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.
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
1
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
2
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.
3
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 Nacetyltransferase; 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.
4
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.
5
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 10subunit 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).
6
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.
7
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).
8
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).
9
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 Nterminal 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).
10
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,
11
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.
12
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).
13
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-NT/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 TATAand 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
14
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 promoterbound 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-
15
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
16
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
17
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).
18
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
19
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, temperaturesensitive 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.
20
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
21
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).
22
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
23
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.
24
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 Cterminal 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).
25
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).
26
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
27
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 proteinbinding 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
28
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 coimmunoprecipitate 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
29
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 basichelix-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
30
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 UVinducible Tyrosinase promoter revealed that USF proteins are crucial for the UVresponsive 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.
31
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 Nterminal 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
32
expressed proteins, and we therefore decided to purify them from mouse EhrlichLettre 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-
33
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
34
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 Sphase specific transcription from the R2 promoter. Therefore, we performed
35
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 TATAbox 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.
36
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
37
promoter probably is mediated through histone-fold interactions between the NFYB-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 NC2mediated 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,
38
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 NFY. 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.
39
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!
40
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