beginning of a gene. The promoter functions as a regulatory unit, determining when and to what level a given gene should be transcribed. General transcription factors can bind to the promoter and recruit the RNA polymerase (RNAP), thereby leading to a low, basal level of transcription. However, quantitative adjustments require the recruitment of DNA-sequence-specific transcription factors (activators or repressor) to a cis-regulatory DNA-element. This thesis addresses how transcription of protein-encoding genes are regulated, in particular at the level of down-regulation (repression) and de-repression.

CHROMATIN

The large quantity of DNA in eukaryotic cell has to be packed in a very efficient structure to be able to fit into the nucleus. In the chromatin structure, DNA is compressed more than 10 000-fold compared to its relaxed form. The fundamental repeating unit of chromatin is the nucleosome (Kornberg and Lorch, 1999; Wolffe and Guschin, 2000). The nucleosome consists of two heterodimers of core histones, H3 and H4, and two heterodimers of core histones, H2A and H2B, which together form a protein-structure around which 146 base pairs of DNA are wrapped. The core histones contain a similar structural motif, the "histone fold", consisting of three helical domains. Conserved arginine residues within the histone fold interact directly with the minor groove of the DNA. Chromatin stability is strongly influenced by the highly basic, amino-terminal parts of the histone. These "tails" protrude from the surface of the nucleosomes and can mediate the contact with adjacent nucleosomes (Luger et al., 1997). In addition, these basic tails associate tightly with the acid backbone of DNA. Importantly, the histone tails are subjected to extensive post- transcriptional modifications, which will affect the stability of this interaction and thereby the accessibility of the transcription machinery to promoter DNA. Chromatin can be further stabilised into higher-order structures by incorporation of histone H1, the "linker histone". However, in contrast to the core histones, H1 is not required for formation of chromatin (Wolffe and Guschin, 2000). Nucleosome positioning is probably not random as they often appear at preferred positions on the DNA. Although it is not known how this positioning is regulated, it is possible that some locations are blocked by DNA sequence-specific transcription factors. In addition, the energy required for bending of specific DNA sequence flanking nucleosome bound DNA might also be important (Kornberg and Lorch, 1999). There is a fundamental difference between gene regulation in prokaryotes and eukaryotes (Struhl, 1999). In prokaryotes, the transcriptional ground state is non-

2 restricted, meaning that the transcription machinery has free access to DNA, unless active measures are taken to prevent this. In contrast, the packing of eukaryotic DNA into chromatin, imposes inhibition of gene expression by preventing access of the basal transcription machinery to the promoter DNA (reviewed in: Grunstein, 1997; Kornberg and Lorch, 1999; Struhl, 1999; Wolffe and Guschin, 2000). However, this theory has recently been called into doubt (Remboutsika et al., 2001; Sekinger and Gross, 2001).

A) C) (I)

H4 H3 (II) H2B H2A

H4 H3

( III)

B) Ac (IV)

Ac HAT Ac (V) HDAC Ac Ac

(VI)

Figure 1. General chromatin organisation. (A) The nucleosome: Two heterodimers of histone H3 and H4 and two heterodimers of H2A and H2B forms an octamer around which 146 base pairs of DNA are wrapped (B) Reversal acetylation of the amino- terminal tails of histones decrease the interaction with DNA creating a more flexible chromatin structure. (C) A model for the many orders of chromatin packing: (II) The double helix of DNA is wrapped around the octamers, forming chromatin. (III) Incorporation of histone H1 results in a more condensed structure known as 30-nm fibre. (IV and V) Further packing results in even more condensed structures. (VI) In the metaphase chromosome structure is DNA condensed approximately 10 000 fold.

Chromatin modifications To express specific genes from an inaccessible chromatin environment (heterochromatin), the chromatin around the promoter has to be relaxed and transformed into an accessible structure (euchromatin). Today, it is generally believed that post-translational modifications such as acetylation, phosphorylation, methylation and ubiquitylation of the histone tails correlate with alterations of the chromatin structure (Jenuwein and Allis, 2001; Marmorstein, 2001; Strahl and Allis, 2000).

3 Acetylation-deacetylation Acetylation of histone tails has been known for a long time to correlate with an active transcriptional state (Braunstein et al., 1993; Hebbes et al., 1988). Acetylation of histone H3 is primarily associated with transcription while acetylation of histone H4 is associated with transcription and chromatin assembly (reviewed in: Strahl and Allis, 2000). The first direct link between histone acetylation and transcriptional regulation came from the observation that the yeast transcriptional activator, Gcn5, was a histone acetyltransferase (HAT) (Brownell et al., 1996) and that Gcn5-induced transcription required HAT-activity (Kuo et al., 1998). This discovery was followed by the demonstration that several previously characterised transcription factors possessed HAT-activity e.g. the co-activators p300/CBP and P/CAF, the basal transcription factor TFIID and the SRC-family of steroid receptor co-activators (reviewed in: Marmorstein, 2001). It has also been shown that the activity and substrate specificity of the HATs are modulated by the composition of the multiprotein complexes with which they interact, suggesting that the promoter recruitment of HATs, through interactions with activators, results in gene-specific effects on transcription (Brown et al., 2000). While HATs are involved in activation of transcription, histone deacetylases (HDACs) are involved in repression (reviewed in: Wade, 2001). Histone deacetylases can be divided into four different classes: the human class I (HDAC1, 2, 3 and 8) and class II (HDAC4, 5, 6 and 7), the Sir2-family and the maize HD2. Human class I and II HDACs share a common catalytic domain while the class II HDACs contain a unique amino-terminal sequence (Grozinger et al., 1999; Kao et al., 2000; Verdel and Khochbin, 1999). Two main co-repressor complexes, Sin3 and Mi-2/NuRD, contain class I HDAC1 and 2. Both complexes contain subunits interacting with repressors or co-repressors that target the deacetylase activity to specific promoters. DNA sequence-specific transcription factors can also directly interact with class I and II HDACs, for example YY1 interacts with HDAC2 (Yang et al., 1996a; Yang et al., 1997) and HDAC4 interacts with MEF2 (Miska et al., 1999). Taken together, the balance between histone acetylation and deacetylation is critical in determining whether a gene is expressed or not. Even if some promoters are acetylated/deacetylaed in a promoter-specific manner, modifications of histones in regions surrounding the promoter (global acetylation/deacetylation) (Vogelauer et al., 2000) might be important in regulating the balance between acetylation/deacetylation. By preventing full acetylation, a default underacetylated state is created, which might inhibit basal transcription by reducing both initiation and elongation. The controlled balance between acetylation/deacetylation might also allow more rapid shifts and prevent irreversible acetylation or deacetylation of chromatin. It is possible that additional mechanisms are involved in controlling the balance between acetylation and deacetylation. In line with this, a multiprotein complex called INHAT (inhibitor of acetyltransferase) was recently shown to inhibit p300/CBP and PCAF-mediated acetylation of histone tails by binding to the tails and thereby masking them from being recognised as substrates for HATs (Seo et al., 2001). Interestingly, some of the class II HDACs (HDAC4, 5 and 6) have been shown to be regulated by their cellular localisation. With the aid of certain transporter proteins, these enzymes actively shuttle between the nucleus and the cytoplasm. Further, their subcellular distribution might be regulated by extra cellular signalling (reviewed in: Wade, 2001).

4 Phosphorylation Phosphorylation of histone H3 has previously been correlated with transcriptional activity (Mahadevan et al., 1991). In addition, a direct link between phosphorylation and acetylation was recently demonstrated, as phosphorylation of serine 10 (Ser-10) in histone H3 resulted in increased Gcn5-dependent acetylation of lysine 14 (Lys-14) (Cheung et al., 2000; Lo et al., 2001; Lo et al., 2000).

Methylation Lysine 9 (Lys-9), at the tail of histone H3, is specifically methylated by the histone methyltransferase (HMTase), SUV39H1 (Rea et al., 2000). The HP1 proteins, previously characterised as heterochromatin adapter molecules involved in gene silencing, specifically recognised SUV39H1-methylated Lys-9 via its chromodomain, suggesting that site-specific methylation could change the chromatin structure (Bannister et al., 2001; Lachner et al., 2001). Furthermore, HP1 interacted with SUV39H1, which could lead to methylation of H3 Lys-9 on adjacent nucleosomes and subsequent spreading the heterochromatic structure over a large region. In addition, the retinoblastoma tumour suppressor (Rb) was necessary for both direct SUV39H1-mediated methylation and recruitment of HP1 to the cyclin E promoter, suggesting that the SUV39H1-HP1 complex might have a role in Rb-dependent repression of euchromatic genes (Nielsen et al., 2001). In contrast, methylation of histone H4 at arginine 3 (Arg-3) by a protein arginine methyltransferase, PRMT1, facilitated subsequent acetylation of H4 tails by p300, suggesting that methylation is also important for activation of transcription (Wang et al., 2001).

Ubiquitylation In yeast, mutation of the conserved ubiquitylation site in H2B, resulted in defects in mitotic cell growth and meiosis (Robzyk et al., 2000). In Drosophila, a mutation that abolished monoubiquitylation of histone H1 by the largest subunit of the TFIID complex, TAFII250, resulted in defects in gene expression during embryonic development (Pham and Sauer, 2000). Together, these observations suggest that reversible histone ubiquitylation is required for disruption of nucleosomal structure, that in turn allows efficient gene expression by making the DNA accessible to the transcription machinery.

The "histone code" The highly basic tail of the core histones can both associate tightly with the acid backbone of DNA and interact with adjacent nucleosomes (Luger et al., 1997). Acetylation of the tail would reduce its ionic strength and as a consequence, chromatin is relaxed, due to a decreased interaction with DNA. However, recent data have indicated that acetylation of histones did not have the predicted large inhibitory effect on the DNA-histone interaction or transcription (Mutskov et al., 1998). Furthermore, the role of histones and chromatin-modifying components have been suggested to be gene specific rather than general (Holstege et al., 1998; Sudarsanam et al., 2000). Together, this suggests that the role of histone modifications in transcription is more complex then previously believed. Distinct histone modification patterns possibly act in combinations, or sequentially, to form a "histone code" that is interpreted to provoke unique downstream transcriptional events (Jenuwein and Allis, 2001; Strahl and Allis, 2000; Turner, 1998). This hypothesis was illustrated by the data from studies on HMTase-mediated methylation of the histone H3 tail in which methylation of Lys-9 by the HMTase SUV39H1 correlated with gene silencing,

5 probably by inhibiting acetylation of Lys-9 and phosphorylation of Ser-10 (Rea et al., 2000). Furthermore, binding of HP1 to the methylated Lys-9 might prevent removal of the methyl groups from the adjacent histones (Bannister et al., 2001; Lachner et al., 2001). In contrast, phosphorylation of Ser-10 increased the acetylation of Lys-14 (Cheung et al., 2000; Lo et al., 2001) which leads to enhanced interaction with the bromodomain of other HATs with different site-specificity (Dhalluin et al., 1999; Marmorstein, 2001). Together, these data suggest that the tails of histone H3 function as a "molecular switch" between repression and activation of transcription, depending on whether the H3 domain is methylated at Lys-9 or phosphorylated at Ser-10.

K9 S10 L14 Histone H3

+HMTase +Kinase

Me P

K9 S10 K14 Histone H3 K9 S10 K14 Histone H3

HP1

Me P Ac

K9 S10 K14 Histone H3 K9 S10 K14 Histone H3

Condensed chromatin (heterochromatin) Accessible chromatin (euchromatin) correlates with silenced transcription correlates with active transcription

Figure 2. Translating the "histone code". Modification of the histone tails might function as markers for downstream modifications, resulting in either condensed hetero- or accessible euchromatin. The heterochromatin associated HP1 protein specifically recognises lysine 9 (K9) of histone H3 that is methylated (Me) by the HMTase SUV39H1. HP1, in turns, interacts with SUV39H1, which could lead to H3 K9 methylation of adjacent nucleosomes, resulting in spreading of a "closed" heterochromatin structure over large regions. In contrast, phosphorylation (P) of H3 serine 10 (S10) increase acetylation (Ac) of lysine 14 (K14), that in turn correlates with accessible euchromatin.

6 Chromatin remodelling Nucleoside structures can also be manipulated by the ATP-dependent chromatin remodelling complexes. These complexes utilise an ATP-dependent mechanism to remove, displace or destabilise nucleosomes at specific chromosomal sites (Aalfs and Kingston, 2000). Two distinct families of chromatin remodelling complexes have been characterised: the SWI/SNF family and the ISWI family. Both families contain an ATPase subunit in addition to several other subunits, forming multiprotein complexes. SWI/SNF can reposition nucleosomes by sliding the histone octamer to new sites on the same DNA molecule (cis-displacement)(Whitehouse et al., 1999). SWI/SNF, and the related complex, RSC, are also capable of transferring the octamer to another DNA molecule (trans-displacement) (Lorch et al., 1999; Owen-Hughes et al., 1996). The general mechanism by which the SWI/SNF family is believed to remodel chromatin involves breaking the DNA-histone contacts and destabilisation, or even complete disruption, of the nucleosomes. In contrast, ISWI enables sliding of the histone octamer along the same DNA molecule without disrupting DNA-histone contacts (Kornberg and Lorch, 1999). Not all promoters are subjected to regulation by SWI/SNF and the sensitivity to SWI/SNF appears to be controlled at the level of individual promoters rather than over chromosomal domains (Holstege et al., 1998; Sudarsanam et al., 2000). In line with this, chromatin remodelling complexes can be recruited to distinct promoters through interactions with activators (Sudarsanam and Winston, 2000). In addition, the bromodomain containing subunit of SWI/SNF has been suggested to mediate recruitment of these complexes to acetylated histones. Chromatin remodelling and modifications of histone tails are most likely coupled but the chronological order of the two processes is not known. Two studies have reported that SWI/SNF could recruit HAT complexes (Cosma et al., 1999; Krebs et al., 1999) while other reports have suggested that acetylation precedes SWI/SNF binding and chromatin remodelling (Hassan et al., 2001; Reinke et al., 2001). Whole-genome mRNA expression studies in yeast have suggested that SWI/SNF might also repress transcription (Holstege et al., 1998; Sudarsanam et al., 2000). SWI/SNF-induced chromatin remodelling possibly facilitates binding of repressor proteins or might even induce inhibitory locations of nucleosomes at a promoter.

TRANSCRIPTION

Eukaryotic gene transcription is a remarkably complicated biochemical process that involves numerous regulatory proteins and several levels of control. The multiple facets of regulation allow a finely tuned expression of specific genes in response to cellular requirements and extracellular signals. Activation of gene expression requires that the promoter is located in a chromatin environment, which is accessible to the transcription machinery. Recruitment of the transcription machinery and regulation of the rate of transcription are controlled by communication between different transcription factors. Binding of general transcription factors and RNAP II to a promoter gives a low basal steady-state level of transcription. Upon binding of activators and recruitment of co-factors, the basal activity can increase several hundred fold. The transcription cycle can be divided into different defined steps. 1) Assembly of the pre-initiation complex at the promoter, 2) initiation, defined by formation of the first phosphodiester bond, 3) promoter clearance and release of

7 RNAP II from the promoter, 4) elongation, 5) termination and 6) modification and recycling of RNAP II, preparing it for a new round of transcription (Lee and Young, 2000). The first step of the transcription cycle will be described below in further detail.

Promoter structure The structure of an eukaryotic RNAP II promoter can be divided into a core element and cis-regulatory elements (reviewed in: Hampsey, 1998). The core promoter includes an initiator (Inr) centred at the start site for transcription (defined as +1) and a TATA-box (an AT-rich sequence) located approximately 30 nucleotides upstream of the start site (see Figure 3). Most promoters contain either a TATA-box or an Inr- sequence, or both of these elements. The core promoter elements define the site for assembly of the pre-initiation complex (PIC). Some genes contain an additional core promoter element called the downstream promoter element (DPE). The DPE is located approximately 30 nucleotides downstream of the initiation site and is supposed to function - together with the Inr sequence - as the site for PIC assembly on promoters lacking TATA.

DNA sequence-specific General transcription factors and transcription factors RNA polymerase II

RNAP II TFIID TFIIH Activator Activator Repressor TFIIE TFIIF DNA- TFIIA TATA TFIIB Inr template

Cis-regulatory element Core promoter element

Figure 3. Schematic illustration of the structure of an RNA polymerase II promoter. The general transcription factors and RNA polymerase II interact with the core promoter, typical containing a TATA-box and an initiator. Cis-regulatory element recognised by DNA sequence-specific transcription factors (activators or repressors) and binding of these transcription factors affect the level of transcription.

Cis-regulatory elements are defined DNA elements which function as binding sites for DNA sequence-specific transcription factors. These factors are divided into two different groups; activators and repressors that will stimulate or inhibit basal transcription respectively. The cis-elements can occur in clusters or as individual factor binding sites. Although some elements act as activator binding-sites or repressor binding-sites, most elements seem to display dual activity capable of conferring both activation or repression of transcription depending on the status of the cell. In higher eukaryotes, these elements can influence transcription independently of their orientation and at variable distances from the start site. Yeast promoters contain similar elements, but they are located upstream of, and proximal to, the core promoter.

8 The basal transcription machinery Basal transcription from a protein-encoding gene requires assembly of the RNAP II holoenzyme at the promoter (Lee and Young, 2000; Roeder, 1996). This complex consists of the RNAP II core enzyme, the general transcription factors (GTFs) ensuring specific promoter recognition and, depending on the purification method, one or several different multisubunit complexes known as co-activators. The RNAP II core enzyme together with the GTFs (named TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH) are sufficient for a basal level of transcription from a promoter driven by a consensus TATA-box. Although it is not fully understood how PIC is assembled (see "Regulation of transcription" and Figure 4.), a stepwise assembly has been demonstrated in vitro (Buratowski et al., 1989). In this process, the GTFs can be identified as stepping stones in the following way: in the first step of PIC formation, the TATA-box in the core promoter is specifically recognised by the TATA-box binding protein (TBP). When bound to the promoter, TBP forms a symmetrical saddle-like structure and its interactions with the minor groove of the TATA-element causes the DNA to bend (Burley and Roeder, 1996; Chasman et al., 1993). The majority of TBP is found in a complex (TFIID), containing several TBP-associated factors (TAFs). TFIIA functions, in part, by interacting with TBP and DNA- sequences upstream of the TATA-box, thereby stabilising the interaction between the core promoter and TBP (Imbalzano et al., 1994). TFIIA also stimulates transcription by promoting the dissociation of TBP homodimers. In vivo, the formation of dimers appears to limit unregulated gene expression and the dissociation of TBP dimers has been described as a rate limiting step for promoter loading of TBP (Coleman et al., 1999; Jackson-Fisher et al., 1999). In addition, TFIIA can function as an anti- repressor by counteracting the activity of TBP inhibitors, like NC2 and hTAFII250 (Pugh, 2000). TFIIB is involved in determining the transcriptional start site (Berroteran et al., 1994; Li et al., 1994; Pinto et al., 1992). Structural studies have demonstrated that TFIIA and TFIIB bind on opposite sides of the DNA-TFIID complex and together with TFIID serve as a platform for the RNAP II core enzyme (Orphanides et al., 1996). TFIIF binds to RNAP II and thereby stabilises the pre- initiation complex but TFIIF also contacts DNA on both sides of the TATA-box, possibly in order to suppress non-specific DNA interaction by RNAP II (Lee and Young, 2000; Orphanides et al., 1996). The interactions between TFIIF and DNA might also be responsible for formation of a structure in which the DNA is tightly wrapped around the PIC. This structure results in DNA bending which may also facilitate unwinding of DNA during TFIIH-dependent formation of an open promoter complex (Conaway et al., 2000; Robert et al., 1998). Like TFIIF, TFIIE and TFIIH also bind RNAP II directly, but in the stepwise assembly model TFIIE might have a function in TFIIH recruitment. TFIIE stimulates the carboxy terminal domain (CTD) kinase and ATPase activity of TFIIH but is also involved in melting promoter DNA. Since the requirement for TFIIE is influenced by the helical stability of the promoter DNA, it is also promoter-specific (Lee and Young, 2000). TFIIH functions both in transcription and nucleotide excision repair (NER) and contains DNA-dependent ATPase-, ATP-dependent helicase- and CTD kinase-activity (reviewed in: Egly, 2001). TFIIH can be separated into two different subcomplexes; the core TFIIH and the CTD kinase subcomplex. Two subunits of the TFIIH core, XPB and XPD, possess opposite helicase activity. The ATP-dependent helicase activity of XPB is required to open the promoter around the start site, which allows productive elongation, while both XPB and XPD are required for NER. In the transition from transcriptional initiation to elongation, the CTD is heavily phosphorylated. This phophorylation

9 requires the TFIIH CTD kinase subcomplex, which includes cdk7 and cyclin H. The ability of TFIIH to activate transcription through its CTD kinase activity can be negatively regulated in higher eukaryotes by a cdk8/cyclin C-containing Mediator complex (Akoulitchev et al., 2000). The last step of initiation of transcription is TFIIH-XPB helicase-induced promoter melting by an ATP-dependent separation of the two DNA strands, which allows the polymerase to escape from the promoter, thereby initiating the transcriptional elongation phase, (see "Elongation").

RNA polymerase II The structure of RNA polymerase II has been resolved at 3.5 Å resolution (Cramer et al., 2000) and shows a remarkable structural similarity to bacterial RNA polymerase. RNAP II core enzyme contains 12-subunits, all with different functions. Specific mutations have shown that subunit-specific functions include start site selection, transcriptional elongation rate and interactions with activators (Hampsey, 1998; Lee and Young, 2000). As mention above, the largest subunit of RNAP II contains a unique CTD, which is highly conserved in higher eukaryotes. The function of the CTD correlates with its phosphorylation status. Hypophosphorylated CTD (RNAP IIA) was found in the initiation complex, while the hyperphosphorylated form (RNAP IIO) was found in the elongation complex, suggesting that RNAP II cycles between the different forms and that RNAP IIA is the form required for PIC formation (reviewed in: Dahmus, 1996). However, data from recent in vivo experiments suggest that the RNAP IIO form is also required for high levels of activated transcription in vivo (Castano et al., 2000). As an additional, but not fully understood, level of control, different serines in CTD are phosphorylated at different stages during initiation/elongation (Komarnitsky et al., 2000). Although a broad repertoire of cellular kinases has been shown to phosphorylate CTD, only one CTD phosphatase (Fcp1) with a general role in transcription, has been found (Archambault et al., 1997; Chambers and Kane, 1996). The activity of Fcp1 is regulated by TFIIB and TFIIF, suggesting that these factors regulate polymerase recycling (Chambers et al., 1995; Cho et al., 1999). Finally, it has recently been shown that cellular factors involved in mRNA capping, polyadenylation and splicing, bind to the CTD of RNAP II, suggesting that these activities are closely linked to each other (reviewed in: Proudfoot, 2000).

DNA sequence-specific transcription factors Transcription of most eukaryotic genes is regulated by DNA sequence-specific transcription factors that either increase (activators) or decrease (repressors) the basal level of expression. These transcription factors usually contain two separate domains: one DNA-binding domain and one regulatory domain. The DNA-binding domain interacts with cis-regulatory DNA elements (binding-sites) and the interaction targets the regulatory activating and repressing domain to distinct promoters. The DNA- binding domains can be grouped into different families of structural motifs, including the so-called helix-turn-helix, zinc fingers and leucine zipper motifs (reviewed in: Nelson, 1995). These domains exhibit a high degree of homology between the members within a particular family. In contrast, very little is known about the structure of the different regulatory domains. The activation domains are classified as acidic, or as being rich in glutamine, proline, or serine/threonine, (reviewed in: Triezenberg, 1995). The activators have been suggested to affect chromatin remodelling, PIC formation, promoter clearance and elongation while repressors are thought to block the activation domain of activators, prevent the association of

10 activators with the promoter and inhibit efficient PIC-formation. In addition, activators and repressors also function by recruiting co-activators and co-repressors, respectively, (see "Regulation of transcription"). Furthermore, several DNA sequence-specific transcription factors can either activate or repress transcription depending on the environment. For example, in the absence of hormones, members of the steroid hormone receptor family can bind to cognate DNA together with a co- repressor complex, thereby leading to transcriptional repression. However, following binding of the hormone-ligand, the conformation of the receptor changes so that the co-repressor complex is exchanged for a co-activator complex leading to gene activation (Glass and Rosenfeld, 2000).

Co-factors The activation of transcription requires a number of co-factors. Two broad classes of global co-factors have been described. The first class acts directly on RNAP II and general transcription factors while the second class affects the chromatin structure. The first class of co-factors includes TAFs, Mediators and upstream stimulatory activity (USA). The second class contains ATP-dependent chromatin remodelling complexes and factors required for modifications of histone tails, (see "Chromatin"). However, the activities of these two classes of co-factors are closely linked to each other and function subsequently to each other.

TAFs The general transcription factor, TFIID, consists of TBP and 8-12 different TBP - associated factors (TAFs). Binding of TBP to a promoter is sufficient to support basal transcription. In contrast, TFIID is required for activated transcription in vitro, initially suggesting that TAFs mediate the function of activators (Hoey et al., 1990; Pugh and Tjian, 1990). However, recent genome-wide expression analyses in yeast demonstrated that the TAFs were not generally required, but rather that individual TAFs were required for the activation of specific subsets of genes (Holstege et al., 1998; Lee et al., 2000). Furthermore, inactivation of yeast TAFII145 (yTAFII145), or the mammalian homologue, TAFII250, resulted in G1/S arrest (Talavera and Basilico, 1977; Walker et al., 1996), suggesting that individual TAFs control cell cycle progression through unique targets. This idea is also consistent with the observation that yTAFII145 was shown to be required for the expression of G1- and B-type cyclins (Walker et al., 1997). A subset of TAFs has structural similarities to the core histones and it is believed that they could form an octamer-like structure within TFIID, although it remains to be determined if such a complex can bind DNA [Burley, 1996 #267]. The histone-like TAFs have also been found in several regulatory complexes besides TFIID, including: SAGA, STAGA, PCAF and the TBP-free TFTC complex, even though the relevance of their presence remains unknown [Albright, 2000 #268]. It has been proposed that TAFs could stimulate transcription by three different mechanisms (Green, 2000). First, specific TAFs might function in recognition and selection of core promoters independently of upstream binding factors. Secondly, the identified intrinsic HAT-activity of yTAFII145 and its mammalian homologue, hTAFII250, suggests an enzymatic function of these TAFs. Finally, TAFs might mediate the contact between upstream activators and the basal transcription machinery. However, these interactions have only been demonstrated for isolated TAFs in vitro and their significance in vivo remains unclear.

11 Mediator In yeast, the Mediator complex consists of three groups of proteins; Mediator proteins, the SRB proteins, and a group of proteins previously found to positively or negatively regulate transcription (Myers and Kornberg, 2000). The Mediator complex can exist alone or as part of the RNAP II holoenzyme and was first isolated from yeast as a non-TAF activity extract that supported activated transcription in vitro (Flanagan et al., 1991; Kelleher et al., 1990). It is believed that the Mediator acts at the interface between DNA sequence-specific transcription factors and the basal transcription machinery. Thus, recruitment of a Mediator-containing RNAP II holoenzyme to a promoter by activators, represents a major regulatory step during initiation of transcription (Malik and Roeder, 2000). In line with this, Mediator was found to function directly through RNAP II and to perform an essential role in transcription from almost all yeast promoters (Holstege et al., 1998; Thompson and Young, 1995). However, the Mediator also stimulated basal transcription and phosphorylation of the CTD of RNAP II by TFIIH kinase (Kim et al., 1994), implying an extended activity pattern. In human cells, several homologues to the yeast Mediator complex have been found, including TRAP/SMCC, DRIP/ARC, NAT, CRSP and Sur2 (Malik and Roeder, 2000; Myers and Kornberg, 2000). In summary, the heterogeneity of the reported Mediator complexes might imply the possibility of extensive tissue- and gene-specific regulatory activities (Lee et al., 1999; Li et al., 1995; Myers et al., 1999).

USA A third of group of general co-factors that can either increase activated transcription or repress basal transcription in cell-free systems, are known by the functional term, upstream stimulatory activity (USA) (reviewed in: Naar et al., 2001). USA consists of positive co-factors (PC) and negative co-factors (NC). PC4, for example, was shown to interact both with TFIIA and the activation domain of the viral activator, VP16, suggesting that PC4 might act by helping activators to recruit and stabilise TFIID at a target promoter (Kaiser and Meisterernst, 1996). NC2 was initially suggested to repress transcription by interacting with TBP, thereby preventing the interaction between TFIIA and the TBP/TATA-complex (Meisterernst et al., 1991). However, it was recently shown that the NC2 homologue in Drosophila (dNC2) was required for activation of transcription from a downstream promoter element (DPE). Nevertheless, dNC2 repressed transcription from a promoter containing a TATA-box, suggesting that dNC2 might be a bi-functional basal transcription factor (Willy et al., 2000).

Co-activators and co-repressors Co-activators and co-repressors do not bind DNA directly, but are recruited to target promoters by activators or repressors, respectively. Generally, these co-factors of transcription are found at the interface between the activators/repressors and the basal transcription machinery, and function by modifying histone tails and/or by targeting other co-regulators to the promoter. Whereas many co-activators contain intrinsic HAT-activity, the co-repressors have not shown this. Instead, many co-repressors can directly recruit the histone deacetylase-containing complexes. The co-activator, p300/CBP, binds several unrelated activators including nuclear hormone receptors, STATs, c-jun, YY1, E2F and c-Fos (reviewed in: Goodman and Smolik, 2000), but also the basal transcription factors, TBP and TFIIB (Kwok et al., 1994; Yuan et al., 1996) as well as RNAP II (Kee et al., 1996). p300/CBP contains intrinsic HAT- activity and recruits other co-factors with HAT-activity (Bannister and Kouzarides,

12 1996; Ogryzko et al., 1996). Taken together, these findings suggest that p300/CBP can stimulate transcription by targeting HAT-activity to a target promoter but also by acting as a "molecular-bridge" between the activators and the basal transcription machinery. The co-repressors, Sin3 and Mi-2/NuRD, both contain a core histone deacetylase complex consisting of HDAC1/2 and the histone binding proteins, RbAP46/48 (reviewed in: Ahringer, 2000). In addition, Mi-2/NuRD specifically contains the ATP-dependent chromatin remodelling activity subunit Mi-2β and the methyl-DNA-binding protein, MBD3 (Knoepfler and Eisenman, 1999). Both co- repressor complexes interact with distinct repressors. Sin3 interacts with, among others, Mad/Mxi1, p53, NCoR and SMRT, while the Mi-2/NuRD interaction partners include Ikaros and HPV-E7 (reviewed in: Burke and Baniahmad, 2000).

Elongation Initiation of transcription is followed by promoter clearance and elongation. The general initiation factors, TFIIE, TFIIF and TFIIH have also been shown to function during elongation (reviewed in: Conaway et al., 2000). By inducing an ATP- dependent structural transition to an open complex, TFIIE and TFIIH suppress arrest of the early elongation complex resulting in an escape-competent transcriptional intermediate. By stimulating the rate of elongation by RNAPII, TFIIF also lowers the frequency of abortive initiation during promoter escape (Price et al., 1989). The transition from initiation to elongation correlates with hyperphosphorylation of RNAP II-CTD (Dahmus, 1996). Two negatively acting transcription elongation factors (N- TEFs) have been identified; NELF (Yamaguchi et al., 1999) and DSIF (Wada et al., 1998). N-TEFs have been suggested to prevent elongation by binding to the hypophosphorylated CTD (Yamaguchi et al., 1999). In contrast, P-TEFb/FACT- mediated phosphorylation of CTD is believed to decrease the affinity for N-TEFs (Marshall and Price, 1995; Price, 2000; Wada et al., 2000). Furthermore, the SII- family of factors reactivate arrested RNAP II by activating cleavage of the terminus of the nascent transcript which brings the terminus to the correct position for re- extension of the transcript (Wind and Reines, 2000). TFIIF, ELL and Elongin belong to a group of elongation factors that suppress cessation of RNAP II activity by stimulating the elongation rate and thereby preventing dissociation of the elongation complex from the DNA template (Conaway and Conaway, 1999). However, factors that accelerate elongation on isolated DNA cannot overcome the inhibitory effect that packaging of DNA into chromatin exerts on elongation (Izban and Luse, 1991; Studitsky et al., 1997). Consequently, a second class of elongation factors, including FACT, DSIF and Spt6, promote elongation by modifying the chromatin structure (reviewed in; Orphanides and Reinberg, 2000). Finally, the Elongator complex, which specifically interacts with the phosphorylated form of the CTD (Otero et al., 1999; Wittschieben et al., 1999), contains a subunit with HAT-activity, suggesting that this complex facilitates elongation by modifications of the histone tails.

How transcription might be regulated Based on available data, regulation of transcription can be regarded as two different main events; ATP-dependent chromatin remodelling (see "Chromatin") and formation of competent transcription machinery. Most likely, chromatin remodelling precedes assembly of the transcriptional apparatus at a promoter. In yeast, the ordered recruitment of an ATP-dependent chromatin remodelling complex (SWI/SNF), a Gcn5 dependent HAT-complex (SAGA) and activators (Swi5p and SBF) to the HO endonuclease promoter has been studied (Cosma et al., 1999; Krebs et al., 1999). This

13 analysis suggested that the first factor to enter the promoter was the activator, Swi5p. Swi5p, in turn, recruited SWI/SNF, leading to ATP-dependent chromatin remodelling over a restricted region of the promoter.

(I)

(II) A1

SWI/ SNF ( III) A1

SWI/ SNF HAT (IV) A1 TATA Inr

SWI/ SNF HAT RNAP II (V) A1 A2 holoenzyme

Fig. 4. Possible mechanism for activation of transcription. Location of the binding-site for an activator (A1) at an accessible chromatin position (I) allows binding of A1 and recruitment of SWI/SNF (II). Recruitment of the SWI/SNF complex (III) results in ATP-dependent chromatin remodelling over a restricted region of the promoter. Chromatin remodelling might create a chromatin structure that is more accessible and allows for subsequent recruitment of a HAT-complex (HAT) (IV). A peak of acetylation over the promoter region, but also increased level of acetylation over the region surrounding the promoter, might in turn create even more accessible chromatin (V). A second activator (A2) binds its unmasked binding-site (VI) and facilitates recruitment of the basal transcription machinery.

14 The subsequent recruitment of the SAGA lead to H3 acetylation, followed by recruitment of a second activator (SBF) and the RNAP II holoenzyme. This suggested that SWI/SNF was recruited to the promoter when an activator binds to positions in the chromatin that were accessible/exposed in its native state. In the next step, the local chromatin structure around the promoter was relaxed by SWI/SNF, allowing additional factors to bind. In contrast, chromatin remodelling and retention of the SWI/SNF complex to the yeast PHO8 promoter required activator-mediated recruitment of the SAGA complex (Hassan et al., 2001; Reinke et al., 2001). These results also suggested that acetylation of specific histones in the PHO8 promoter functioned as a transient signal for, and a determinant of, the domain for nucleosome remodelling (Reinke et al., 2001). In summary, activation of the HO and PHO8 promoter required activators to recruit chromatin modifying complexes like SWI/SNF and SAGA although the order of recruitment might be specific for each individual promoter. The idea that the requirement of ATP-dependent chromatin modulating activities is specific for individual promoters was further supported by whole-genome mRNA expression studies in yeast. These studies have suggested that expression of only 3% of yeast genes appeared to depend on SWI/SNF or the HAT-activity of Gcn5 or both (Holstege et al., 1998; Sudarsanam et al., 2000). In addition, it was suggested that SWI/SNF remodelling controlled the recruitment of Gcn5 HAT-activity to promoters expressed during late mitosis, but that, in contrast, Gcn5 HAT-activity was recruited independently of SWI/SNF to promoters expressed in interphase (Krebs et al., 2000). This suggests that the SWI/SNF activity is globally required only when genes are expressed from a condensed chromatin e.g. in the expression of mitotic genes. Gene expression can be cell –type-, tissue- or gene-specific. Simply stated, such discrete expression patterns may arise due to the specificity of the activators, which recruit the transcription machinery to the target promoters. Although these activators are thought to increase the rate at which the transcription machinery is assembled at a specific promoter, the precise mechanism is not fully understood and different models have been suggested. Association between TBP and the TATA-box in the core promoter is the first step in the assembly process. Recruitment of TBP (TFIID) to a promoter is stimulated by activators even if a stable interaction might also require TFIIA and additional factors (Keaveney and Struhl, 1998). The next step in the assembly process is less clear. TFIID and TFIIA, when bound to the core promoter, can recruit the remaining GTFs and the RNAP II core enzyme in an ordered process in vitro (Buratowski et al., 1989). However, different pre-assembled RNAP II- containing complexes, lacking TBP, are found in vivo (Koleske and Young, 1994). In addition to RNAP II, the different complexes contain different subsets of GTFs (except TFIID and TFIIA) and Mediator complexes of varying compositions. It has been suggested from these different results that the transcription apparatus is either assembled in stepwise order on TBP (TFIID)/TFIIA, or that the RNAP II holoenzyme is recruited to promoter-bound TBP (TFIID)/TFIIA in one step. However, several arguments counter both models. Firstly, it would be very time consuming if the RNAP II holoenzyme, containing more than 40 subunits, would have to be assembled de novo and at every single round of transcription. On the other hand, recruitment of RNAP II holoenzyme to a promoter by a two-step mechanism, would limit the possible levels of regulation and create a less dynamic system acting more like a "on- off switch". Instead, it has recently been suggested that the transcription machinery might be assembled by a mixed mechanism (Lemon and Tjian, 2000). In this model, some of the components of the core machinery could be pre-assembled to distinct

15 complexes, functioning like building blocks that are assembled into different and relatively simple combinations. Distinct complexes could then be added or removed to the core-complex, which would result in different possibilities of response to specific requirements. Assembly by this model would require a limited number of recruitment steps without dramatically decreasing the flexibility of the system. Importantly, this would allow activators to stimulate transcription in a synergistic way by recruiting different subcomplexes of the transcription apparatus.

TATA Inr

TATA Inr TATA Inr

TATA Inr

TATA Inr TATA Inr TATA Inr

Figure 5. Assembly of the pre-initiation complex (PIC) on an RNA polymerase II promoter. The PIC might be assembled on TBP (TFIID)/TFIIA according to a stepwise model (left), through recruitment of distinct pre-assembled sub-complexes (middle) or by recruitment of the hole RNAP II holoenzyme to the promoter in a single step (right).

Transcription can also be repressed at a gene-specific level by several different mechanisms (reviewed in: Roberts, 2000). Firstly, repressors can prevent the interaction between activators and the target DNA, thereby precluding recruitment of the transcription machinery. For example, binding of the mad/max heterodimer to its binding-site prevents the interaction between the same binding-site and the transactivating myc/max heterodimer. Secondly, repressors can inhibit transcription by binding to, and inactivating the regulatory domain of specific activators. Thirdly, repressors can inhibit gene expression through distinct repressor domains that have a negative effect on assembly of the transcription machinery. As an example, Rb binds to the activator, E2F, and directly blocks its trans-activating activity. In addition, Rb

16 also actively represses transcription from adjacent promoter elements by recruiting a histone deacetylase (Harbour and Dean, 2000b), illustrating that different repressor activities can overlap each other. Finally, a repressor can inhibit gene expression by a general mechanism, by directly preventing assembly of the transcription machinery. In Drosophila, for example, the repressor, Eve, inhibits transcription by blocking the interaction between TFIID and the TATA-box independently of sequences outside the core promoter (Austin and Biggin, 1995; Li and Manley, 1998).

Regulation of transcription factor activity The function of DNA sequence-specific transcription factors is essential for the regulation of transcription. It is therefore logical that these factors themselves can be regulated by different mechanisms. So far, regulation at the level of stability, cellular localisation, DNA binding, and activity have been characterised, and examples will be given below. In general, it seems that a regulatory event that ultimately alters gene expression, can involve several of these mechanisms. The regulation of the activity of the transcription factor NFκB is an example of such regulation by multiple mechanisms (see "Phosphorylation and dephosphorylation").

Protein stability Protein degradation via the ubiquitin-26 S proteasome pathway is a dynamic mechanism by which the steady-state level of proteins can be controlled (reviewed in: Weissman, 2001). Rapid degradation of regulatory factors allows the system to quickly respond to environmental stimuli. Many transcription factors that are only required for a short time during the cell cycle have been shown to be regulated by ubiquitin-mediated proteolysis. The transactivation domain (TAD) of several transcription factors, including c-myc, p53, E2F and VP16, overlaps the region signalling for ubiquitin-mediated proteolysis and the sensitivity to degradation appears to correlate with the potency of the activation domain (Molinari et al., 1999; Salghetti et al., 2000). Interestingly, ubiquitylation might also positively and negatively regulate transcription via pathways other than protein degradation. For example, ubiquitylation of VP16 TAD is not only required for degradation but also in order to enable VP16 to activate transcription (Salghetti et al., 2001). Non- ubiquitylated VP16 was stable and inactive but ubiquitylation of the TAD activated the transcriptional function of VP16 and simultaneously primed it for proteasome- mediated degradation. In contrast, ubiquitylation of the yeast activator, Met4, inhibited its trans-activating potential (Kaiser et al., 2000). Since the inhibition was not due to blocked promoter recruitment of Met4, the result suggested that ligation of ubiquitin to the activator prevented efficient assembly of the transcription apparatus. In addition, several proteins with sequence similarities to ubiquitin have been identified, e.g. SUMO (small ubiquitin-related modifier). Like ubiquitin, SUMO is conjugated to other proteins and sumoylation might modulate subcellular compartmentalization and enhance the stability of the target protein (reviewed in: Muller et al., 2001). For example, Mdm2 targets p53 for ubiquitin-mediated proteolysis, however, Mdm2 itself is regulated by the ubiquitin-proteasome system. Ubiquitylation and sumoylation act on the same lysine residue of Mdm2 and sumoylation of Mdm2 has been suggested to prevent ubiquitin-mediated degradation of Mdm2. Stabilisation of Mdm2 subsequently resulted in increased ubiquitylation and degradation of p53. It is also important to note that both ubiquitination and sumoylation are reversible.

17 Cellular localisation The activity of certain transcription factors is regulated by their cellular localisation. The transforming growth factor-β (TGF-β) family of cytokines is part of the cell-cell signalling that controls cell proliferation, differentiation and death. The TGF-β pathway is mediated by the SMAD-family of transcription factors (reviewed in: Massague, 2000). Receptor-regulated SMADs (R-SMADs) are maintained in an inactive state in the cytoplasm by binding to the cytoplasmic domain of the TGF-β membrane receptor. R-SMADs are phosphorylated upon ligand binding and activation of the receptors. Phosphorylation results in release of R-SMAD from the receptor, and following binding of co-SMADs, the newly formed complex is trans-located into the nucleus where it can regulate expression from specific genes.

DNA binding The DNA binding affinity of DNA sequence-specific transcription factors is regulated by phosphorylation/dephosphorylation, acetylation/deacetylation and oligomerisation. For example, phosphorylation of the carboxy terminus of c-jun, close to the DNA- binding domain, decreases the affinity of c-jun for DNA. However, this inactive form of c-jun can rapidly by re-activated by extracellular signalling via site-specific dephosphorylation of the carboxy-terminus (Boyle et al., 1991). In contrast, phosphorylation of the amino-terminal activation domain results in induction of c-jun- mediated transactivation (Pulverer et al., 1991). Acetylation of the transcription factor, E2F1, close to the DNA-binding domain, is multifunctional since it increases DNA-binding affinity but also increases the half-life and the activation potential (Martinez-Balbas et al., 2000). Importantly, acetylation, and therefore also the increased activity is reversed upon Rb-mediated recruitment of HDAC1 and deacetylation of E2F1. DNA binding of certain DNA sequence-specific transcription factors requires dimerisation or even oligomerisation. The activator, c-myc, for example, does not bind DNA as a monomer but does so with high affinity after heterodimerisation with the max-protein (Grandori et al., 2000).

Phosphorylation and dephosphorylation Phosphorylation has long been regarded as a major mechanism by which protein activities are controlled, a dogma confirmed by the fact that regulation of stability, cellular localisation, DNA binding and the activity of specific transcription factors are tightly controlled by phosphorylation (reviewed in: Whitmarsh and Davis, 2000). That the phosphorylation level can affect cellular localisation and DNA binding affinity has been discussed above. The first transcription factor shown to be regulated by phosphorylation was the cyclic AMP (cAMP) response element (CRE)-binding protein, CREB (reviewed in: Mayr and Montminy, 2001). Accumulation of the second messenger cAMP, due to growth factor, mitogen or stress signalling, activates protein kinase A (PKA) which then diffuses into the nucleus and phosphorylates CREB. Phosphorylation of the activation domain of CREB is required for recruitment of the co-activator p300/CBP and the subsequent transcriptional activation of specific genes. It is interesting to note that phosphorylation of CREB is a relatively fast reaction, which allows the cell to respond to e.g. growth factor signalling at the level of specific gene expression, in less than 30 minutes, post stimulus. Furthermore, phosphorylation is also reversible and dephosphorylation of CREB by protein phosphatase 1 (PP1) and PP2A, results in decreased transactivation potential of CREB.

18 Phosphorylation also indirectly regulates the activity of the transcription factor, NFκB (reviewed in; Whitmarsh and Davis, 2000). The inhibitor, IκB, keeps NFκB in the cytosol by masking the nuclear localisation signal (NLS) of NFκB. Upon cytokine stimulation, IκB is phosphorylated and subsequently targeted for ubiquitin- mediated degradation. The release of NFκB from its inhibitor exposes the NLS and the transcription factor can then enter the nucleus and activate transcription of specific genes. Together, these results demonstrate that post-translational modifications of DNA sequence-specific transcription factors can cause rapid trans-location, altered activity or DNA binding affinity, which in turn affects transcription of specific genes. Importantly, the link between these regulatory pathways and cellular signalling allows the cell to quickly respond to environmental changes with adjustments of transcriptional levels.

THE ADENOVIRUS MODEL SYSTEM

Since its discovery, adenovirus has been intensively and successfully used as a model for the study of regulatory pathways in eukaryotic cells. Adenovirus as an experimental system is based on several different characteristics of the virus; firstly, it depends on the host cells for its replication and has consequently evolved specific factors to modify the function of cellular key proteins that regulate gene expression and replication. These viral-specific regulatory factors can be used to study the cellular processes with which they interfere. Secondly, the viral genome, in contrast to that of the host, is small and is therefore relatively easy to manipulate. Finally, adenovirus efficiently infects cultured cell lines and can easily be grown to high-titer virus stocks. Split genes and RNA splicing were discovered in adenovirus (Berget et al., 1977; Chow et al., 1977). Indeed, studies on adenovirus have led to many important insights into the general mechanisms involved in transformation and cell- cycle control as well as transcriptional and translational control (reviewed in: Shenk, 1996).

Adenovirus Adenovirus is a double-stranded DNA virus with a linear genome of approximately 35000 base pairs. They are non-enveloped, icosahedral particles, 70-100 nm in diameter, with fibers projecting from each vertex. Human adenoviruses were first isolated in the early 1950s by two independent research groups (Hilleman and Werner, 1954; Rowe et al., 1953). Adenovirus can cause respiratory, gastrointestinal and eye infections and today more than 100 adenovirus serotypes, infecting a wide range of mammalian and avian hosts, have been identified. In 1962, it was shown that human adenovirus type 12 induced malignant tumours in newborn hamsters (Trentin et al., 1962). This was the first demonstration that a human virus could induce oncogenesis. Although adenovirus can cause tumours in rodents, there is no evidence for any link between cancer and adenovirus infection in humans.

The adenovirus life cycle Adenovirus can infect most cells. Attachment to the host cell is mediated by the fiber knob that binds to the primary cellular adenovirus receptor (CAR) (Bergelson et al., 1997; Hong et al., 1997; Tomko et al., 1997). Interaction between the penton base protein and the secondary cellular receptor (Wickham et al., 1993) promotes the

19 uptake of the virus particle by receptor-mediated endocytosis. Subsequently, the virus escapes the endosome into the cytoplasm and is transported to the nuclear membrane. During transport, the virion is disassembled in a strictly ordered, stepwise process (reviewed in: Greber et al., 1994). At the nuclear membrane, the viral DNA is injected into the nucleus, through the nuclear pore complex, together with the viral DNA- associated protein VII (Greber et al., 1997). During the early phase of an adenovirus infection, a constitutively active promoter ensures the expression of the early region 1A gene (E1A). E1A protein forces the cell to enter S-phase and initiate DNA synthesis by modulating the function of key regulatory proteins (see "The role of E1A in cellular transformation"). Moreover, by activating transcription from all of the other early transcription units (E1B, E2, E3 and E4), E1A ensures expression of numerous early viral regulatory proteins. Briefly, proteins encoded from the E1B region counteract E1A-induced growth arrest and apoptosis; the E1B-55K protein represses transcription induced by the tumour suppressor protein p53 by binding to it, whereas the E1B-19K protein functions as a homologue of the cellular anti-apoptotic Bcl-2 protein, preventing both p53-dependent and p53-independent apoptosis (reviewed in: Roulston et al., 1999). The E2 region encodes the DNA polymerase, the terminal protein and the DNA binding protein (DBP), all needed for efficient viral replication (reviewed in: Van der Vliet, 1995). Proteins encoded from the E3 region are important in protecting the infected cell from the anti-viral defence strategies of the host organism. The E3- gp19K protein forms a complex with the major histocompatibility complex (MHC) class I molecule in the endoplasmic reticulum, thereby blocking further transport to the cell surface. Furthermore, the E3-14.7kD and E3-10.4K/14.5K proteins have been shown to inhibit the cytotoxic effect of tumour necrosis factor-α (TNF-α) (Wold et al., 1995). Finally, alternative splicing generates at least seven different proteins from the E4 region which are important regulators of transcription, splicing, mRNA transport and transformation (reviewed in: Leppard, 1997). At the completion of the early phase of infection, viral DNA replication is initiated and at the same time, the major late transcription unit (MLTU) is expressed. The MLTU generates a pre-mRNA that is processed into at least 20 different mRNAs by alternative splice site selection and poly(A) site usage (reviewed in: Akusjarvi, 1999). The mRNAs produced from the MLTU encode structural proteins. During the late phase of the infection cycle, modifications of cellular pathways regulating mRNA export and translation allow preferential synthesis of viral proteins, (reviewed in: Shenk, 1996). After translation, the structural proteins are imported into the nucleus and assembled into capsids (Sundquist et al., 1973) which subsequently incorporate the viral chromosome. The adenovirus life cycle ends with lysis and release of progeny virus particles.

Early region 1A The primary transcript from E1A generates at least 5 different mRNAs by alternative splicing (Stephens and Harlow, 1987; Ulfendahl et al., 1987). The 13S and 12S mRNAs are translated into two proteins of 243 and 289 amino acids (243R and 289R), respectively. Sequence comparison of E1A from different adenovirus serotypes has identified three conserved regions (CR), CR1, CR2 and CR3 (Kimelman et al., 1985), of which the CR3 is unique to the 289R protein. Genetic studies have shown that most activities ascribed to the multifunctional E1A depend on the interaction between the conserved regions in E1A and host cell regulatory proteins (Fig. 6) (Moran, 1994). Through these interactions, E1A can activate and repress transcription, force the cell to enter S-phase and induce DNA synthesis, induce

20 apoptosis, immortalise rodent cells and cause full transformation together with a co- operating oncogene (Jones, 1995; Nevins, 1995).

TFx DBD p300 / CBP Rb Mediator CtBP CR1 CR2 CR3 E1A-289R CR1 CR2 E1A-243R

Exon 1 Exon 2

Figure 6. Most functions of E1A are mediated by the interaction with cellular regulatory proteins. Three conserved regions (CR1, CR2 and CR3), located in the first exon of E1A (exon 1), are required for the interactions with several different regulatory proteins (a selection of these interactions are shown above). In contrast, CtBP is the only protein known to interact with the second exon (exon 2) of E1A.

Activation of transcription by E1A E1A displays great flexibility as transactivating protein and can activate transcription from almost any promoter. Several different mechanisms for E1A- dependent activation of transcription have been described including direct interference with cellular transcription factors, displacement of inhibitory co-repressor complexes or modulation of the activity of cellular transcription factors by phosphorylation. CR3 is required for induced activation of cellular transcription factors and for the efficient expression of other early viral genes. CR3 consists of two functional domains: an activation domain, including an amino-terminal zinc finger, and a carboxy-terminal promoter localisation domain. E1A does not bind DNA by it self. Instead, E1A is recruited to promoters through the interaction between the DNA binding domain of different cellular transcription factors and the promoter localisation domain in CR3 (Lillie and Green, 1989; Liu and Green, 1994). Upon promoter targeting, CR3 is believed to stimulate transcription by acting as a "molecular bridge" between up-stream binding transcription factors and the basal transcription machinery. This idea is supported by the ability of E1A to recruit a human homologue of the yeast Mediator complex, SUR-2 (Boyer et al., 1999). In addition, CR3 has been shown to interact directly with individual TAFs (Chiang and Roeder, 1995; Geisberg et al., 1995; Mazzarelli et al., 1997) and this interaction has been shown to be required for CR3-mediated transactivation in vitro (Zhou et al., 1992). Despite its promiscuous transactivating capacity, CR3 is not the only E1A region engaged in activation of transcription. CR1 is a strong transactivator of gene expression when tethered to the promoter through the heterologous Gal4 DNA- binding domain (Bondesson et al., 1994), and is also essential for the induction of transcription by the 243R protein (Kannabiran et al., 1993) via the CREB-CBP pathway (Lee and Mathews, 1997). Most of the CR3-independent activation by E1A involves the disruption of inhibitory complexes formed on cellular transcription factors. Perhaps the best characterised interaction is the direct binding between E1A protein and the retinoblastoma tumour suppressor (Rb), thereby disrupting its inhibitory interaction with the transcriptional activator, E2F (reviewed in: Cress and Nevins, 1996; Dyson and Harlow, 1992). E1A can also prevent the negative co-factor, NC2, from competing with TFIIA in binding to the TBP/TATA complex (Inostroza et

21 al., 1992; Kraus et al., 1994). By binding to E1A, NC2 is displaced from TBP/TATA and efficient formation of the pre-initiation complex can occur (Kraus et al., 1994). Whereas the interaction with Rb requires CR1 and CR2 (Fattaey et al., 1993; Ikeda and Nevins, 1993), the interaction with NC2 is mediated by the extreme amino- terminal region of E1A. Finally, sequences in the second exon of E1A have also been suggested to activate transcription by a displacement mechanism. As demonstrated in papers I and II, E1A can target the repressor protein, CtBP, and thereby relieve repression mediated by CtBP. This activity may also explain a previous observation that the second exon of E1A can activate transcription from early viral genes (Mymryk and Bayley, 1993).

A) Mediator ( hSur-2 )

E1A- 289R TFx TATA Inr

Basal transcription machinery

B) Rb

Rb E1 A E1 A

E2 F E2 F

C) E4F P E1 A E1 A P P E4F

Figure 7. E1A can activate transcription by different mechanisms. (A) E1A activates transcription by binding to the DNA binding domain of different transcription factors (TFx) and the human Mediator complex (hSur-2) thereby creating a more active transcription complex, (B) Dissociating of inhibitory transcription factor complexes (C) Inducing phosphorylation of cellular transcription factors.

The third mechanism that is used by E1A to activate transcription is to induce phosphorylation of cellular transcription factors. Although E1A does not contain any intrinsic kinase activity, it is suspected to modulate the activity of cellular kinases. E1A is known to interact with a multiprotein complex containing cdk 8 (Boyer et al., 1999; Gold et al., 1996) and to associate with cdk2 (Faha et al., 1993; Giordano et al., 1991; Herrmann et al., 1991; Kleinberger and Shenk, 1991). The significance of these interactions, however, is unknown. Nevertheless, E1A has been shown to induce phosphorylation of the DNA binding domain of E2F and E4F (Bagchi et al., 1989;

22 Raychaudhuri et al., 1989) and the transactivation domain of c-jun (Hagmeyer et al., 1993), and thus to increase the transactivation potential of these factors.

Repression of transcription by E1A E1A proteins, in addition to activating transcription, can also repress transcription. Molecular cloning and functional analysis of the E1A-associated 300 kDa protein (p300) revealed its function as a transcriptional co-activator (Eckner et al., 1994). E1A can repress transcription stimulated by p300 (Eckner et al., 1994) and also by the closely related co-activator, CBP (Arany et al., 1994). Repression may involve multiple mechanisms. Firstly, E1A blocks the intrinsic HAT-activity of p300/CBP (Chakravarti et al., 1999). Secondly, E1A prevents the suggested interaction between p300/CBP bound to activators and the basal transcription machinery (Nakajima et al., 1997). Finally, E1A can displace additional co-factors with HAT activity from p300/CBP (Kurokawa et al., 1998; Yang et al., 1996b). However, on the contrary, E1A has been shown to stimulate the HAT-activity of CBP at the G1/S boundary of the cell cycle (Ait-Si-Ali et al., 1998) and to be required for efficient recruitment of p300/CBP to the E2 promoter from adenovirus serotype 12 (Fax et al., 2000). The conflicting data might partly be due to the finding that p300 and CBP have distinct roles in cell differentiation (Kawasaki et al., 1998). The interaction between E1A and p300/CBP requires the amino-terminus of E1A and CR1 (Arany et al., 1995; Lundblad et al., 1995). The amino-terminus of E1A has also been shown to repress transcription in vitro, possibly by preventing the interaction between TBP and the TATA-box (Lu et al., 2000; Song et al., 1995a; Song et al., 1997; Song et al., 1995b) and for repression of the HIV long terminal repeat promoter in vivo (Tsang et al., 1996).

The role of E1A in cellular transformation Since adenovirus normally infects quiescent cells, the virus must have the capability to force the host cell to enter the S-phase of the cell cycle thereby creating an optimal environment for viral DNA replication. This function is carried out by the E1A proteins, which, as described above, target a number of cellular proteins with a key function in cell cycle regulation. Continuous E1A expression thus leads to deregulation of the cell cycle, which may immortalise primary cells (Houweling et al., 1980). Progression through the cell cycle is regulated by different cyclin-dependent protein kinases (cdk) which induce phosporylation of down-stream acting regulatory proteins. Activation of cdks requires the interaction with one of several so called cyclins, which also specifies the targets for phosphorylation. The activity of cyclin/cdk complexes is controlled by cdk inhibitors (cki) and different cyclin/cdk complexes are active during different stages of the cell cycle (reviewed in: Morgan, 1995). The Rb protein is an important target for phosporylation by cyclin/cdk complexes. In its hypophosphorylated form, Rb arrests the cell in mid to late G1-phase of the cell cycle (Harbour and Dean, 2000a; Harbour and Dean, 2000b; Weinberg, 1995) by binding to E2F and blocking E2F-dependent transcriptional activation. Phosphorylation of Rb, initially by cyclinD/cdk4/6, disrupts the inhibitory Rb/E2F complex and increases the pool of free E2F (Harbour and Dean, 2000b). The E2F family of transcription factors is important for cell cycle progression and transient expression of E2F1 can induce quiescent cells to enter S-phase (Harbour and Dean, 2000b; Johnson et al., 1993). Targeting of E1A to Rb, and the related p107 and p130, relieves E2F and permits transcription from genes, the products of which are needed for DNA replication (Nevins, 1992; Whyte et al., 1988a). Although E1A relieves the

23 requirement for Rb phosphorylation it also directly targets the activities of additional cell cycle regulatory complexes. Cyclin A/cdk2 and cyclin E/cdk2 are active when bound to p107 and p130 and E1A retains their activities by stabilising the complexes via direct interactions (Faha et al., 1993). E1A can also interact directly with the cdk- inhibitor, p27 (Mal et al., 1996) thereby preventing inhibition of the cyclin E/cdk2 complex which is needed for progression through late G1-phase (Morgan, 1995). It is postulated that the p300/CBP co-activators are required for the G1 arrest observed in differentiated cells, thereby preventing DNA synthesis (Kolli et al., 2001). Repression of p300/CBP by E1A would therefore be essential for the induction of S-phase by preventing differentiation (see "Repression of transcription by E1A" and (Goodman and Smolik, 2000)). Depending on the target cell, E1A induction of S- phase might therefore require inactivation of p300/CBP or the Rb family of proteins or both (Howe and Bayley, 1992; Howe et al., 1990). In addition, it has recently been shown that E1A is associated with a SWI/SNF-containing complex and that the chromatin remodelling activity is required for E1A-mediated transformation (Fuchs et al., 2001; Miller et al., 1996). In co-operation with a second oncogene such, as E1B or activated ras, E1A can fully transform rodent cells (Ruley, 1983). The second exon of E1A contains immortalisation and nuclear localisation functions, both of which are required for co- transformation with E1B (Douglas and Quinlan, 1995; Subramanian et al., 1991). In contrast, the second exon is dispensable for co-transformation with activated ras (Whyte et al., 1988b) and in fact encodes functions that down regulate E1A+ras mediated transformation, tumorigenesis and metastasis (Schaeper et al., 1995). Reduction in the transforming ability partly correlates with binding of the cellular co- repressor protein, CtBP, to the second exon of E1A (Boyd et al., 1993). In addition, it has recently been shown that deletion of sequences predominantly outside the CtBP interacting domain, which also results in an E1A+ H-ras hypertransforming phenotype, can alter the expression of at least two members of the Rho family of GTPases (rac/cdc4) (Fischer and Quinlan, 2000).

CtBP, A CELLULAR CO-REPRESSOR OF TRANSCRIPTION

C-terminal Binding Protein (CtBP) was first identified as a cellular protein that binds to the carboxy terminus of E1A. CtBP binds to the highly conserved CtBP binding motif, PXDLS, first identified in the extreme carboxy-terminus of E1A-243R (between amino acids 233 and 237) (Schaeper et al., 1995). However, the overall secondary structures and amino acids flanking the PXDLS motif is important for the interaction with CtBP (Molloy et al., 2000; Molloy et al., 2001; Molloy et al., 1998). In addition, p300 and P/CAF acetylation of a conserved lysine residue adjacent to the core binding site (Lys-239) in E1A blocks CtBP interaction (Zhang et al., 2000). Similarly, acetylation by p300/CBP of the corresponding lysine residue in the nuclear hormone receptor co-repressor, RIP140, disrupts the interaction with CtBP and decrease RIP140 repressor activity (Vo et al., 2001). Mammalian cells contain at least two distinct CtBP proteins, CtBP1 and CtBP2, encoded by 2 different genes (Katsanis and Fisher, 1998). A third protein, CtBP3, has been cloned in the rat, but it is unclear whether this represents an additional gene or a splice variant of CtBP1. Homologues to mammalian CtBP have been found in Drosophila (Nibu et al., 1998b; Poortinga et al., 1998) and Xenopus (Brannon et al., 1999) in which two additional CtBP proteins have also been

24 described (CtBP1 and XCtBP) (Brannon et al., 1999). Furthermore, the pre-mRNA from the gene encoding CtBP in Drosophila is alternatively spliced into at least 4 different mRNAs that are dynamically expressed (Poortinga et al., 1998). CtBP is a ubiquitously expressed phosphoprotein (Boyd et al., 1993; Schaeper et al., 1995) and the level of phosphorylation changes during the cell cycle with a peak at mitosis (Boyd et al., 1993). Endogenous CtBP is located both in the cytoplasm and in the nucleus (Nibu et al., 1998b; Weigert et al., 1999). The localisation of exogenous CtBP is cell type-specific and furthermore, co-expression of a mainly nuclear CtBP-interacting protein relocates CtBP from the cytoplasm to the nucleus (Criqui-Filipe et al., 1999). These results might suggest that CtBP is predominantly cytoplasmic and that it is re-localised to the nucleus upon interaction with proteins that function to impart a nuclear import signal. The first indication that CtBP functions as a co-repressor of transcription came from analyses of the CR1 domain in E1A. The CR1 domain functions as a strong activator of transcription when tethered to the promoter through the heterologous Gal4 DNA-binding domain. However, when the CR1 domain was fused to the carboxy-terminal region of E1A, containing the binding site for CtBP, transcription was efficiently blocked (Sollerbrant et al., 1996) and paper II. These results suggested that CtBP is a repressor of transcription when recruited to the promoter. The prediction was later confirmed when CtBP was shown to specifically interact with a broad range of transcription factors and to repress transcription in several organisms when recruited to the promoter (reviewed in: Turner and Crossley, 2001).

CtBP interacting proteins The list of proteins demonstrated to interact with CtBP is rapidly increasing. To date, CtBP has been shown to interact with repressors, co-repressors, HDACs and HDAC- interacting protein, members of the polycomb family of transcription factors, viral proteins and CtIP. Some of these proteins are presented in table 1.

Table 1. Examples of CtBP-interacting proteins

Family of protein CtBP-interacting protein Reference

Repressor Knirp, Krüppel, Snail (Nibu et al., 1998b) Hairy (Poortinga et al., 1998) Net (Criqui-Filipe et al., 1999) ZEB (Postigo and Dean, 1999) KLF8 (van Vliet et al., 2000) Ikaros (Koipally and Georgopoulos, 2000) MITR (Zhang et al., 2001) Co-repressors FOG (Fox et al., 1999) pRb/p130 (Meloni et al., 1999), TGIF (Melhuish and Wotton, 2000) Evi1 (Izutsu et al., 2001) RIP140 (Vo et al., 2001) Polycomb proteins Pc2 (Sewalt et al., 1999) HDACs and HDAC interacting HDAC1 (Paper I) proteins HDAC2, (Koipally and Georgopoulos, 2000) HDAC4, 5, 7 (Zhang et al., 2001) mSin3A (Koipally and Georgopoulos, 2000) Viral proteins E1A (Schaeper et al., 1995) EBNA3C (Touitou et al., 2001) Other proteins CtIP (Schaeper et al., 1998)

25 The transcription factors interact with CtBP via a sequence similar to the conserved PXDLS motif (Turner and Crossley, 2001). However, HDAC1, 2, 5, 7 and mSin3A interact with CtBP even though these factors do not contain a recognisable CtBP- binding motif.

Mechanisms of CtBP mediated repression CtBP acts as a transcriptional repressor following recruitment to target promoters through repressors or other co-repressor proteins. However, the contribution of CtBPs to the level of repression differs depending on the nature of the recruiting factor. For example, mutation of the CtBP binding sites in the repressor protein, ZEB, totally blocks its repressor activity (Postigo and Dean, 1999), while MITR-mediated repression of MEF2-dependent transcription only partly depends on recruitment of CtBP (Zhang et al., 2001). The mechanism by which CtBP represses transcription is not known. Mammalian CtBP interacts with class I and class II histone deacetylases (Koipally and Georgopoulos, 2000; Zhang et al., 2001) and papers I and III and with the co- repressor mSin3A (Koipally and Georgopoulos, 2000). The requirement for HDAC activity in co-repressor mSin3A (Koipally and Georgopoulos, 2000). The requirements for HDAC activity in CtBP-mediated co-repression is, however, under question. Analysis of transcriptional repression by a Gal4CtBP fusion protein have generated different results concerning the sensitivity to a histone deacetylase inhibitor (Criqui-Filipe et al., 1999; Izutsu et al., 2001; Koipally and Georgopoulos, 2000), papers II and III. Furthermore, dCtBP does not interact with HDACs (Phippen et al., 2000) and the short-range repressors in Drosophila, the activities of which depend on dCtBP (Nibu et al., 1998a; Nibu et al., 1998b), are fully functional in embryos carrying a mutated Rpd3 histone deacetylase gene (Mannervik and Levine, 1999). It has been suggested that the interaction between CtBP and HDACs has structural importance, since CtBP can be recruited to the MEF2 transcription factor via interaction with the class II HDACs (HDAC-4, 5 and 7) or by interaction with MITR (a transcriptional repressor with homology to the non-catalytic domain of class II HDACs) (Zhang et al., 2001). In summary, the inconsistent results might be due to the fact that CtBP can repress transcription by both HDAC-independent and - dependent mechanisms and/or that CtBP recruits different protein complexes in different cell types. In Drosophila, CtBP-mediated repression is distance dependent and dCtBP interacts with several short-range repressor proteins; Knirp, Krüppel and Snail (Nibu et al., 1998a; Nibu et al., 1998b). However, dCtBP also interacts with the long-range repressor, Hairy, but the relevance of this interaction remains unclear since the repressor activity of Hairy does not depend on dCtBP and, further, mutations of dCtBP suppress the Hairy phenotype (Poortinga et al., 1998). Specific short- and long-range repressors are not defined in mammalian cells, however, results in paper III indicate that CtBP-mediated repression in mammalian cells is also distance dependent.

The biological role of CtBP Transcriptional repression, mediated by repressors and co-repressors, is required for proper regulation of cell growth and development. Rb/p130 regulates cell cycle progression by actively repressing E2F-mediated transcription (Harbour and Dean, 2000a; Harbour and Dean, 2000b). Another tumour suppressor, BRCA1, activates transcription from the p21 promoter (a cdk inhibitor, see "The role of E1A in cellular

26 transformation") in response to DNA damaging agents. Significantly, both the RB/p130 and the BRCA1 tumour suppressors have been found to interact with CtBP through the bridging factor, CtBP interacting protein (CtIP) (Meloni et al., 1999; Schaeper et al., 1995; Yu et al., 1998). Whereas the possible recruitment of CtBP- CtIP to Rb/p130 might provide an additional mechanism by which Rb/p130 represses E2F-mediated transcription (Meloni et al., 1999), the interaction between CtBP-CtIP and BRCA1 probably blocks BRCA1-dependent transactivation (Li et al., 1999; Yu and Baer, 2000). These data suggest that transcriptional repression, involving CtBP, participates in the regulation of cell cycle progression. In Drosophila, transcriptional repression by CtBP is essential for proper regulation of development. Three different short-range repressors require the repressor activity of dCtBP during embryonal development (reviewed in: Mannervik, 2001). It is most likely that a similar molecular scenario will be found in mammals. Several of the CtBP-interacting repressor proteins are required for proper development and differentiation e.g. ZEB regulates lymphocyte and muscle differentiation (Postigo and Dean, 1999), Ikaros is essential for lymphocyte development (Koipally and Georgopoulos, 2000), FOG is required for differentiation of red blood cells (Deconinck et al., 2000) and XTcf-3 is important throughout development in Xenopus (Brannon et al., 1999). Furthermore, CtBP has been shown to interact with Pc2 (Sewalt et al., 1999), a member of the Polycomb family of transcription factors that are needed to maintain an absence gene expression during development (Francis and Kingston, 2001). Recently, it has been found that CtBP also represses transcription through Evi-1 (by blocking TGF-β signaling) (Izutsu et al., 2001; Palmer et al., 2001) and AP-2rep (by blocking AP-2 mediated regulation of gene expression programs during embryonic development) (Schuierer et al., 2001). Importantly, deregulation of both these processes has been shown to occur in natural tumours (Izutsu et al., 2001; Palmer et al., 2001; Schuierer et al., 2001). As noted earlier (see "The role of E1A in cellular transformation"), the interaction between CtBP and E1A correlates with down regulation of transformation, tumorigenesis and metastasis of cells transformed with both E1A and H-ras (Boyd et al., 1993; Schaeper et al., 1995). In contrast, the second exon of E1A, containing the binding site for CtBP, is required for co-transformation with E1B (Subramanian et al., 1991; Whyte et al., 1988b). EBNA3C from Epstein-Barr virus has been suggested both to have similar immortalising properties to E1A and, like E1A, to interact with CtBP (Parker et al., 1996; Touitou et al., 2001). EBNA3C also shows an increased capacity to co-operate with H-ras, upon the interaction with CtBP, in a transformation assay (Touitou et al., 2001). In summary, these results indicate that CtBP-mediated repression is possibly important in several different pathways regulating cell growth and development. It is therefore possible that inactivation of CtBP, or almost any factor, in any pathway in which it participates, might result in aberrant cell cycle regulation that possibly results in the induction of transformation. However, further studies, including knock-out experiments of individual CtBP genes are required to conclusively establish the biological role of CtBP.

Additional characteristics of CtBP In addition to its function as a co-repressor of transcription, one CtBP family protein, CtBP3/BARS (closely related to CtBP1 (Turner and Crossley, 2001)), has been shown to be involved in inducing fission in the Golgi tubular network (Weigert et al., 1999). An intrinsic acyltransferase activity of CtBP/BARS was shown to cause

27 acylation of specific membrane lipids resulting in formation and release of vesicular fragments from the Golgi stacks. All CtBP species are highly homologous to NAD-dependent D-isomer specific 2-hydroxyacid dehydrogenase enzymes although neither dehydrogenase activity nor binding to NAD+ has been observed (Schaeper et al., 1998). Moreover, mutation of a conserved histidine residue that appears to correspond to the active site in the corresponding enzyme D-isomer dehydrogenase catalytic domain does not effect the repressor activity of mCtBP2 or dCtBP. Nevertheless, the same mutation disrupts the ability of dCtBP to activate transcription in a Gal4 tethering assay (Phippen et al., 2000; Turner and Crossley, 1998). Based on these observations, it has been suggested that the conserved domain functions as an interface for protein-protein interactions and possibly mediates the formation of CtBP homodimers (Poortinga et al., 1998; Schaeper et al., 1995; Turner and Crossley, 1998).

PRESENT INVESTIGATION

Paper I: The carboxy-terminal region of adenovirus E1A activates transcription through targeting of a C-terminal binding protein-histone deacetylase complex As described in the introduction, the capability of adenovirus E1A to regulate transcription has strong implications for the its capacity to induce progression into S- phase and cellular transformation. Deregulation of the cell cycle requires the physical interaction of the amino-terminus, CR1 and CR2 of E1A with cellular proteins controlling cell-cycle progression. Targeting of cellular regulatory complexes can result in transcriptional activation, as exemplified by E1A induced release of E2F from RB, or in inhibition of transcription, as exemplified by inhibition of the co- activator p300/CBP by direct targeting of E1A (see "The role of E1A in transformation"). In addition, it has been shown that CR1 functions as a strong activator of transcription when tethered to the promoter through the heterologous Gal4 DNA-binding domain (Bondesson et al., 1994). However, in the context of full- length E1A (Gal4E1A-243R), CR1 transactivation was totally inhibited. E1A deletion mapping identified a region located between amino acids 225 and 238 in the carboxy- terminus of E1A-243R as responsible for the inhibition of CR1 activation (Sollerbrant et al., 1996). The same region was shown to correspond to the binding site for CtBP (Boyd et al., 1993; Schaeper et al., 1995). These results suggest that recruitment of CtBP to a promoter silences CR1-dependent transactivation. In Paper I, we further investigated the interaction between E1A and CtBP and focused on its relevance for regulation of CR1-dependent gene expression by the native E1A protein. For this purpose we chose to study gene expression from the naturally E1A-243R responding PCNA-promoter (Kannabiran et al., 1993). E1A-243R activation of the PCNA promoter requires the PCNA-E1A responsible element (PERE) and targets the CREB- CBP pathway (Lee and Mathews, 1997). We showed that efficient E1A-243R mediated activation of expression from a reporter driven by the proximal PCNA promoter element (-87 to +60) required the CtBP interacting domain (CID) of E1A (amino acids 225-238 of E1A-243R) in addition to CR1. This result can be interpreted in either of two ways: CtBP is a positive co-factor for CR1 mediated induction of the PCNA-promoter, or alternatively, CtBP represses transcription from the promoter and efficient expression requires sequestering of CtBP by E1A. The latter model was supported by the finding that co-expression of a CtBP-binding competitor, encoding a 100 amino acid fragment in the second exon of E1A (E1Aexon2) restored the

28 defective transactivtion capacity of an E1A-243R mutant lacking the CID (E1A- 243R∆CID). Importantly, co-expression of a competitor lacking the capability to interact with CtBP (E1Aexon2∆CID) had no effect on expression from a reporter driven by the PCNA promoter. It has been shown that redundant sequences in the second exon of E1A can activate transcription from several viral genes (Mymryk and Bayley, 1993).

Ct BP

TFx

E1A exon2 E1A Ct BP E1A exon2 exon2 E1A exon2

TFx + + +

Figure 8. Possible models for CtBP-mediated repression of transcription and E1A mediated de-repression. CtBP might be recruited to the promoter through the CtBP interaction domain (CID) of different DNA-bound repressors. At the promoter, CtBP contributes to repression of transcription from the target promoter. Over-expression of a CtBP binding competitor, e.g. E1Aexon2, might relive repression mediated by CtBP due to displacement of CtBP from the promoter, by binding of CtBP to the CID of E1Aexon2.

In paper I, we demonstrated that second exon-mediated activation was also observed in the PCNA promoter. Importantly, whereas induction of the PCNA promoter by CR1 requires the PERE element (Lee and Mathews, 1997), de-repression by E1Aexon2 was shown to be mediated through the core promoter, downstream and independently of the PERE sequence. The promoter element responding to E1ACID contains an initiator element but lacks binding sites for transcription factors known to respond to E1A. In line with these results, we speculate that CtBP not only has the capability to repress transcription induced by CR1 but also to repress transcription in general, when recruited to the promoter. Furthermore, as a first attempt to address the mechanism by which CtBP represses transcription, we investigated whether CtBP could interact with a histone deacetylase (HDAC), since deacetylation of histones correlates with transcriptional repression. CtBP was indeed shown to interact with HDAC1 both in vitro and in vivo and the result suggested that CtBP could repress transcription by recruiting deacetylase activity to a promoter. From paper I, we conclude that CtBP is a repressor of transcription when recruited to the promoter and that targeting of CtBP by E1A de-represses transcription, possibly by preventing localization of CtBP to the promoter.

29 Furthermore, we conclude that E1A-mediated de-repression of CtBP repression requires the CID in the second exon and is independent on functions ascribed to the amino-terminal half of E1A. Finally, since E1Aexon2 could activate transcription through a core promoter sequence, this suggested that CtBP represses transcription via the basal transcription machinery.

Paper II: Functional knock-out of the co-repressor, CtBP, by the second exon of adenovirus E1A relieves repression of transcription In paper II, we started to address the specificity of CtBP-mediated repression. Recruitment of CtBP to a promoter, either through interaction with a Gal4 fusion protein expressing a 44 amino acid, CID-containing fragment of E1A (Gal4ctE1A), or by directly using a Gal4CtBP fusion protein resulted in a similar repression of transcription. As a control, a Gal4ctE1A mutant lacking the capacity to interact with CtBP (Gal4ctE1A∆CID) did not repress transcription. Together, these data strongly supported the conclusion from paper I, that CtBP functions as a repressor of transcription when targeted to the promoter. Co-expression of a CtBP-binding competitor (E1Aexon2) blocked transcriptional repression by CtBP recruited by Gal4ctE1A, but did not effect Gal4CtBP repression. Surprisingly, the E1Aexon2 competitor was able to de-repress basal transcription from the reporter construct driven by the tk promoter (G5tkLuc) but not from the construct driven by the SV40 enhancer (SV40G5Luc), also in absence of exogenous Gal4ctE1A or Gal4CtBP, implying that the tk promoter is negatively regulated by endogenous CtBP. To further identify the specificity of CtBP-mediated repression, the response of a panel of unrelated promoters to the E1Aexon2 were analysed. This included the major late- and E4-promoters from adenovirus, the human Hsp70- and PCNA-promoters and an artificial promoter containing five binding sites derived from a c-myc responsive element. Co-expression of E1Aexon2 gave a 4 to 11 fold de-repression of all tested promoters. No effect was observed upon co-expression of E1Aexon2∆CID. Furthermore, over-expression of CtBP gave a dose-dependent repression of the MLP-, E4- and the Hsp70-promoters, which was reverted by co-expression of E1Aexon2. In summary, these results suggested that CtBP functions as a general repressor of transcription. To further explore this possibility, we used inducible expression of E1Aexon2 as a 'molecular trap' for CtBP. Based on the Tet-On system, we constructed cell lines harbouring inducible genes for E1Aexon2 and E1Aexon2∆CID. The changes in endogenous gene expression in the cell lines were measured by using a macro-array technique. Following induction with doxycylin, E1Aexon2 specifically altered expression from approximately 7% of the genes. Induction of E1Aexon2∆CID had significantly less effect, giving an altered expression of only 1% of the arrayed genes. Although a wide variety of genes were found to be regulated, the most pronounced effect of CtBP sequestering was observed on genes involved in cell cycle regulation, apoptosis and intracellular communications. An analysis of these genes might help to explain the capacity of the second exon of E1A to restrict the level of tumorigenicity of E1A-transformed cells (Boyd et al., 1993; Linder et al., 1992), possibly by reducing cell growth and promoting apoptosis. The gene array also identified genes that were exclusively regulated by E1Aexon2∆CID, suggesting that other regions in the second exon of E1A might contribute to the observed E1A- hypertransformed phenotype. Importantly, expression of E1Aexon2∆CID specifically induced expression of the RhoGTPase Rho6 and the dissociation inhibitor, GDIγ. This is interesting, as these factors have been suggested to be involved in transformation (Aspenstrom, 1999) but also because studies of hyper-transforming E1A second exon

30 mutants have implicated members of the Rho family of GTPases as effectors during E1A+H-ras hyper-transformation (Fischer and Quinlan, 2000). In paper I, we showed that CtBP interacts with HDAC1 both in vitro and in vivo and we speculated that CtBP might repress transcription by recruiting deacetylase activity to the promoter. In paper II, we expanded the study and showed that a GSTCtBP fusion protein interacts with HDAC2 and 3 translated in vitro. Nevertheless, addition of the histone deacetylase inhibitor, TSA, did not relieve repression mediated by Gal4CtBP. In contrast, the TSA sensitivity measurements on the E1ACID sensitive PCNA-promoter in the E1A-inducible cell lines gave different results. Induction of E1Aexon2 or addition of TSA, increased the expression from the PCNA-reporter several fold when used independently, but simultaneous treatment with both doxycylin and TSA did not give a significant additional increase in expression from the reporter. A possible explanation for the different results might be that HDAC is required for formation of a native CtBP repression complex upon binding to DNA sequence-specific transcription factors on natural target promoters. On the other hand, when a Gal4CtBP fusion protein is artificially tethered to a promoter, the normal promoter recruitment function of CtBP might become redundant.

Paper III: Molecular characterisation of the transcriptional co-repressor, CtBP In this paper, the initial mechanistic characterisation of CtBP as a co-repressor of transcription is presented. In agreement with previously published results (Criqui- Filipe et al., 1999; Koipally and Georgopoulos, 2000; Meloni et al., 1999; Phippen et al., 2000) and paper II, a Gal4CtBP fusion protein was shown to repress transcription from two promoters, where basal transcription was ensured by relatively strong, but unrelated promoter elements. Importantly, Gal4CtBP was also shown to repress basal transcription driven from either the minimal adenovirus E1B TATA-box or from the initiation sequence (-46 to +62) from the human PCNA promoter. This suggested that CtBP could repress transcription through the basal transcription machinery. The possibility that Gal4CtBP was able to repress transcription by targeting the basal transcription machinery raised the question whether repression required a promoter proximal location of Gal4CtBP. Our data showed that the capacity of Gal4CtBP to repress transcription significantly decreased when the Gal4 binding sites were moved away from the promoter. This finding was in agreement with previous results demonstrating that dCtBP belongs to the group of 'short range' repressors of transcription (Nibu et al., 1998b). A reporter lacking Gal4 binding sites was found to be relatively resistant to repression by Gal4CtBP. However, when very high amount of transfected Gal4CtBP were used, a weak inhibition was observed, suggesting that Gal4CtBP could be recruited to the core promoter independently of DNA sequence-specific transcription factors binding upstream. The mechanism for this repression remains unclear, but might relate to the ability of CtBP to repress basal transcription from a minimal promoter. Together with the results from the gene array analysis in paper II, indicating that CtBP may function as a general transcription repressor, we should like to hypothesise that CtBP can repress transcription by targeting a general component of the basal transcription machinery. Using mutational analysis of the Gal4CtBP fusion protein, the repressor activity was mapped to the amino-terminal 173 amino acids. A central domain of CtBP, spanning amino acids 284 to 374, demonstrated a significant capacity to activate transcription. However, in the full-length Gal4CtBP fusion protein the

31 activation function was masked. The question, however, remains: does CtBP contain a 'real' activation domain? An alternative explanation might be that amino acids 284 to 374 of CtBP can interact with components of the basal transcription machinery. When expressed as a Gal4 fusion protein, this region would theoretically be able to recruit or stabilise the PIC. In summary, we conclude that when the activity is analysed by a Gal4 fusion protein assay, at least two separate domains with distinct protein interaction interfaces are found within CtBP. CtBP has been shown to interact in vivo with over-expressed Class I (Koipally and Georgopoulos, 2000), paper I and II HDACs (Zhang et al., 2001). In paper III we showed that endogenous CtBP can interact with endogenous HDAC1 and that this correlates with the ability of CtBP to recruit TSA-sensitive deacetylase activity. Nevertheless, addition of TSA is unable to relieve the repression activity mediated by Gal4CtBP. These results show that even if endogenous CtBP has the capacity to recruit deacetylase activity this activity is not needed for repression when CtBP is artificially targeted to the promoter.

Conclusions and perspectives The work presented in this thesis shows that: 4) CtBP is a repressor protein capable of repressing transcription from a broad range of endogenous genes and that Gal4CtBP represses both basal- and activated transcription, possibly by interfering with the basal transcription machinery. 5) Repression mediated by Gal4CtBP requires proximal promoter localisation. 6) E1A can relieve CtBP-mediated repression, possibly by preventing promoter recruitment of CtBP. 7) CtBP has the capacity to interact with HDAC1, 2 and 3 and to recruit deacetylase activity. 8) CtBP represses transcription independently of histone deacetylase activity when artificially recruited to a promoter through the heterologous Gal4 DNA binding domain. Although not experimentally proven, the results presented in papers I, II and III support the idea that CtBP has the capacity to repress basal transcription. Firstly, co- expression of E1Aexon2 de-repressed transcription from a promoter driven by only the initiator sequence from PCNA promoter (paper I). Secondly, Gal4CtBP was shown to efficiently repress transcription from a reporter driven by a minimal promoter, paper III. Thirdly, the results from the gene array analysis in paper II showed that a relatively high number of genes were regulated by induction of E1Aexon2, suggesting that CtBP could act as a wide-range co-repressor of transcription or possibly take part in a global repressor complex. Together, these findings indicate that CtBP can repress transcription driven by a wide repertoire of unrelated transcription factors or that CtBP represses transcription by interfering with a more general component of the basal transcription machinery. In the latter scenario, we speculate that CtBP represses transcription by preventing efficient assembly of the PIC at the core promoter. This type of repression has been suggested for NC2, which competes with TFIIA for binding to the TBP/TATA-complex and thereby prevents efficient formation of the PIC (Meisterernst et al., 1991). Alternatively, CtBP might repress transcription by inhibiting promoter clearance or/and elongation. It has recently been shown that c-myc expression, induced by the FUSE binding protein (FBP), was down-regulated by the FBP Interacting Repressor (FIR). FIR represses transcription by interfering with the ATP- dependent helicase activity of TFIIH after initiation of transcription and thereby inhibits promoter escape by RNAP II (Liu et al.,

32 2001). To better understand the mechanism by which CtBP represses transcription it will be important to determine if CtBP can directly interact with components of the basal transcription machinery. Co-expression of the CtBP binding region of E1A-243R de-represses transcription from the PCNA promoter independently of CR1. However, the mechanism by which E1Aexon2 activates transcription is unclear. When recruited to the promoter through Gal4ctE1A or directly tethered to a promoter using Gal4CtBP (papers II and III), CtBP clearly represses expression from several different promoters. However, co-expression of a CtBP binding competitor (E1Aexon2) only relieves repression mediated by Gal4ctE1A and not by Gal4CtBP (paper II). This suggests that E1Aexon2 de-represses transcription by preventing promoter localisation of CtBP and not by blocking a repressor domain of CtBP. Alternatively, it is possible that E1A can only interfere with the function of wild type CtBP, by e.g. preventing homodimerasation, but that Gal4CtBP is insensitive to the regulatory function of E1A. CtBP interacts with HDACs (Koipally and Georgopoulos, 2000; Zhang et al., 2001) and papers I, II and III. Nevertheless, Gal4CtBP shows different dependency on deacetylase activity for efficient repression of transcription (Criqui-Filipe et al., 1999; Izutsu et al., 2001; Koipally and Georgopoulos, 2000) and papers II and III. This might be due to the existence of different cellular- or promoter specific-repressor complexes containing CtBP. Similarly, it is possible that HDACs fulfil a structural rather than enzymatic activity in CtBP mediated repression. According to this model, CtBP's interaction with HDAC offers a pathway by which CtBP can be recruited to the promoter. This would allow a more general ability for CtBP to repress transcription. In line with this, it was recently shown that CtBP could be recruited to the MEF2 transcription factor through interaction with the Class II HDACs (HDAC4, 5 and 7) or by interaction with MITR (a transcriptional repressor with homology to class II HDACs) (Zhang et al., 2001). Nevertheless, the results from paper II, suggesting that sequestering of endogenous CtBP diminished the effect of TSA on CtBP-repressible promoters, support the idea of a requirement for functional HDACs in CtBP-mediated repression. It is interesting to speculate that CtBP might be involved in E1A-mediated repression of p300/CBP-induced transcription. E1A represses transcription induced by p300/CBP by different mechanisms, all of which require CR1 and the amino- terminus of E1A (see "Repression of transcription by E1A"). However, it is possible that recruitment of E1A/CtBP to specific promoters, via an interaction with p300/CBP, represents a novel mechanism by which E1A might repress p300/CBP- induced transcription. It might, for example, be possible that CtBP recruits histone deacetylase to these promoters to counteract the HAT-activity associated with p300/CBP. In this respect, it is worth noting that E1A mutants lacking the second exon of E1A repress p300/CBP induced transcription less efficiently than wild type E1A (C. Svensson personal communication) although the direct involvement of CtBP has not been investigated. The involvement of CtBP in repression of p300/CBP might offer an extra dimension to E1A's capacity to induce G1-S-phase transition, although this activity may not necessarily be needed for completion of a successful virus infection in rapidly growing target cells. Support for its relevance stems from the result that the effect of the second exon on E1A transformation differs depending on co-operating oncogenes and the origin of the primary cells (Fischer and Quinlan, 2000).

33 Future studies of CtBP should, in my opinion, include three major studies. First, it would be interesting to find out if CtBP physically interacts with components of the basal transcription machinery and if so to possible target(s) should be identified. This would be the first step in an investigation trying to describe the exact molecular mechanism by which CtBP repress transcription. Secondly, although endogenous CtBP recruits histone deacetylase activity, Gal4CtBP mediated repression is insensitive to HDAC inhibitors, suggesting that the enzymatic activity per se is not required for repression. Thus, it would be interesting to study if CtBP mediated recruitment of histone deacetylase activity affect the activity, protein-stability or binding affinity of any other proteins interacting with CtBP. This would be in agreement with Rb mediated recruitment of HDAC resulting in deacetylation of E2F- 1 and decreased activity, protein-stability and binding affinity of E2F-1 (Martinez- Balbas et al., 2000). Finally, as it is known that the phosphorylation status of CtBP changes during the cell cycle (Boyd et al., 1993), it would be exciting to map the phosphorylation site in CtBP and investigate whether the activity or stability of CtBP in itself is regulated by phosphorylation. DNA tumour viruses that infect quiescent cells have to force the host cell to enter the S-phase of the cell cycle in order to induce DNA-synthesis. This is achieved by modulation of the function of cellular cell cycle regulatory proteins including Rb and p53. In agreement with this, Rb has been shown to be targeted by, for example, adenovirus E1A, simian virus 40 (SV40) large T antigen and human papilloma virus (HPV) E7, and p53 by adenovirus E1B, SV40 large T antigen and HPV E6. These findings demonstrate that by targeting key-player of regulatory pathways early regulatory viral proteins have the capacity to modulate cell cycle progression in favour of the virus. Due to its interaction with E1A (see "CtBP interacting proteins") it is possible that CtBP should be added to the list of important cellular targets. Although, the define function of CtBP remains unclear, accumulating results suggest CtBP to be involved in regulation of cell growth and development (see "The biological role of CtBP"). The data available today define CtBP as a co-repressor of transcription. Co-repressors are supposed to be recruited to the promoter by repressors and to contribute in different degree to repression. This means that inactivation of CtBP results only in secondary effects. Nevertheless, a functional inactivation of CtBP, by over-expression of E1Aexon2, altered expression from approximately 7% of the tested genes and the most pronounced effect was observed on genes involved in cell cycle regulation, apoptosis and intracellular communications (see "Present investigation"). Taken together, these results, even if they are indirect, might suggest that CtBP is important and commonly used as a co-repressor of transcription by repressors regulating cell-growth and development. In line with this idea, we might further speculate that E1A can modulate the effect of these repressors fast and simply by targeting the common co-repressor CtBP instead of each individual repressor. The relevance of this kind of inactivation of CtBP by E1A for a successful adenovirus infection is not known and the importance might be difficult to investigate. Since CtBP is a co-factor inactivation of CtBP might not results in drastic effects. Moreover, inactivation of CtBP in cell lines where principle pathways regulating cell cycle progression are already disturbed might be small and difficult to assess. However, inactivation of CtBP in primary cells, i.e. during infection in organisms, might be critical for a productive adenovirus infection. Possibly it might turn out that the importance of inactivation of CtBP depends on the type or proliferation status of the host cell. Taken together this suggests that future investigation concerning CtBP should focus on cell cycle regulatory events and not on transcription per se.

34 ACKNOWLEDGMENTS

This study was carried out at the Department of Medical Biochemistry and Microbiology, Uppsala University. The projects were supported by grants from the Swedish Cancer Society, The Swedish Medical Research Council and The Swedish Society of Medical Research. A number of people have entertained and supported me during my years as a PhD-student. Now, I would like to express my sincere gratitude to you: First of all, I would like to thank all my friends who, due to limited space, are not in the list below. Thankyou for your friendship and happy memories - you are not forgotten!

My love Mette, for being my best team-mate and supporter.

Catharina Svensson, my supervisor, for recruiting me to your group and the field of transcription, for endless patience, for letting me mature both as an individual and as a "researcher" at my own pace. Thank you for encouraging me and for allowing me to do my "own experiments", to stand by me whenever I have needed support, for careful reading and valuable comments on my thesis and finally, for providing me with a second home for many years.

Göran Akusjärvi for showing us, occasionally in a stuffy seminar room, the spirit and the beauty of real science and for trying to inspire us to do "interesting science". Vita for friendship and for entertainment during pipetting, Tanel for always having an idea, Camilla and Christina for all the years together in the lab, Cecilia for always bringing me down to earth again, Peter for all the true stories, Erika for helping me to conquer the enormous amount of bureaucracy, Josef and Tony for nice lunches outside the lab and everyone else making the Adeno-lab a nice place to be at: Martin, Anette, Magnus, Lisa, Saiedeh, Regine, Bin Hongxing and Raj. Thankyou also to Göran Magnusson and Stefan Schwarz and the people working in their research groups.

"Uppsala’s Transcription Group" for good seminars and discussions. Special thanks to Johan Ericsson, Eva Grönroos, Gunnar Westin and Lars-Gunnar Larsson for the kind gifts of research material.

My parents (the best in the world), Maud and Bo. I will always be enormously grateful to you for your love, support and for having confidence in me; you are my best friends. The same for the best brothers in the world; Magnus, Björn and Erik. Also, lots of love to Anne-Catherine, Clara and Ingrid - take care of my brothers! I am also grateful to my relatives in Enköping, Eva, Sven, Alexander and Uncle Sven.

Everyone I have met at Göteborgs Nation and for everything I have experienced thanks to you. Skål för glädjen på Göteborgs Nation!

My friends in Enköping for all the times in different racing yachts, kayaking, skating, skiing and all the other good times together. To the shipowner-families: Axelsson and Dyrevik for the years onboard GG 158 Sunnanland as a "Fångstman i Silljagarflottan". To Peter and Patrik onboard S/Y Pink Gin for approximately 8500 n.m. on the Atlantic Ocean. To Captain Martinez and the rest of the crew onboard M/V KOHO for bringing me home…

Alan McWhirter for excellent linguistic revision.

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