UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New series No 849 ISSN 0346-6612 ISBN 91-7305-500-X

From the Department of Medical Biochemistry and Biophysics,

Umeå University, SE 901 87, Umeå, Sweden

Purification of General RNA Polymerase II Transcription

Factors from Mouse for Studies of Proliferation-Specific

Transcription

Irina Kotova

AKADEMISK AVHANDLING som med vederbörligt tillstånd av rektorsämbetet vid Umeå Universitet för avläggande av doktorsexamen i medicinsk vetenskap kommer att offentligen försvaras i hörsal KB3A9, KBC-huset, Umeå Universitet fredagen den 12:e september, kl. 13.00. Fakultetsopponent: Professor Lars-Gunnar Larsson, Institutionen för växtbiologi och skogsgenetik, SLU, Uppsala, Sweden.

UMEÅ 2003

Purification of General RNA Polymerase II Transcription Factors from Mouse for Studies of Proliferation-Specific Transcription

Irina Kotova, Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87, Sweden

Accurate initiation of transcription by RNA polymerase II depends on general transcription factors (GTFs), which include the TATA-binding protein (TBP) and the transcription factors (TF) IIB, IIF, IIE and IIH. In order to reconstitute mouse transcription in vitro, we cloned the genes encoding mouse TFIIB, and both subunits of TFIIE and TFIIF from a mouse cDNA library. TBP and TFIIB were expressed in E.coli, while both subunits of TFIIE and the two subunits of TFIIF were expressed in a baculovirus system. All these factors were purified to > 90% homogeneity. The more complex transcription factors, TFIIH and RNA polymerase II, were purified more than 1000-fold and to near homogeneity, respectively, from tissue cultured mouse ascites cells. We have shown that the purified mouse transcription factors are active in a reconstituted RNA polymerase II in vitro transcription assay. The transcription reaction was inhibited by α-amanitine, and dependent on the addition of all the GTFs. Ribonucleotide reductase is a key enzyme in deoxyribonucleotide synthesis. It consists of two subunits, R1 and R2, which are both required for the enzyme activity. Transcription of the R1 and R2 genes is restiricted to the S-phase of the cell cycle, but the mechanisms that control this coordinated expression remain to be identified. We have studied initiation of transcription from the mouse R2 gene using a combination of in vivo reporter gene assays and in vitro transcription assays with crude nuclear extracts or with purified transcription factors. This promoter has an atypical TATA-box and a CCAAT-box that binds the transcription factor NF-Y. We found that a mutation in the R2 CCAAT-box had no effect on the transcription level in in vitro transcription assays reconstituted with pure transcription factors. However, it significantly decreased the level of transcription in similar experiments using crude nuclear extract. We also found that the sequence downstream from the R2 transcription start site (5´-UTR) (from +1 to +17 base pairs relative to transcription start site) is essential for initiation of transcription from this promoter. The presence of the wild type 5´-UTR made the R2 TATA-box redundant. On the other hand, the R2 5´-UTR had a repressing effect on transcription from the mouse R2 promoter. This region contains a palindrome sequence that covers 10 base pairs, and it is partially conserved in the human R2 promoter. Gel shift assays and in vitro transcription experiments using antibodies against mouse TAF4 (=TAF135) demonstrate that TAF4 is a component of the protein complex that interacts with this palindrome region, and suggest involvement of this component of the TFIID complex in negative regulation of the R2 promoter. The Adenovirus Major Late (AdML) promoter is commonly used as a model for studies of transcription initiation and regulation. It is a TATA-box dependent promoter, which also contains an initiator (Inr) element, a CCAAT-box interacting with transcription factor NF-Y, and an E-box binding the upstream stimulatory factor (USF). Using gel shift assays with recombinant NF-Y, USF, and immunopurified human TFIID, we show that binding of USF1 and NF-Y to DNA is not cooperative and that both factors independently facilitate binding of TFIID to the core promoter. The activation domains of NF-Y are expendable for this effect. Negative cofactor (NC2) comprises two subunits, which have a histone-fold structure similar to NF-Y, and represses transcription through formation of an inhibitory complex with TBP. Using an in vitro transcription system based on crude nuclear extracts, we show that NC2 has a negative effect on transcription in the presence of NF-Y or USF1, indicating that the two activators do not act as antirepressors. In vitro transcription using highly purified transcription factors efficiently reproduces repression of transcription by NC2. However, USF1 was inactive and NF-Y had a repressing effect in this system, which suggests that the activator functions of USF and NF-Y depend on cofactors.

1

Table of contents

Abstract ..................................................................................................3

Abbreviations ........................................................................................4

List of papers .........................................................................................5

Part I. Introduction ...............................................................................6

Eukaryotic RNA polymerases ...........................................................6

RNA polymerase II ..........................................................................6

The RNA polymerase II general transcription factors .........................7

Two models of preinitiation complex formation .................................8

General transcription factors and their functions during transcription

initiation..........................................................................................9

Topology of the preinitiation complex ...............................................12

Structure of Pol II promoters.............................................................13

Core promoter elements....................................................................13

Transcriptional coregulators .............................................................15

Chromatin-mediated repression of transcription .................................15

Acetylation of histones .....................................................................16

ATP-dependent chromatin remodelling .............................................17

Mediator..........................................................................................17

TFIID ..............................................................................................19

Negative cofactor 2.................................................................................21

The eukaryotic cell cycle ..................................................................22

Cyclins and cyclin-dependent kinases in cell cycle regulation .............23

Regulation of transcription during cell cycle progression....................24

Ribonucleotide reductase..................................................................25

Transcription factor NF-Y .............................................................28

Upstream stimulatory factors.........................................................30

2

Part II. Results.......................................................................................32 Reconstitution of a mouse RNA polymerase II transcription system

from purified transcription factors (Paper I) .......................................32 Sequences downstream of the transcription initiation site are important

for proper initiation and regulation of mouse ribonucleotide reductase

R2 gene transcription (Paper II) ........................................................34

Mechanisms of transcriptional activation of the AdML promoter by

NF-Y (Paper III)...............................................................................37

Acknowledgements................................................................................40

References ..............................................................................................41

Papers I-III ............................................................................................60

3

Abstract Purification of General RNA Polymerase II Transcription Factors from Mouse for Studies of Proliferation-Specific Transcription. Accurate initiation of transcription by RNA polymerase II depends on general transcription factors (GTFs), which include the TATA-binding protein (TBP) and the transcription factors (TF) IIB, IIF, IIE and IIH. In order to reconstitute mouse transcription in vitro, we cloned the genes encoding mouse TFIIB, and both subunits of TFIIE and TFIIF from a mouse cDNA library. TBP and TFIIB were expressed in E.coli, while both subunits of TFIIE and the two subunits of TFIIF were expressed in a baculovirus system. All these factors were purified to > 90% homogeneity. The more complex transcription factors, TFIIH and RNA polymerase II, were purified more than 1000-fold and to near homogeneity, respectively, from tissue cultured mouse ascites cells. We have shown that the purified mouse transcription factors are active in a reconstituted RNA polymerase II in vitro transcription assay. The transcription reaction was inhibited by α-amanitine, and dependent on the addition of all the GTFs. Ribonucleotide reductase is a key enzyme in deoxyribonucleotide synthesis. It consists of two subunits, R1 and R2, which are both required for the enzyme activity. Transcription of the R1 and R2 genes is restiricted to the S-phase of the cell cycle, but the mechanisms that control this coordinated expression remain to be identified. We have studied initiation of transcription from the mouse R2 gene using a combination of in vivo reporter gene assays and in vitro transcription assays with crude nuclear extracts or with purified transcription factors. This promoter has an atypical TATA-box and a CCAAT-box that binds the transcription factor NF-Y. We found that a mutation in the R2 CCAAT-box had no effect on the transcription level in in vitro transcription assays reconstituted with pure transcription factors. However, it significantly decreased the level of transcription in similar experiments using crude nuclear extract. We also found that the sequence downstream from the R2 transcription start site (5´-UTR) (from +1 to +17 base pairs relative to transcription start site) is essential for initiation of transcription from this promoter. The presence of the wild type 5´-UTR made the R2 TATA-box redundant. On the other hand, the R2 5´-UTR had a repressing effect on transcription from the mouse R2 promoter. This region contains a palindrome sequence that covers 10 base pairs, and it is partially conserved in the human R2 promoter. Gel shift assays and in vitro transcription experiments using antibodies against mouse TAF4 (=TAF135) demonstrate that TAF4 is a component of the protein complex that interacts with this palindrome region, and suggest involvement of this component of the TFIID complex in negative regulation of the R2 promoter. The Adenovirus Major Late (AdML) promoter is commonly used as a model for studies of transcription initiation and regulation. It is a TATA-box dependent promoter, which also contains an initiator (Inr) element, a CCAAT-box interacting with transcription factor NF-Y, and an E-box binding the upstream stimulatory factor (USF). Using gel shift assays with recombinant NF-Y, USF, and immunopurified human TFIID, we show that binding of USF1 and NF-Y to DNA is not cooperative and that both factors independently facilitate binding of TFIID to the core promoter. The activation domains of NF-Y are expendable for this effect. Negative cofactor (NC2) comprises two subunits, which have a histone-fold structure similar to NF-Y, and represses transcription through formation of an inhibitory complex with TBP. Using an in vitro transcription system based on crude nuclear extracts, we show that NC2 has a negative effect on transcription in the presence of NF-Y or USF1, indicating that the two activators do not act as antirepressors. In vitro transcription using highly purified transcription factors efficiently reproduces repression of transcription by NC2. However, USF1 was inactive and NF-Y had a repressing effect in this system, which suggests that the activator functions of USF and NF-Y depend on cofactors.

4

Abbreviations

The abbreviations used are: snRNA, small nuclear RNA; Pol II, RNA polymerase

II; CTD, C-terminal domain of the largest RNA polymerase II subunit; GTF,

general transcription factor; TBP, TATA-binding protein; TAF, TBP associated

factor; bp, base pair; CDK, cyclin dependent kinase; CAK, CDK activating

complex; BRE, TFIIB recognition element; Inr, initiator element; DPE,

downstream promoter element; NC2 negative cofactor 2; HAT, histone

acetyltransferase; HDAC, histone deacetylase; GNAT, Gcn-5 related N-

acetyltransferase; SAGA, SPT-ADA-GCN5-acetyltransferase complex; ADA,

HAT complex containing the ADA (adapter) proteins; PCAF, P300/CBP

associated factor; CBP, CREB (cyclic AMP response element binding protein)

binding protein; EKLF, erythroid Krüppel-like factor; SWI/SNF, nucleosome

remodeling complex containing the products of genes involved in mating type

switching (SWI) and/or sucrose fermentation (SNF, sucrose nonfermenting); RSC,

complex remodeling the structure of chromatin; NURD, nucleosome remodeling

and histone deacetylase complex; NURF, nucleosome remodeling factor; ACF,

ATP-utilizing chromatin assembly and remodeling factor; CHRAC,

chromatin accessibility complex; ts, temperature sensitive; TFTC, TBP-free TAFII

complex; DHFR, dihydrofolate reductase; TK, thymidine kinase; Rb,

retinoblastoma; RNR, ribonucleotide reductase; dNDP, deoxyribonucleotide

diphosphate; rNDP, ribonucleotide diphosphate; NF-Y, nuclear factor Y; USF,

upstream stimulatory factor; bHLH-zip, basic-helix-loop-helix-leucine zipper;

USR, USF-specific region; 5´-UTR, 5´-untranslated region.

In this thesis, TAFs are named according to the new unified nomenclature (1). The

former names are indicated in parenthesis.

5

List of papers This thesis is based on the following publications, which will be referred to by their

roman numbers.

I. Kotova, I., Holfslagare, A. L., Segerman, B., Flodell, S., Thelander, L., and

Björklund, S. (2001) A mouse in vitro transcription system reconstituted

from highly purified RNA polymerase II, TFIIH, and recombinant TBP,

TFIIB, TFIIE and TFIIF. Eur. J. Biochem. 268, 4527-4536.

II. Kotova, I., Holfslagare, A. L., Lobov, S., Thelander, L., and Björklund, S.

(2003) Sequences downstream of the transcription initiation site are

important for proper initiation and regulation of the mouse ribonucleotide

reductase R2 gene transcription. Eur. J. Biochem. 270, 1791-1801.

III. Frontini, M., Kotova, I., Björklund, S., and Mantovani, R. Mechanisms of

transcriptional activation of the AdML promoter by NF-Y. Manuscript.

6

Part I. Introduction

Eukaryotic RNA polymerases

Transcription in eukaryotic cells is carried out by three classes of RNA

polymerases, which are responsible for transcription of different sets of genes.

RNA polymerase I transcribes ribosomal genes encoding large pre-rRNA

transcripts that are later processed into mature 5.8S, 18S, 28S ribosomal RNAs.

RNA polymerase II is responsible for transcription of all protein encoding genes

and some small nuclear (sn)RNAs. RNA polymerase III transcribes 5S ribosomal

RNA, snRNAs, and all tRNAs. Although RNA polymerase I and RNA polymerase

III are involved in transcription of a very limited set of genes, their activities

dominate cellular transcription and catalyse more than 80% of the total RNA

synthesis in growing cells. All three RNA polymerases have a complex

multisubunit structure and consist of 12-14 subunits. They share five common

subunits, have several homologous and a few unique subunits (reviewed in 2).

RNA polymerase II

In eukaryotic cells, transcription of all protein encoding genes is catalysed by RNA

polymerase II (Pol II), a multisubunit enzyme with a total mass of > 0.5 MDa. It

consists of 12 subunits which are highly conserved among phylogenetically diverse

organisms, and at least 10 of the yeast Pol II genes can be substituted by their

human counterparts (reviewed in 3). RNA polymerase II has been isolated in two

forms, as a 12-subunit “complete” enzyme and a 10-subunit enzyme. The 10-

subunit form of Pol II is sufficient for elongation, but initiation of transcription

requires the presence of the two additional RNA polymerase II subunits, Rpb4 and

Rpb7. These two subunits form a heterodimer that associates reversibly with the

10-subunit form of the enzyme (4). The X-ray structures of the yeast 10-subunit

and the 12-subunit forms of RNA polymerase II have been resolved (5, 6, 7).

7

The largest subunit of RNA polymerase II (Rpb1) has a unique carboxy-terminal

domain (CTD) consisting of a heptapeptide repeat with the consensus sequence

YSPTSPS (8). This sequence is conserved among eukaryotes, but the number of

repeats varies between species. For example, the CTD of yeast Pol II comprises 26

heptapeptide repeats while the human Pol II has a longer CTD of 52 repeats. The

CTD can be phosphorylated at serine residues, and the phosphorylation plays an

important regulatory role in transcription (9, 10). It was shown that Pol II enters the

preinitiation complex in the nonphosphorylated form, and that the CTD then

becomes extensively phosphorylated during initiation of transcription. The CTD

phosphorylation is required for transition from initiation to elongation and for

recruitment of capping enzymes (11). In addition, later stages of mRNA processing

including splicing and polyadenylation are also affected by CTD-phosphorylation

(reviewed in 12). After termination of transcription, a CTD phosphatase recycles

Pol II by reversing the CTD phosphorylation.

The RNA polymerase II general transcription factors

Although Pol II has a very complex multisubunit structure, it requires several

accessory proteins termed the general transcription factors (GTFs) for accurate

initiation of transcription. They include transcription factors (TF) IIA, IID, IIB, IIF,

IIE and IIH, which are conserved between eukaryotes (Reviewed in 13, 14).

Figure 1. Schematic representation of the Pol II preinitiation complex.

8

Efficient initiation of transcription requires assembly of the GTFs together with Pol

II at the core promoter in a certain order to form a preinitiation complex (Fig. 1).

Preinitiation complex formation is the first step in transcription initiation, which is

then followed by promoter opening, synthesis of the first phosphodiester bond and

promoter clearance (14).

Two models of preinitiation complex formation

In vitro studies demonstrated that transcription initiation complexes could be

assembled at promoters by a stepwise recruitment of GTFs and Pol II. In the first

step, TFIID binds sequence specifically to the promoter. In the second step, TFIIB

recognizes and binds the TFIID/DNA complex. In the third step, Pol II is recruited

to this complex by TFIIF. Finally, TFIIE enters the complex and recruits TFIIH,

which completes the formation of preinitiation complex (Reviewed in 13, 14).

However, the stepwise recruitment model has been challenged by the finding that

Pol II can exist as a preformed holoenzyme complex also in the absence of

promoter DNA. The composition of this megadalton complex varies depending on

the isolation conditions (15-17). It can include TFIIF, TFIIE and TFIIH, as well as

cofactor complexes (Mediator, SWI/SNF components) (see below). The

holoenzyme form of Pol II responds to transcriptional activators, and therefore it

has been proposed to represent the form of polymerase that initiates transcription in

vivo (15, 18). However, quantification of Pol II, GTFs and Mediator subunits in

yeast cells does not support the idea of holoenzyme recruitment. The measurements

showed comparable amounts of RNA polymerase II, TFIIE, and TFIIF, lower

levels of TBP and TFIIB, and much lower levels of Mediator and TFIIH, consistent

with their involvement only in transient interactions with Pol II at the promoters

during initiation of transcription (19).

9

General transcription factors and their functions during transcription

initiation.

As mentioned before, binding of TFIID to the promoter initiates the formation of a

transcription preinitiation complex. TFIID is a multisubunit complex composed of

the TATA binding protein (TBP), and more than ten additional proteins called the

TBP associated factors (TAFs) (20, reviewed in 21). TBP is responsible for the

TATA-box recognition and is highly conserved between eukaryotes. The human

TBP has a molecular weight of 38 kDa and is by itself sufficient to initiate

formation of the preinitiation complex at promoters containing a TATA-box,

whereas recognition of other core promoter sequences requires TAFs. Like TBP,

TAFs are also conserved between eukaryotes and most of the yeast TAFs have

mammalian homologues. Almost all of the yeast TAFs are essential for

viability (1, 22, 23). TBP is also essential for transcription by RNA polymerases I

and III, and can therefore be considered as a universal eukaryotic transcription

factor (24, 25). The crystal structure shows that TBP is a saddle-shaped molecule.

It binds sequence specifically to the TATA-box through contacts between its

concave surface and the minor groove of DNA thus inducing a 90 degree bending

of the DNA (26, 27).

The TBP-TATA-box complex is stabilized by TFIIA, which directly binds TBP

and DNA upstream of the TATA-box through non-specific contacts with the

phosphate backbone of DNA (28). The α and β subunits derive from a single

polypeptide as a result of proteolytic cleavage (29, 30). The role of TFIIA in

transcription is controversial. The binding of TFIIA significantly stabilizes the

TFIID- and TBP-promoter complexes, although it is not absolutely essential for

initiation of transcription (31, 32). The requirement of TFIIA for transcription

depends on both the core promoter and the regulatory regions (33). In addition, it is

believed to act as an antirepressor, preventing NC2-TBP interactions (reviewed in

34).

10

Similarly to TFIIA, TFIIB recognizes and stabilizes the TBP-DNA complex

through direct binding to TBP and to DNA, but unlike TFIIA, it is absolutely

required for formation of preinitiation complex (35). Human TFIIB is composed of

a single subunit with a molecular weight of 35 kDa (35). The N-terminal region of

TFIIB has a zinc ribbon domain, and the C-terminal domain (TFIIBc) includes

cyclin A-like repeats. In addition, TFIIB has a conserved sequence that links the N-

terminal and the C-terminal parts (36-39). The zinc ribbon interacts with Pol II,

while the C-terminal domain interacts with the TBP-DNA complex. A co-crystal

structure of TBP, TFIIB, and DNA shows that human TFIIBc binds to the promoter

asymmetrically through base-specific contacts in the major groove upstream, and in

the minor groove downstream of the TATA-box. This asymmetric binding by

TFIIB results in a unidirectional assembly of the preinitiation complex, which is a

prerequisite for directional transcription (40). The TBP-induced bending of the

DNA allows TFIIB to interact with DNA both upstream and downstream of the

TATA-box by binding underneath of the TBP-DNA complex (41). The structural

studies of TFIIB and Pol II demonstrated that TFIIB functions as a bridge between

TFIID at the promoter and the Pol II/TFIIF complex, thus directing the

transcription start site to a position 30 bp downstream from the TATA-box (see

below) (42).

Mammalian TFIIF is an α2β2 heterotetramer. The small subunit (TFIIFβ/RAP30)

has two regions of sequence homology to bacterial σ-factors indicating that these

transcription factors are functionally related (43). Interestingly, RAP30 alone is by

itself able to recruit Pol II to the promoter (44). The large subunit of TFIIF

(TFIIFα/RAP74) is phosphorylated at several sites, and phosphorylation is

important both for the initiation and elongation activities of TFIIF (45). The fact

that both subunits of TFIIF contain cryptic DNA-binding domains, which become

activated during interaction with Pol II, suggests that TFIIF undergoes dramatic

conformational changes upon binding to Pol II (46).

11

TFIIF has several functions important for specific transcription initiation. First, it

prevents non-specific binding of Pol II to DNA. Second, it is able to remove Pol II

from non-specific DNA sites, thereby significantly decreasing non-specific

transcription. TFIIF also delivers Pol II to the promoter and stabilizes the

preinitiation complex through interactions with TFIIB and DNA (47, 48). In

addition, TFIIF is required for the entry of TFIIE and TFIIH into the preinitiation

complex, for subsequent open complex formation catalysed by the TFIIH, for

synthesis of the first phosphodiester bond of nascent transcripts, and for the activity

of TFIIH during promoter escape (49). Finally, TFIIF is the only GTF that remains

bound to Pol II after promoter escape, and it is also required for transcriptional

elongation and for association with the CTD phosphatase (47, 50).

Like TFIIF, TFIIE is a heterotetramer containing two large (TFIIEα) and two small

(TFIIEβ) subunits (56 kDa and 34 kDa in human, respectively) (51). TFIIE is

important for recruitment of TFIIH to promoters and has a stimulatory effect on

both the ATPase and kinase activities of TFIIH, thus playing a role in transition

from initiation to elongation (52).

TFIIH is a complex factor composed of nine subunits. Unlike the other GTFs, it

has defined enzymatic activities. TFIIH can be resolved into two subcomplexes: a

core and a CDK-activating complex (CAK) (53, 54). The core TFIIH contains two

ATP-dependent helicases (ERCC2/XPD and ERCC3/XPB) of opposite polarity,

which play different roles during transcription initiation (55). The ERCC3/XPB

helicase activity is crucial for melting of a ∼10 bp region just upstream from the

transcription start site, for formation of the first phosphodiester bond and for

promoter escape, whereas ERCC2/XPD is important for promoter escape and plays

an important structural role in the association of core TFIIH with the CAK

complex (56). The CAK complex comprises three subunits: CDK7, cyclin H and

MAT1. This cyclin–kinase complex is involved in phosphorylation of the Pol II

CTD, of the general transcription factors TFIIFα and TFIIEα, and of some

transcriptional regulators such as nuclear receptors (52, 54, 57-59). Interestingly,

12

phosphorylation of CTD occurs in a TFIIE-dependent manner. In addition to its

role in Pol II transcription, TFIIH is also involved in ribosomal RNA transcription

and in nucleotide excision repair (60, 61).

Topology of the Pol II preinitiation complex

Photocrosslinking experiments demonstrated that Pol II interacts with promoter

DNA within a region extending from –40 to +13 bp relative to the transctiption

start site. This 53 nucleotides long sequence represents 180 Å of B-form DNA,

which is longer then the diameter of Pol II in its largest dimension (140 Å). This

finding suggested that promoter DNA is wrapped around Pol II, thus allowing

promoter contacts both upstream of the TATA-box and downstream of the

transcription start site (Fig. 2) (62, 63). In addition, TFIIF and TFIIE were

crosslinked at several positions both upstream of the TATA-box and downstream

of the transcription start site, which can be possible only if DNA would be wrapped

around the Pol II initiation complex.

Figure 2. Proposed structure of the RNA polymerase II preinitiation complex

(adapted from Robert et al., 1998 (63)). DNA is wrapped around the (POL II)

preinitiation complex. The general transcription factors TBP, TFIIB (B), TFIIFα

(F74), TFIIFβ (F30) and TFIIEβ (E34) and their contacts with DNA are indicated

in the figure.

13

These extensive Pol II-DNA interactions were dependent on RAP74, which

suggests that TFIIF is important for inducing the conformational changes that lead

to the wrapping of promoter DNA around the preinitiation complex (62). These

findings can explain the broad range of functions that TFIIF has during initiation of

transcription. In agreement with photocrosslinking data, the X-ray structure

analysis and electron microscope images of initiation complex on promoter DNA

also suggest that promoter DNA is wrapped around Pol II (64).

Structure of Pol II promoters

RNA polymerase II promoters are composed of core and regulatory regions.

Regulatory regions are gene specific and highly variable. They can be located near

or at great distance downstream or upstream from the transcription start site. They

bind different regulatory proteins, which can have positive or negative effect on

transcription. In contrast, core promoter elements are located in a region of 40 bp

around the transcription start site and are recognized by components of the general

transcription machinery (Fig. 3).

Core promoter elements

There are several core promoter elements including the TATA-box, the TFIIB

recognition element (BRE), the Initiator (Inr), and the downstream promoter

element (DPE) (Fig. 3). The TATA-box has the consensus sequence

TATA(A/T)A(A/T) and is located 25-30 bp upstream of the transcription start site

(reviewed in 13, 65). The TATA-box is a key element of many Pol II promoters

and it is recognized by the TBP subunit of the TFIID complex. In some promoters,

the TATA-box is immediately preceded by a TFIIB recognition element with the

consensus sequence (G/C)(G/C)(G/A)CGCC. This sequence increases the affinity

of TFIIB to the promoter through direct interactions (66).

14

Figure 3. Schematic representation of TFIID binding to core promoter elements.

The initiator element (Inr) was originally identified as a sequence that is sufficient

to direct accurate initiation of transcription in the absence of a TATA-box.

However, Inr elements are present in both TATA containing and TATA-less

promoters. In mammalian promoters the Inr consensus sequence is Py-Py-A+1-N-

T/A-Py-Py (where A+1 is the transcription start site). The Inr element is recognized

by two components of TFIID, TAF1 (= TAF250) and TAF2 (=TAF150) (67, 68).

The downstream promoter element (DPE) is as common as the TATA-box and has

the conserved consensus sequence G-(A/T)-C-G. The DPE is usually located 30 bp

downstream from the transcription start site, and it has been found in a variety of

promoters from Drosophila to humans. The DPE functions cooperatively with the

Inr to bind to TFIID and to direct accurate initiation of transcription.

Photocrosslinking studies in Drosophila revealed that DPE interacts with TAF11

(=TAF40) and TAF6 (=TAF60) (69, 70). Interestingly, the TATA-box and the

DPE have similar functions in basal transcription and they are both recognized by

components of the TFIID complex. Promoters that contain either a TATA or a DPE

motif are typically completely dependent on these elements for transcriptional

activity and the addition of a consensus DPE sequence at its normal downstream

position to a TATA-box dependent core promoter can compensate for a mutation

of the TATA-box in basal transcription. However, it has been shown that TATA-

and DPE-containing promoters are regulated in different ways. For example,

negative cofactor 2 (NC2), which represses TATA-dependent transcription, has

been found to activate DPE-dependent promoters (71). In addition to this, it has

been found that some enhancers show specificity for TATA- or DPE-containing

15

promoters (72). These data suggest that core promoter sequences are important not

only for the initiation of transcription by the basal transcription machinery, but that

they also can influence the response to transcriptional regulators.

It is most likely that various combinations of core promoter elements, as well as

variations of the exact DNA sequences of these elements in natural promoters have

a significant influence on both the overall efficiency of gene transcription and on

the ability of individual genes to respond to transcriptional activators.

Transcriptional coregulators

As already mentioned, the minimal set of transcription factors required for

reconstitution of eukaryotic transcription in vitro includes Pol II, TBP, TFIIB, IIE,

IIF and IIH. Such in vitro transcription systems are able to recognize a TATA-box

containing promoter and to support correctly initiated transcription on naked DNA

templates (so-called basal transcription). However, these systems fail to respond to

regulatory proteins. Transmission of signals from sequence-specific promoter-

bound regulatory proteins to the general transcription machinery requires an

additional set of cofactors that do not bind DNA directly. The transcriptional

coregulators can be divided into two broad classes: those that function through

RNA polymerase II and/or associated proteins and those that affect the chromatin

structure. The transcriptional coregulators are usually recruited to promoters

through direct interactions with regulatory proteins and/or cofactor-cofactor

interactions (73).

Chromatin-mediated repression of transcription

DNA in the eukaryotic cell is packed into chromatin, which has a general

repressing effect on basal transcription (reviewed in 74, 75). The primary unit of

chromatin is the nucleosome that comprises a histone octamer that contains a

histone H3-H4 heterotetramer and two histone H2A-H2B dimers wrapped by 146

bp of DNA (76). The histone proteins contain folded globular core domains, which

are mainly responsible for structural organization, and externally located N-

16

terminal tails, that are subject to covalent posttranslational modifications, including

acetylation, methylation, phosphorylation and ADP-ribosylation (reviewed in 77,

78). The diversity and complexity of covalent histone modifications and the

functional specificity associated with distinct patterns of modifications has led to a

“histone code” hypothesis suggesting that these patterns are read by other proteins

or protein modules (79).

Packaging of DNA in nucleosomes represses transcription at several levels. First,

nucleosomes restrict access of DNA-binding proteins to DNA. Second, they

interact with additional chromosomal proteins, thus leading to repression of gene

expression of entire chromosomal domains (reviewed in 78, 80). To overcome this

repression and to make DNA available for regulatory proteins, the cell contains

numerous chromatin-modifying complexes that function by loosening and opening

up the chromatin structure. These complexes can be divided into two major groups:

those that alter chromatin structure through posttranslational modifications, and

those that alter the nucleosome structure or position in an ATP-dependent manner

(81, 82).

Acetylation of histones

Acetylation of lysine residues is one of the best studied histone modifications, and

has been shown to be positively linked to transcriptional activation. The acetylation

level is regulated by two opposing groups of cofactors; histone acetyltransferases

(HATs) and histone deacetylases (HDACs). To date, numerous HATs have been

described and classified into several families (reviewed in 83). The GNAT family

(for Gcn-5 related N-acetyltransferase) includes the well characterised Gcn5

containing complexes such as the yeast SAGA and ADA complexes and the highly

related human PCAF complex (reviewed in 83, 84). Another family comprises two

of the most widely studied HATs in transcriptional regulation: p300 and CBP.

These closely related proteins act as positive cofactors in transcription and are

recruited to particular promoters through interactions with DNA-bound

17

transcription factors such as Gcn4, c-Jun, c-Fos, E2F1, c-Myb, nuclear hormone

receptors and NF-Y (reviewed in 85). In addition to their HAT activity, p300 and

CBP have also been shown to acetylate transcription factors, such as p53, E2F and

EKLF (86-88). The largest subunit of TFIID, TAF250, and one of the subunits of

yeast mediator, Nut1, also has HAT activity (89).

It is interesting, that particular histones and particular sites within histones become

acetylated during specific cellular processes (90, 91). In addition, distinct HATs

show different substrate and acetylation site preferences (92-94). Taken together,

these findings suggest that different HAT complexes have distinct functions and

regulate specific subsets of genes.

ATP-dependent chromatin remodeling

The second group of chromatin remodeling complexes includes factors that use the

energy of ATP hydrolysis to affect chromatin in a more transient way, thus making

it more accessible to regulatory factors (reviewed in 95, 96). This class includes a

diverse group of complexes (e.g., SWI/SNF, RSC, NURD, NURF, ACF and

CHRAC) that are able to induce ”sliding” of intact histone octamers to adjacent

DNA segments. These more or less random, short range continuous nucleosome

movements make short DNA regions more accessible to regulatory proteins within

a certain time frame, while maintaining the overall packaging of DNA (reviewed in

97).

Mediator

The Mediator was first purified from yeast as a coactivator that enables the basal

transcription machinery to respond to gene-specific transcriptional activator

proteins in an in vitro transcription system reconstituted from purified transcription

factors (98, 99). The yeast Mediator is a large multisubunit complex that comprises

20 subunits. Genetic studies revealed two classes of Mediator genes: one that

encodes Pol II-interacting proteins and another that interacts with gene-specific

18

transcription factors, thus pointing to the role of Mediator as an intermediary

molecule between transcriptional regulators and Pol II (100-104). Biochemical

studies of purified Mediator revealed three major functional activities. Besides

mediating the response to activators, it also stimulates basal transcription

approximately 10-fold and stimulates TFIIH-dependent phosphorylation of the Pol

II CTD 30- to 50-fold (98). The electron microscopic imaging and biochemical

studies of yeast Mediator revealed that this complex has a modular structure and

can be divided into three major modules (105-108). Purification from yeast yields

both free Mediator and Mediator associated with Pol II referred to as the

holoenzyme complex (98, 103). The Mediator-associated Pol II has a

hypophosphorylated CTD, whereas the elongating form of Pol II, which has a

heavily phosphorylated CTD is not associated with Mediator. These findings

suggest a model in which CTD phosphorylation dissociates Mediator from Pol II

and allows it to be recycled by binding to hypophosphorylated polymerase to

initiate new rounds of transcription (109). In addition, in vitro studies indicate that

Mediator and a subset of the GTFs are left behind at the promoter during the

transition from initiation to elongation suggesting that Mediator may function by

facilitating reinitiation (110).

Following the discovery of yeast Mediator, several Mediator-like multiprotein

complexes have been identified in metazoans (reviewed in 111). The composition

of these complexes differs slightly, which might reflect different strategies used for

purification or differences in cell types or growth conditions. However, all reported

complexes are rather similar and most likely represent different forms of the same

complex. Sequence comparisons of genes encoding yeast and metazoan Mediator

subunits revealed that this complex is conserved among eukaryotes and all yeast

Mediator proteins have homologues in metazoan Mediator complexes (112).

Structural studies of yeast and mammalian Mediator complexes also demonstrate

that these complexes have a very similar three-modular structure (105, 106).

19

TFIID

Another cofactor important for regulation of transcription is a component of the

general transcription machinery, namely TFIID. In addition to its key role in

promoter recognition, TFIID was originally shown to support activator-dependent

transcription in Drosophila and human in vitro transcription systems, which

suggested that TAFs function as coactivators targeted by DNA-binding

transcriptional factors (113-115). In agreement with these findings, TAFs have

been shown to interact directly with multiple activators, which resulted in

transcriptional synergy in vitro (116-118). In yeast, 14 TAFs have been identified,

and 13 of these subunits are required for viability. The role of individual TAFs in

vivo has been studied using genome-wide expression analysis of yeast temperature

sensitive (ts) TAF mutants. The percentage of genes that required an individual

TAF ranged from 3% (TAF2) to 59–61% (TAF9), indicating that none of the 13

essential yeast TAFs is required for global transcription (119). Considering the

requirements for TAFs, yeast genes can be classified into three groups, those that

are dependent on upon all or almost all TAFs for their transcription, those that do

not require any TAFs and those that are dependent only on a subset of TAFs (119,

120). Studies of the TAF-dependent promoters suggest that TAFs are important for

recruitment of TBP to these promoters. In contrast, TBP is dispensable for the

recruitment of TAFs, which is consistent with the hypothesis that TAFs, but not

TBP, are targeted by activators (119, 120). However, there is a discrepancy

between the results of the genome-wide expression analyses and the analyses of

total poly(A)+ mRNA levels of ts TAF mutants. The latter demonstrate rapid

decrease of total poly(A)+ mRNA levels upon inactivation of the yeast histone-fold

TAFs, (TAF9, TAF6, TAF12 (=TAF17, =TAF60, =TAF68/61, respectively), as

well as TAF10 (=TAF25) and TAF11(=TAF40), which suggests a central role for

these factors in transcription (121-123). Moreover, the inactivation of each of these

TAFs affected the stability of TFIID and resulted in degradation of multiple TAFs

and even of TBP (121-124). The histone-fold TAFs and TAF10 are shared by the

TFIID and SAGA complexes, which could explain their broad effect on

20

transcription. However, TAF11 is TFIID-specific, and its inactivation does not

affect the integrity of SAGA complex, which demonstrates that its broad effect on

transcription is due to TFIID function (123).

The role of TAFs in transcription is not limited to recruitment of TFIID to the

promoter but seems to be more complicated. For instance, mammalian TAF1

(=TAF250) has two enzymatic activities: a histone acetyl transferase activity which

can be involved in chromatin modifications at the core promoter, and a protein

kinase activity which was shown to phosphorylate the RAP74 subunit of TFIIF in

vitro (89, 125). A hamster cell line, carrying a ts mutation in the TAF1 HAT

domain rapidly arrests in G1-phase and undergoes apoptosis at the restrictive

temperature (126). Accordingly, it was shown that this mutation affects expression

of the cyclin A, D1, D3 and cdc2 genes, but not the c-fos promoter, at the

restrictive temperature (127, 128). Interestingly, in hamster cells, temperature-

sensitive mutations in the HAT domain and the kinase domain of TAF1 affect

different subsets of genes, which indicate that these enzymatic activities function in

a promoter-selective manner (129).

It has also been shown that some TAFs have tissue-specific homologues, which are

required for the expression of cell-type specific subsets of genes. For example,

mammalian TAF4 (=TAF130) has a cell type-specific homologue TAF4b

(=TAF105), which is selectively expressed in testis, ovary and B cells and is

important for ovary development (130). The mammalian TAF7 (=TAF55) has a

germ-cell-specific paralogue TAF7L, which is expressed during spermatogenesis

(131). The can gene of Drosophila encodes a paralogue of dmTAF5 (=TAF80)

which is expressed only in primary spermatocytes and is required for transcription

of genes involved in gametogenesis (132). The discovery of cell type–specific

TAFs suggests that the composition of TFIID may be different in distinct cell

types, and that these changes may play a role in cell differentiation and in

regulation of tissue-specific programs of gene expression.

21

Taken together, these data demonstrate that TFIID has a complex role in

transcription. It can serve both as a general transcription factor important for core

promoter recognition, and as a cofactor targeted by gene-specific transcriptional

activators. In addition, it can also function as a tissue-specific cofactor that is

important for cell differentiation and for tissue-specific regulation of transcription.

Furthermore, the presence of several TAFs in other TBP-lacking multiprotein

complexes such as the histone acetyltransferase complex PCAF/SAGA and the

TBP-free TAFII complex (TFTC) suggests a multifunctional role of TAFs in

transcription (reviewed in 21).

Negative cofactor 2

Negative cofactor 2 (NC2, also called Bur6-Ydr1 in yeast) was initially isolated as

an activity that repressed basal transcription by Pol II in a reconstituted in vitro

transcription system (133, 134). NC2 is composed of two subunits, NC2α and

NC2β which contain histone fold motifs that resemble the histones H2A and H2B,

respectively. NC2 is highly conserved between eukaryotes (135). Experiments in

vitro demonstrated that NC2 interacts with promoter-bound TBP. This NC2-TBP

association prevents interactions with TFIIA and TFIIB and thereby inhibits

formation of the preinitiation complex (134). The crystal structure of NC2 bound to

the TBP-DNA transcription complex confirmed a model for NC2 repression that

was based on the in vitro studies. It demonstrated that NC2 binds to the underside

of the TBP-DNA complex interacting with both the TBP and the DNA, thereby

blocking the entry of TFIIB into the preinitiation complex (136). Genetic studies in

yeast showed that both bur6 and ydr1 mutations were found to suppress mutation in

SRB4, which suggested that NC2 functions as a general repressor of transcription

in vivo (137, 138). However, several lines of evidence suggest that NC2 plays more

complex role in transcription and is involved in both positive and negative

regulation of transcription. First, it was shown that ydr1 mutations reduce

transcription from the HIS3 and HIS4 TATA-less promoters, although it is not

known if this effect is direct (139). Second, NC2 was shown to stimulate

22

transcription in vitro from Drosophila TATA-less promoters containing

downstream promoter elements, whereas it was shown to have a negative effect on

transcription from TATA-box containing promoters (71). Third, chromatin

immunoprecipitation studies demonstrate that NC2 is associated with

transcriptionally active promoters in vivo and that it is recruited to the promoters

upon induction. Interestingly, NC2 association correlates with formation of a Pol II

preinitiation complex (140). Fourth, genome wide expression analysis of yeast

bur6 (encoding the NC2α subunit) temperature-sensitive strain revealed that

expression of 17% of all yeast genes were affected at the restrictive temperature,

and an equivalent numbers of genes were affected positively and negatively (140,

141). Interestingly, the activating function of NC2 is also mediated through

formation of NC2/TBP/DNA complex and requires the same domain of TBP that is

necessary for repressing function of NC2 (141). Together, these data suggest that

NC2 has a complex role in transcription regulation and is involved in both positive

and negative regulation of transcription.

The eukaryotic cell cycle

The life of a eukaryotic cell can be represented as a repeating sequence of cell

growth and division, which is traditionally presented as a cycle (Fig. 5). The cell

cycle consists of several phases. During the first G1-phase, the cell grows in size

and prepares for DNA synthesis. DNA replication occurs during the S-phase, when

newly synthesized DNA immediately gets packed in chromatin. At the end of this

process, chromosomes consist of two chromatids which are attached to each other

at the centromere. During the G2-phase the cell corrects errors that occurred during

DNA replication and prepares for cell division. During the cell division (mitosis,

M-phase), the duplicated chromosomes segregate equally and the cell divides.

After mitosis the two daughter cells enter a new G1-phase and the cell cycle is

completed. In multicellular organisms however, cell division is a very tightly

controlled process and most tissues contain terminally differentiated cells that

never divide (reviewed in 142).

23

Figure 5. Schematic representation of eukaryotic cell cycle.

The G1-phase is the interval in which cells respond to extracellular signals, such as

the presence of stimulatory or inhibitory growth factors, which determine whether

cells will make the decision to enter another round of DNA replication or,

alternatively to exit the cell cycle into a quiescent state, the so-called G0-phase. If

cells make the decision to begin DNA replication, they are irreversibly committed

to complete the cycle (reviewed in 142).

Cyclins and cyclin-dependent kinases in cell cycle regulation

In general, progression through the cell cycle is regulated by a conserved family of

protein kinases, the cyclin-dependent kinases (CDKs). The CDKs are inactive as

monomers and need to bind to a cyclin partner to become activated. Different

cyclin-CDK complexes are assembled and activated to control progression through

different cell cycle phases (reviewed in 143). Progression through the G1 phase is

regulated by the D-type cyclins (D1, D2 and D3), which interact with two catalytic

partners, CDK4 and CDK6. The CDK4 and CDK6 are long-lived proteins while

the D-type cyclins are unstable, and both their synthesis and assembly with their

catalytic partners depend on persistent mitogenic signalling. The activity of cyclin

E-CDK2 increases in the end of the G1-phase and peaks at G1-S transition, after

24

which cyclin E is rapidly degraded and replaced by cyclin A. Cyclin E controls the

entry into the S-phase, whereas cyclin A-CDK2 regulates progression through the

S-phase. Finally, cyclin B in a complex with CDK1 regulate the entry into mitosis

(reviewed in 143).

Regulation of transcription during cell cycle progression

Progress from one phase of the cell cycle to the next requires activation and

repression of certain genes, such as genes encoding cyclins, transcriptional

regulators and proteins involved in processes that are characteristic for the specific

phases of the cycle.

The entry into the S-phase of the cell cycle requires the coordinated induction of a

number of genes, whose products are involved in nucleotide metabolism and DNA

synthesis. These include the genes encoding dihydrofolate reductase (DHFR),

thymidine kinase (TK), both subunits of ribonucleotide reductase (R1 and R2),

DNA polymerase α, and genes encoding proteins involved in cell cycle

progression. The expression of several of these genes is controlled by the

transcription factor E2F. The activity of E2F is regulated by its cell cycle

dependent association with the retinoblastoma tumour suppressor protein (Rb) and

with the related proteins p107 and p130 (reviewed in 144). Rb proteins interact

with the carboxy-terminal region of E2F, which also contains a transcriptional

activation domain, and thereby interfere with the function of the activation domain.

In addition, Rb proteins recruit histone deacetylases (145). The association with Rb

results in downregulation of E2F mediated transcription and cell cycle arrest. It has

been shown that The Rb-E2F interaction depends on the phosphorylation status of

Rb. During the G1/S transition, cyclin D-CDK complexes initiate phosphorylation

of Rb which leads to disruption of Rb-E2F complexes and a subsequent activation

of E2F-dependent genes. One of the E2F targets is the cyclin E gene.

Phosphorylation of Rb leads to induction of cyclin E and an accumulation of cyclin

E-CDK2 complexes, which is required for S-phase entry.

25

In mammalian cells, E2F is a complex family of transcription factors, which

includes at least seven distinct E2F proteins that form heterodimers with one of the

two members of the DP family. E2F and DP proteins are structurally related and

both contain highly conserved DNA binding and dimerization domains (reviewed

in 146). The complexity of the E2F family suggests that individual proteins

perform distinct functions. The E2F family can be divided into three distinct

groups based both on sequence/structural similarity as well as on functional roles

(147). One group, including E2F1, E2F2 and E2F3a is related by both sequence

homology as well as expression pattern. The expression of these proteins is tightly

regulated during the cell cycle with essentially no expression in quiescent cells and

a large induction when cells are stimulated to grow. This subclass of E2F is

thought to act as transcriptional activators (148, 149). In contrast, a second group

of E2Fs, which includes the E2F4 and E2F5, as well as a recently described

alternate version of E2F3 termed E2F3b, is not regulated by cell growth and is

involved in G1 repression (150, 151). Finally, the third group is defined by the

E2F6 protein. E2F6 has only weak homology to other E2F proteins, it lacks the C-

terminal activation domain that is common for the all other E2F proteins and does

not interact with the retinoblastoma family members (152). E2F6 is involved in

transcriptional repression, which is mediated through its ability to recruit the

polycomb transcriptional repressor complex (153).

Ribonucleotide reductase

Ribonucleotide reductase (RNR) is essential for the synthesis of all four

deoxiribonucleotide triphosphates required for DNA replication. It catalyses the

synthesis of deoxyribonucleotide diphosphates (dNDPs) from the corresponding

ribonucleotide diphosphates (rNDPs) (Fig. 6) (reviewed in 154, 155).

26

Figure 6. Ribonucleotide reductase catalyses the synthesis of all four dNDPs. The mammalian ribonucleotide reductase is a heterotetramer consisting of two

homodimeric subunits, proteins R1 and R2, which are both required for the enzyme

activity. Accurate DNA replication requires maintaining of the proper levels of

dNTPs, which is achieved through allosteric regulation of the activity and of the

substrate specificity of ribonucleotide reductase (156). In addition, the RNR

activity is regulated during the cell cycle. In mammalian cells, transcription of both

the R1 and R2 genes is restricted to the S-phase of the cell cycle (157). However,

the R1 protein is present in excess in proliferating cells and its level remains almost

constant during the cell cycle, because this protein has a long half-life (158).

Therefore, the cell cycle-dependent activity of ribonucleotide reductase is

controlled by the limiting levels of the R2 protein (159). Although transcription of

the R1 and R2 genes is coordinated during the cell cycle, sequence comparisons of

their promoters did not reveal any significant sequence homologies. Sequence comparisons of the human and mouse R1 promoters revealed four highly

conserved regions (Fig. 7). One of them is an Inr element that was shown to bind

the TFII-I transcription factor (160). The other two regions, the α element (-98 bp

to -76 bp, relative to the transcription start site) and the β element (-189 bp to –167

bp), were shown to bind proteins by DNase footprinting assays (161).

27

Figure 7. Schematic representation of the mouse ribonucleotide reductase R1 and

R2 promoters. The arrows indicate the transcription start sites.

Luciferase reporter assays has demonstrated that the α and β elements are

important for promoter strength, and gel shift experiments identified that the

transcription factor YY1 is a component of a protein complex binding to this

region (162). The fourth conserved region, the γ element (from +34 to +61 bp,

relative to the transcription start site), together with the Inr element are important

for the S-phase specific regulation of the R1 promoter (160, 162).

In contrast, the mouse R2 promoter contains a TATA-box with the noncanonical

sequence TTTAAA and an upstream CCAAT-box that binds transcription factor

NF-Y (Fig. 7). It has been shown that NF-Y interaction with the R2 promoter

CCAAT-box is important for promoter strength (163, 164). The DNase I

footprinting analysis of the R2 promoter revealed an upstream protein-binding

region located at –672 bp to –527 bp, relative to the R2 transcription start site.

Deletion of this sequence or of a part of it (–623 bp to –597 bp, relative to the

transcription start site) resulted in a decrease of the R2 promoter activity by 80%.

The protein(s) that recognize this region still remain to be identified. Mutational

analysis of potential E2F binding sites in the R2 promoter showed that two

28

proximal elements located in the regions from –112 bp to –107 bp and from -47 bp

to -42 bp, relative to the transcription start site, have a repressing effect on

transcription from this promoter. Interestingly, the repressing effect was more

profound during the G1 phase, which suggests that these two elements are involved

in the cell-cycle regulation of the R2 promoter. Chromatin immunoprecipitation

assays showed that E2F4, but not E2F1, binds to the R2 promoter in vivo (Chabes

A. L., manuscript in preparation). E2F4 is known to be involved in the repression

of transcription and is constitutively expressed during the cell cycle, which

correlates with the expression pattern from R2 promoter constructs carrying

mutations in the putative E2F binding sites. Interestingly, mutations of both the

upstream protein-binding region and the putative E2F binding sites led to a similar

80% decrease of promoter activity as the single mutation of the upstream protein-

binding region, which demonstrate that this region is important for the repressing

effect of E2F4 binding. A mutation of the CCAAT-box in combination with

mutations of the putative E2F4 binding sites does not have a repressing effect on

the R2 promoter activity, but instead, similarly to the mutations of the E2F binding

sites, leads to an up-regulation of transcription from the R2 promoter.

Transcription factor NF-Y

Nuclear factor Y (NF-Y) is an ubiquitous transcription factor which recognizes the

CCAAT-box. The CCAAT-box is one of the most common cis-acting regulatory

sequences and is present in 30% of all eukaryotic promoters. The position of the

CCAAT-box is highly conserved, with a strong preference to the region from –60

bp to –100 bp relatively to transcription start site (165, 166). The CCAAT-boxes

can also be present in several copies, both in direct and in reversed orientations.

NF-Y is composed of three subunits termed NF-YA, NF-YB and NF-YC. Each

subunit contains an evolutionary conserved part that is important for subunit

interactions and CCAAT binding (167, 168) In particular, NF-YB and NF-YC

contain conserved histone-fold motifs that are homologous to those of H2B and

H2A, respectively. These histone-fold motifs are important for NF-YB/NF-YC

29

dimerization, which is a prerequisite for association with NF-YA. The association

of all three subunits is necessary for DNA binding (169, 170). Mammalian NF-YA

and NF-YC contain activation domains rich in glutamines and hydrophobic

residues. Mutation analysis showed that glutamine residues are crucial for

activation (171, 172).

The CCAAT-box is often located in the close vicinity of other promoter elements,

and in many cases NF-Y has been shown to be involved in cooperative interactions

with adjacent transcription factors (173-176). In addition, several lines of evidence

suggest that NF-Y directly interacts with TFIID. First, NF-YB was shown to co-

immunoprecipitate together with TAF5 (=TAF100) from Hela cell nuclear extracts.

In addition, it was demonstrated that the NF-YB/NF-YC dimer can bind to TBP in

solution. Furthermore, the histone-fold containing TAFs (TAF12 (=TAF20),

TAF11 (=TAF28), and TAF13-TAF11 (=TAF18-TAF28) were shown to interact

with the NF-YB-NF-YC dimer, and TAF9-TAF6 (=TAF31-TAF80) were shown to

interact with the NF-Y trimer. Finally, NF-Y was shown to facilitate the binding of

TFIID to the major histocompatibility complex class II Ea promoter (177, 178).

The fixed position of the CCAAT-box relative to the transcription start site also

suggests the possibility of functional interplay between NF-Y and TFIID. Similarly

to other gene-specific transcriptional activators, the activating function of NF-Y is

mediated, at least in part, through its influence on the chromatin structure. In

agreement with this, NF-Y was shown to interact with several HATs including

p300, PCAF and hGCN5 (179-181). Interestingly, p300 was shown to acetylate

NF-YB, although the function of this modification remains to be determined (181).

NF-Y is involved in transcriptional regulation of several cell cycle regulated

promoters. Chromatin immunoprecipitation studies demonstrate that NF-Y is

present at these promoters only during the specific phases of the cell cycle when

these promoters are active and the binding of NF-Y correlates with the recruitment

of p300. In contrast, PCAF and hGCN5 were often recruited to promoters before

NF-Y binding (182). Interestingly, the activity of NF-Y is regulated during the cell

30

cycle, and this regulation is mediated by the NF-YA level, which is maximal in the

middle of the S-phase and decreases in G2/M, whereas the levels of NF-YB and

NF-YC remain constant (183).

Upstream stimulatory factors

Upstream stimulatory factors (USF) is a family of evolutionarily conserved basic-

helix-loop-helix-leucine zipper (bHLH-zip) transcription factors. It comprises two

ubiquitously expressed proteins, USF1 (43 kDa) and USF2 (44 kDa) that form

either homo- or heterodimers and bind as dimers to a canonical CACGTG DNA

sequence (termed an E-box) (184, 185). The C-terminal parts of USF1 and USF2

contain highly conserved bHLH-zip domains which are also shared by other

bHLH-zip transcription factors. The helix-loop-helix motif and the leucine zipper

motif allow these proteins to dimerize while the basic region is important for DNA

binding. In addition, USF1 and USF2 both have a small conserved domain, termed

the USF-specific region (USR), which is apparently unique to the USF proteins and

is located just upstream of the basic region. The N-terminal sequences of USF

proteins are much more divergent (185). Mutational analysis of the USF1 and

USF2 revealed that the USR is essential for transcriptional activation. Interestingly,

the USR of USF2 was required and sufficient for proper activation only at

promoters containing both a TATA-box and an initiator element, which indicates

that the activating properties of this domain are dependent on the core promoter

context (186). The strong evolutional conservation of USR suggests that this region

is crucial for the function of USF.

The major form of USF present in most tissues and cell lines is the USF1/USF2

heterodimer. USF1 homodimers are less abundant and USF2 homodimers are

usually barely detectable. The biological functions of USF proteins in vivo were

studied by using a gene targeting approach. USF1- and USF2-deficient mice are

viable, whereas combined Usf1-/- and Usf2-/- mutations resulted in embryonic

lethality, which suggests that functions of these transcriptional activators are, at

31

least partially, redundant (187). Identification of USF target genes is complicated

by the fact that other helix-loop-helix proteins, including all of the Myc and TFE3

family members, have a similar DNA-binding specificity (188). In particular, it

was shown that at least a subset of c-Myc target sites could be recognized by USF

and that E-box flanking sequences influence the affinity of these factors to

particular binding sites (reviewed in 189).

USF is involved in transcriptional regulation of different subsets of genes,

including tissue-specific, developmentally regulated, cell cycle regulated and

stress-inducible genes (190-192). It is noteworthy that, although the USF proteins

are ubiquitously expressed, they may not be transcriptionally active in all cell

types. For example, it was shown USF activity was lost in several breast cancer cell

lines and in the Saos-2 osteosarcoma cell line. Interestingly, the DNA binding

properties of USF in these cell lines were not affected and the loss of USF function

in Saos-2 cell line was linked to inactivity of the USR. These results suggest the

existence of a USF-specific coactivator that interacts with the USR domain to

mediate the transcriptional activity of USF1 and USF2 in cells such as HeLa and is

either absent or somehow inactivated in Saos-2 cells (193, 194). Studies of the UV-

inducible Tyrosinase promoter revealed that USF proteins are crucial for the UV-

responsive transcriptional activation of this promoter. Although USF1 appears to

be constitutively bound to this promoter, the ability of USF1 to activate

transcription is activated through phosphorylation by the p38-dependent pathway,

which is strongly stimulated by UV irradiation and other stress-inducing agents

(192). It was also shown that USF1 can be phosphorylated by protein kinases C

and A in vitro, and that this phosphorylation stimulated the DNA-binding activity

of USF in gel shift assays (195). Taken together, these data suggest that although

USF transcriptional factors are ubiquitously expressed, their transcriptional activity

is tightly regulated through posttranslational modifications and probably also by

USF-specific cofactors.

32

Part II. Results

Reconstitution of a mouse RNA polymerase II transcription system

from purified transcription factors (Paper I)

Unregulated transcription of protein encoding genes can be reconstituted in vitro

from purified RNA polymerase II and five general transcription factors (GTFs),

termed the TATA-binding protein (TBP), and the transcription factors IIB (TFIIB),

IIE, IIH and IIF. At the time when we started this project, these transcription

factors had been purified from yeast S.cerevisiae, rat liver and human HeLa cells,

and proved to be sufficient for proper initiation of transcription. Using sequences

from conserved regions of the corresponding human cDNAs we selected primers

for synthesis of probes to be used for cDNA cloning of the corresponding mouse

transcription factors.

The cDNAs encoding TFIIB, the small and large subunits of TFIIF and both TFIIE

subunits were cloned from an Okayama-Berg, full-length mouse cDNA library.

The cDNA encoding mouse TBP was a kind gift from Dr. T. Tamura (Chiba

University). After cloning of the cDNAs, expression vectors including six N-

terminal histidine codons were made.

The transcription factors TBP and TFIIB, which consist of single polypeptides

were expressed in Escherichia coli. Our attempts to express the two-subunit

transcription factors IIF and IIE in bacteria were unsuccessful, probably because of

the need for translational modifications. We therefore adopted a baculovirus

expression system, which also allowed us to coexpress both subunits of each factor.

Mouse TBP, TFIIB, TFIIEα, TFIIEβ, TFIIFα and TFIIFβ were then purified to

near homogeneity.

The mouse TFIIH and RNA polymerase II consist of 9 and 12 subunits,

respectively. They are too complex to be reconstituted from recombinantly

33

expressed proteins, and we therefore decided to purify them from mouse Ehrlich-

Lettre ascites cells grown in tissue culture.

A fraction enriched for the elongating form of RNA polymerase II was prepared by

polyethyleneimine precipitation of DNA-bound proteins from nuclear extract. The

precipitated proteins were collected by centrifugation and proteins were extracted

by addition of ammonium sulphate to a final concentration of 0.2M. This extract

was then fractionated by chromatography on DE52 and UNO Q-1 followed by gel

filtration on a Bio-Silect 400 column. Mouse TFIIH was purified from one of the

side fractions from the polyethyleneimine extract preparation by chromatography

on phosphocellulose P11, UNO S-2 and UNO Q-1.

The activity and purity of the transcription factors was tested in in vitro

transcription experiments performed in the presence of all factors or in reactions

where the individual factors were omitted one by one. These experiments

demonstrated that all purified factors were active, and that the mouse in vitro

transcription system was dependent on the addition of all transcription factors to

the reaction mixture. However, a low level of transcription product was detected in

reactions where TFIIE was omitted, probably because our TFIIH fraction contains

trace amounts of TFIIE sufficient to support transcription. Inhibition of

transcription by α-amanitine, a specific inhibitor of RNA polymerase II, confirmed

that the in vitro synthesized RNA transcripts were produced by RNA polymerase

II.

Previous studies of the mouse R2 promoter revealed a proximal CCAAT element

which binds the transcription factor NF-Y (163, 164). The importance of the

CCAAT-box for R2 promoter strength and S-phase specific activation was

analysed in vivo using different R2 promoter-luciferase reporter constructs.

Transient transfection experiments with the luciferase reportergene constructs

showed that a CCAAT→CTAGT mutation in the mouse R2 promoter decreased its

activity to 20% of the wild type level. However, the mutation did not affect the S-

34

phase specific expression from the R2 promoter. In vitro transcription experiments

using crude nuclear extracts and R2 promoter G-less cassette reporter gene

constructs showed a similar decrease of transcription efficiency from the mutated

R2 promoter compared to the wild type. In contrast, the transcription levels from

the wild type and the CCAAT-box mutated R2-promoters were the same in in vitro

transcription reactions reconstituted from purified transcription factors, indicating

that NF-Y and/or essential coactivators are absent in the pure in vitro transcription

system.

Sequences downstream of the transcription initiation site are important

for proper initiation and regulation of mouse ribonucleotide reductase

R2 gene transcription (Paper II)

The TATA-box of the mouse R2-promoter has a noncanonical sequence

(TTTAAA) that differs significantly from the consensus sequence (163). In order

to study the binding of TBP to the R2 TATA-box, we obtained a polyamide

specific for a sequence at the 5´-end of and immediately upstream from the mouse

R2 TATA-box. Polyamides are synthetic polymers that can be designed to bind

DNA in a sequence-specific way thereby preventing protein-DNA interactions, and

they can efficiently penetrate the plasma membrane (196). DNase 1 footprinting

assays showed that the synthesized polyamide bound specifically to its target

sequence immediately upstream from the mouse R2 TATA-box with a KD of 1 nM.

In collaboration with Anna Lena Chabes and Lars Thelander we performed gel

shift experiments using recombinant human TBP and an oligonucleotide

corresponding to the region around the R2 TATA-box. These experiments showed

that human TBP binds specifically to the mouse R2 TATA-box and that the

polyamide interferes with this binding. However, the polyamide had no effect on

expression from the mouse R2 promoter in vivo when it was added to Balb/3T3

cells stably transfected with an R2-luciferase reporter construct. Interestingly,

transient transfection experiments using R2-luciferase reporter constructs also

35

showed that a mutation of the TATA-box had a very limited effect on the R2

promoter strength.

In contrast to these in vivo results, in vitro transcription experiments using purified

transcription factors demonstrated that both the wild type mouse R2 TATA-box

and TBP are absolutely required for transcription from the R2 promoter. This

discrepancy could be explained by the lack of an essential transcription factor in

the reconstituted in vitro transcription system. To test this hypothesis, we

performed in vitro transcription experiments using crude nuclear extracts. These

experiments also showed that transcription from mouse R2 promoter is dependent

on its TATA-box.

The differences in the requirement of the TATA-box could still reflect differences

between the in vivo and in vitro transcription systems. However, we also realized

that the templates used for the two systems differed slightly from each other. The

R2-promoter luciferase reporter constructs used for the in vivo assays contained a

17 bp sequence from the region immediately downstream from the transcription

start site of R2 promoter (5´-UTR), while the R2 promoter G-less cassette

constructs used for the in vitro transcription experiments lacked this sequence. In

order to compare the in vivo and in vitro systems and to test if this sequence was

important for initiation of transcription from the R2 promoter, we made new

reporter gene constructs; both G-less cassette constructs including the R2 5´-UTR,

and luciferase constructs where the R2 5´-UTR was replaced by sequences from

the G-less cassette and the luciferase 5´-UTR. For each construct we also made a

corresponding version with a mutated R2 TATA-box. Both, the in vivo

experiments using the luciferase reporter constructs and the in vitro transcription

experiments with crude nuclear extracts showed that the presence of the 5´-UTR

made the TATA-box redundant for the activity of the R2 promoter. However, this

sequence also had a repressing effect on transcription from the R2 promoter.

We next wanted to test if the TATA-box or the R2 5´-UTR is important for the S-

phase specific transcription from the R2 promoter. Therefore, we performed

36

transient transfection experiments in synchronized Balb/3T3 cells. We found that

that a mutation of the R2 TATA-box and/or a mutation of the R2 5´-UTR had no

effect on the S-phase specific regulation of this promoter.

Using primer extension experiments, we also demonstrated that mutations in the

R2 5´-UTR did not affect the transcription start position. In contrast, the TATA-

box mutations, both alone and combined with a 5´-UTR mutation, resulted in a

shift of transcription start site to a position a few base pairs upstream from the

normal position. However, this change in transcription start site did not correlate to

the difference in promoter strength between the different constructs.

Sequence analysis of the mouse R2 promoter revealed a 10 bp palindrome

sequence that overlaps with the R2 transcription start site. This sequence is also

partially conserved between the human and mouse R2 promoters, which suggests

that it is functionally relevant. Overall, the region downstream from the TATA-box

into the first 30 nucleotides downstream from the R2 transcription start site shows

a 60% identity between mouse and humans. This is much higher than the 30

nucleotides immediately upstream or downstream from this region which show less

than 20% identity. Together, these results suggest that the R2 5´-UTR contains

sequences that bind protein(s), which assist or replace TBP in the formation of a

functional preinitiation complex. To test this hypothesis we performed gel shift

assays using crude nuclear extracts and a probe corresponding to –8 bp to +23 bp

in the R2 promoter relative to the major transcription start site. These experiments

identified the formation of a specific protein–DNA complex which could be

competed by incubation of the nuclear extract with TAF4 (=TAF135) antibodies.

Gel shift experiments using mutated probes demonstrated that the palindrome

region is important for formation of the specific DNA-protein complex. Finally, in

vitro transcription experiments with the wild type R2 promoter and antibodies

against TAF4 showed a 4-fold increase in the promoter activity in the samples

treated with antibodies, which suggests the involvement of components of TFIID

complex in the negative regulation of the mouse R2 promoter.

37

Mechanisms of transcriptional activation of the AdML promoter by

NF-Y (Paper III)

The adenovirus Major Late (AdML) promoter is a TATA-box dependent promoter,

which is widely used as a model promoter for studies of transcriptional regulation.

In addition to the TATA-box, the AdML promoter contains an Inr element, and

two proximal elements; a CCAAT-box and an E-box, which bind NF-Y and USF,

respectively (197-199). Furthermore, transcription from the AdML promoter is

downregulated by NC2. In this paper we study functional interactions between the

regulatory transcription factors NF-Y and USF1, the general repressor NC2 and the

general transcription factor TFIID.

Previous studies demonstrate that both NF-Y and USF often function cooperatively

with neighboring transcriptional activators in different promoters (166, 200, 201).

Using off-rate gel shift assays we demonstrate that binding of NF-Y and USF1 to

the AdML promoter is not cooperative. We next tested if NF-Y and/or USF are

important for recruitment of TFIID to the AdML promoter. Using on rate gel shift

experiments, we found that both NF-Y and USF independently facilitate binding of

TFIID to the AdML core promoter. In contrast, NF-Y and USF had no effect on

binding of TBP and TFIIA, which indicates that the effect on TFIID is mediated

through TAFs. NF-Y consists of three subunits called NF-YA, NF-YB and NF-YC.

Subunits NF-YB and NF-YC dimerize through their histone-fold motifs and this

dimerization is a prerequisite for binding of NF-YA in formation of the complete

NF-Y complex. Both the NF-YC and NF-YA protein subunits have Q-rich

activation domains (171, 172). In order to map the regions of NF-Y that are

responsible for TFIID recruitment, we performed gel shift experiments with a

deletion mutant of NF-Y (YA9/YB4/YC5) that that lacks activation domains and

contains only the histone-fold domains of the NF-YB-NF-YC dimer and the

evolutionary conserved part of NF-YA. Interestingly, this mutant was also able to

promote TFIID recruitment, which indicates that the activation domains of NF-Y

are dispensable for this effect and that the TFIID recruitment to the AdML

38

promoter probably is mediated through histone-fold interactions between the NF-

YB-NF-YC subunits of NF-Y and TAFs.

NC2 represses basal transcription by interacting with DNA-bound TBP, thereby

preventing recruitment of TFIIB and Pol II (133, 134). It has also been

demonstrated that some transcriptional activators function by counteracting NC2-

mediated repression (202, 203). Interestingly, the structure of the NF-YB-NF-YC

dimer is very similar to the structure of NC2, which also contains histone-fold

domains (170). In addition, they both were shown to interact with TBP and to bind

to a similar or overlapping region in TBP (177). We were therefore interested to

test if NF-Y functions as an antirepressor, by interfering with the binding of NC2

to TBP. In order to test this hypothesis we used an in vitro transcription assays

based on crude nuclear extracts isolated from mouse cells. Initially, we tested the

NF-Y dependency of the AdML promoter in in vitro transcription experiments. We

found that addition of antibodies against NF-Y to the in vitro transcription

reactions significantly reduced transcription from the AdML promoter. The

inhibiting effect of NF-Y antibodies was not as pronounced as the effect of

addition of TBP antibodies, but it was clearly stronger compared to the addition of

TFIIB antibodies. Heating of the extracts to +48˚C for 10 min abolished

transcription, which was restored addition of immunopurified human TFIID but not

NF-Y or USF. This demonstrated that in vitro transcription from the AdML

promoter is dependent on TFIID. In vitro transcription experiments using

recombinant, bacterially expressed NF-Y, USF, NC2 and immunopurified human

TFIID showed that transcription from the AdML promoter is inhibited by NC2,

both in the presence and in the absence of NF-Y and USF. We conclude that these

transcriptional factors do not act as antirepressors.

The crude nuclear extracts contain endogenous NF-Y, USF and NC2 activities, as

well as additional transcription factors and cofactors that could influence our

results. We therefore performed experiments with an in vitro transcription system

reconstituted from our purified mouse general transcription factors TFIIB, TFIIE,

39

TFIIF, TFIIH, RNA polymerase II, and immunopurified human TFIID. We found

the NC2 inhibited transcription from the AdML promoter in this in vitro

transcription system. More surprisingly, NF-Y also inhibited transcription in this

system, and addition of NF-Y together with NC2 had an additive negative effect on

transcription. The opposite effect of NF-Y found in the crude and purified in vitro

transcription systems can be explained by the lack of important cofactor(s) in the

purified system. In contrast, the addition of the NF-YB/NF-YC dimer had no effect

on transcription, suggesting that inhibition requires a functional DNA binding NF-

Y. Similarly, addition of USF alone or in combination with NC2 had no effect on

transcription using the purified in vitro transcription system suggesting that the

activating function of USF requires cofactors.

40

Acknowledgements

I would like to thank everybody at the Department of Medical Biochemistry and Biophysics for the support during these six years and for creating a nice working atmosphere. I would especially like to thank:

Stefan Björklund, my supervisor: for the great help with everything, for your patience and for valuable discussions. You always gave me support whenever I needed help or advice.

Lars Thelander: for nice collaboration, for recommendation letters, for ideas and discussions.

Anna-Lena Chabes, Bo Segerman, Sergei Lobov: for all the help, for fruitful collaboration and discussions.

Magnus Hallberg, Gena Polozkov: for sharing buffers and reagents with me, for help and discussions.

Margareta Thelander, Kerstin Hjortsberg, Anders Hofer, Eric Johansson, Richard Lundmark: for help and discussions.

Artur Fijolek, Pelle Håkansson: for “sänka skepp” battles.

Urban Backman: you made teaching much easier.

Ingrid Råberg: for helping me with all the papers.

Lola Fredricsson: for your help, for making all the buffers that we needed, and for keeping all the glassware clean.

Olga Shilkova and Nasim Sabouri: for making me a company in dancing.

Jinan Li: I learned from you a lot about Chinese culture and, especially, movies.

Jürgen Schleucher, Gerhard Gröbner, Angela Augusti, Tatiana Nicol, Sara Flodell, Heather Reese, Olivier Guittet, and Burkhart for all the nice time and fun that we had together. Thank you for being good friends, for your cakes, for saunas and for volleyball games!

Jenny and Bernd van Buuren, as well as Tilla and Miep for bringing some light and warmth during long dark winters.

The Wushu team and the samba course people for the great training sessions and inspiration.

I also want to thank all my Russian friends. Victor: for being a great friend, for biking trips and for enormous help with English. Lena and Valia: for a nice company during swimming and for support in all the creasy ideas. Olga and Oxana: you are great friends and travel companions. Kirill: for the nice art lessons. Dima and Natasha: for nice lunches and for enormous help with computer.

My parents and sister: for your love and support!

41

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