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Review
Transcriptional repression in eukaryotes:
repressors and repression mechanisms
K. Gaston and P.-S. Jayaraman*
Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD (United Kingdom),
Fax + 44 117 9288274, e-mail: sheela.jayaraman@bristol.ac.uk
Received 26 September 2002; accepted 16 October 2002
Abstract. For many, if not most genes, the initiation of
transcription is the principle point at which their expres-
sion is regulated. Transcription factors, some of which
bind to specific DNA sequences, generally either activate
or repress promoter activity and thereby control tran-
scription initiation. Recent work has revealed in molecu-
lar detail some of the mechanisms used by transcription
factors to bring about transcriptional repression. Some
transcriptional repressor proteins counteract the activity
of positively acting transcription factors. Other repressors
CMLS, Cell. Mol. Life Sci. 60 (2003) 721–741
1420-682X/03/040721-21
DOI 10.1007/s00018-003-2260-3
© Birkhäuser Verlag, Basel, 2003
CMLS
Cellular and Molecular Life Sciences
inhibit the basal transcription machinery. In addition, the
repression of transcription is often intimately associated
with chromatin re-organisation. Many transcriptional
repressor proteins interact either directly or indirectly
with proteins that remodel chromatin or can themselves
influence chromatin structure. This review discusses
the mechanisms by which transcriptional repression is
achieved and the role that chromatin re-organisation
plays in this process.
Key words. Transcription; repression; gene expression; chromatin; histone; gene silencing; RNA polymerase.
Introduction
When transcriptional repression mechanisms in eukary-
otes were first described, gene-specific repression was
often thought to be either direct or indirect. Indirect or
passive repression mechanisms were thought to be of two
kinds: repression by competition between an activator
and a repressor for a common binding site, or repression
by the sequestration of an activator by a repressor into an
inactive complex. In contrast to passive repression, direct
or active repression was thought to involve direct in-
hibitory contacts between the repressor and components
of the basal transcription machinery. However, over re-
cent years, there has been a huge increase in the number
of characterised repression mechanisms and few of them
can easily be placed into these two categories. Any clas-
* Corresponding author.
sification of repression mechanisms is complicated by
the complexity and diversity of protein-protein interac-
tions involved in the regulation of transcription. Further-
more, a great many repressors are now known to act via
several mechanisms and the mechanism used by a partic-
ular repressor is often promoter dependent. For example,
the mammalian cell cycle regulator and tumour supressor
protein Rb uses multiple mechanisms to repress tran-
scription and represses different promoters using differ-
ent combinations of mechanisms [1–8]. In this review,
we will first outline how transcription is regulated and
describe the various classes of repressor proteins.
We will then attempt to divide the myriad of repression
mechanisms that have been identified into one of three
categories: inhibition of the basal transcription machin-
ery, ablation of activator function and remodelling of
chromatin. We will describe the repression mechanisms
that have been studied in detail and we suggest that
this analysis of repressors and their mechanisms of ac-
tion leads to a more coherent understanding of repressor
function.
The regulation of transcription
In higher eukaryotes, transcription initiation at promoters
recognised by RNA polymerase II is brought about by the
concerted action of general transcription factors (GTFs)
and the RNA polymerase core enzyme, an assembly of
around ten RNA polymerase core subunits. The GTFs are
recruited to promoters in an ordered fashion in vitro and
aid the binding of RNA polymerase to promoter DNA
(fig. 1A). The first GTF to bind in vitro is TFIID, a com-
plex containing the TATA box-binding protein (TBP) and
a number of TBP-associated factors (TAFs). The assem-
bly of GTFs on naked DNA templates forms the pre-ini-
tiation complex (PIC) and in the presence of NTPs, the
PIC can initiate transcription and begin transcription
elongation. The low level of transcription directed by
these proteins is known as basal transcription [reviewed
in ref. 9]. In cells, RNA polymerase II is in fact loosely
associated with a variety of proteins and these complexes
are referred to as the RNA polymerase II holoenzyme.
Holoenzyme complexes may be recruited to promoters
without the prior ordered assembly of GTFs (fig. 1B)
[10]. In yeast and mammalian cells, one set of factors that
are associated with RNA polymerase II are the Srb/Med
proteins. These proteins allow RNA polymerase to re-
spond to both activators and repressors. In mammalian
cells, in addition to Srb/Med proteins, a number of pro-
teins that alter the structure of chromatin (chromatin re-
modelling factors) are components of holoenzyme com-
plexes [reviewed in refs 9, 11].
Activators of transcription are often defined as sequence-
specific DNA-binding proteins that stimulate transcrip-
tion initiation or elongation. Many activators interact
with proteins known as co-activators; these proteins do
not have DNA-binding activity but they help the activator
to perform its function. One set of co-activators are the
TAF proteins present in TFIID, although these proteins
can also be regarded as GTFs [reviewed in ref. 12]. Most
activators and many co-activators act by stimulating the
formation of the PIC (fig. 1C). This is often brought
about by direct interactions between these proteins and
various components of the PIC. In addition, both activa-
tors and co-activators can activate transcription by pro-
moting the alteration of chromatin structure in the vicin-
ity of the promoter (fig. 1D). Three classes of protein as-
sociated with the RNA polymerase II holoenzyme are
involved in this remodelling of chromatin: histone-modi-
fying enzymes, chromatin-binding proteins, and ATP-de-
pendent nucleosome-remodelling proteins [reviewed in
ref. 9]. Activators and co-activators can recruit one or
more of these proteins to a promoter and the resulting
722 K. Gaston and P. S. Jayaraman Transcriptional repression mechanisms
chromatin remodelling can alter histone-DNA interac-
tions, nucleosome-nucleosome interactions, and/or re-
position nucleosomes relative to transcription factor-
binding sites [reviewed in ref. 13]. These changes in chro-
matin structure regulate transcription by altering the local
accessibility of the DNA to transcription factors, RNA
polymerase II, and other components of the PIC.
Repression
Transcriptional repression is of two types: general or
global repression and gene-specific repression. General
repression occurs when a repressor protein or complex ei-
ther sequesters or modifies a central component of the
PIC or a component of RNA polymerase II, so that it is
unavailable for transcription. Thus general repression
will down-regulate the expression of all the genes tran-
scribed by this RNA polymerase. For example, phospho-
rylation of a core subunit of RNA polymerase II by the
herpes virus (HSV-1) proteins ICP22 and UL13 kinase
generates a non-functional form of polymerase resulting
in general repression [14]. Nucleosomes also generally
repress promoter activity by sequestering promoter DNA
into chromatin [reviewed in ref. 15]. In contrast, gene-
specific repression occurs when the transcription of a
particular gene or set of genes is controlled by the activ-
ity of a gene-specific repressor or co-repressor. Repres-
sors can bring about gene-specific repression by decreas-
ing the concentration of a functional activator/co-activa-
tor at the promoter or by counteracting the stimulatory
effect of these proteins on transcription. In addition, some
repressors inhibit transcription by interacting in a pro-
moter-specific fashion with components of the PIC or by
recruiting chromatin-remodelling proteins.
Gene-specific repressor proteins often bind either di-
rectly or indirectly to DNA and they can regulate tran-
scription from binding sites proximal to, or at a distance
from, the promoter [reviewed in ref. 16]. Repression that
is effected by distally located repressor proteins is often
known as ‘long-range’ repression and these proteins are
sometimes known as ‘long-range’ repressors. Distally
bound repressors may repress promoter activity by re-
modelling chromatin at or near the promoter. Alterna-
tively, they may contact transcription activators and/or
components of the PIC by looping out the intervening
DNA [17, and references therein]. In contrast to long-
range repression, ‘short-range’ repression occurs when a
repressor protein acts locally. In this case, the repressor
down-regulates the activity of nearby activator proteins or
components of the PIC but does not affect the activity of
distally located activators. Proteins that bring about this
‘short-range’ repression are sometimes referred to as
‘short-range’ repressors. However, these two types of re-
pression are not necessarily mutually exclusive and a re-
CMLS, Cell. Mol. Life Sci. Vol. 60, 2003 Review Article 723
Figure 1. Pre-initiation complex formation and the activation of transcription. (A) GTFs bind in an ordered fashion to form the preinitia-
tion complex (PIC). The TATA box and initiator element are indicated by TATA and INR, respectively. The transcription start site is repre-
sented by the bent arrow. (B) PIC formation may involve the recruitment of an RNA polymerase II holoenzyme. The holoenzyme com-
prises RNA polymerase, GTFs, Srb/Mediator proteins (Srb/Med), and chromatin-remodelling factors (CRFs). (C) The activation of tran-
scription in the context of chromatin. Activators often stimulate the formation of the PIC by making direct contacts (red lines) with GTFs
(A1). Activators bound at promoter-proximal locations (A2) or at promoter distal locations (A3) can recruit co-activators (CoAct). (D) Sev-
eral co-activators as well as some components of the TFIID complex have histone acetyltransferase activity (red arrows) and the acetyla-
tion of histones (red dots) brings about changes in chromatin structure. Activators and co-activators can also bring about changes in chro-
matin structure by recruiting a variety of CRFs.
AB
C
D
pressor may bring about both long- and short-range re-
pression depending on the context of its binding site and
its exact mode of action [18, 19]. For example, the re-
pressor protein Hairy can bring about both long- and
short-range repression [20, 21]. Furthermore, one should
note that the terms long and short range do not refer to
any particular molecular mechanisms that bring about re-
pression.
Repressor proteins
Gene-specific repressor proteins are a large group of di-
verse proteins that negatively regulate transcription, and
they are not easily defined. Repressor proteins have been
categorised in a number of ways. They have been divided
into proteins that bring about short- or long-range repres-
sion [19]. Repressors have also been divided into groups
that can or cannot recruit histone deacetylases to promot-
ers. In addition, they have been categorised as either se-
quence-specific DNA-binding repressors or non-DNA-
binding co-repressors. There are, however, many exam-
ples of repressors that do not fit into any of these
categories. In addition, there are a rapidly expanding
number of ‘context-dependent’ transcription factors that
bind DNA and are capable of positively or negatively reg-
ulating transcription depending on the context of their
binding sites, the complement of protein interactions they
can make and other environmental cues.
To categorise the different kinds of repressor proteins we
have defined three main classes (table 1). However, we
would like to point out that the members of each class can
repress via multiple mechanisms and that some repres-
sors fall into more than one category. Class I repressors
are DNA-binding proteins that negatively regulate the
transcription of specific genes. These repressor proteins
are of two types: sequence-specific DNA-binding pro-
teins (class IA) and proteins that bind to methylated DNA
(class IB). Examples of the former are the Drosophila
zinc-finger protein Krüppel [22, 23] and homeodomain
protein Engrailed (En) [24]. Examples of the latter are
MeCP2 [25] and MBD2 [26, 27]. In contrast to class I re-
pressors, class II repressors are proteins that do not bind
DNA directly. Instead, they are recruited to promoters by
other proteins. Class II repressors can be divided into two
subclasses. Class IIA repressors interact with ‘dedicated’
repressor proteins and can be considered to be true co-re-
pressors. Examples of this type of repressor are Dnmt3
and MBD3; these proteins repress transcription by inter-
acting with the class I repressors RP58 and MBD2, re-
spectively [28, 29]. Unlike the class IIA repressors,
class IIB repressors interact with both ‘dedicated’ class I
repressors and ‘context-dependent’ transcription factors,
proteins that will in other situations activate transcription.
Class IIB repressors include the SMRT [30] and NCoR
[31] proteins and members of the Groucho/Tup1 and
CtBP families [18, 19, 32]. Class IIB repressors also in-
clude proteins such as the GAL80 repressor protein from
yeast and the human MDM2 tumour supressor protein
which bring about repression by protein-protein interac-
tions with the activator proteins GAL4 and p53, respec-
tively [33, 34]. Subunits from a number of repressive
chromatin-remodelling complexes can also be considered
as class II repressors. For example, the Sin3 and MBD3
proteins present in the SIN and NuRD chromatin-remod-
elling complexes can also be considered to be class II re-
pressor proteins, because they can bind directly to DNA-
binding proteins such as Ikaros and MBD2, respectively
[35, 36].
Class III repressors are proteins that do not necessarily
bind to DNA directly or indirectly. These repressors often
target activators, co-activators or components of the PIC
and usually reduce the amount of functional protein avail-
able to regulate transcription. Class IIIA repressors often
724 K. Gaston and P. S. Jayaraman Transcriptional repression mechanisms
Table1. Classes of Repressor Proteins.
Class Defining feature Examples
Class I: DNA-binding proteins
A sequence-specific DNA binding PRH, Eve, Krüppel, TGIF,
Mad, IRF-2, RP58, E2F-6
B methyl-CpG-binding proteins MeCP2, MBD2
Class II: bind to DNA-binding proteins
A proteins (co-repressors) that Dnmt3, MBD3, Tup1,
bind to dedicated repressors Groucho
B proteins that bind to context- Groucho, CtBP, TGIF,
dependent transcription factors NcoR, Rb, MDM2
Class III: other repressors
A bind to activators, I
k
B, Mot1, FIR, GAL80,
co-activators, or PIC E1A243R, Rb, PHO80,
B post-translationally modify OGT, PHO80, CARM-1,
activators, co-activators or PIC Srb10, CCK-II
sequester these proteins into inactive complexes, or alter
their stability or nuclear localisation without post-transla-
tional modification. An example of this group is the I
k
B
protein which sequesters the activator NF
k
B activator
protein in the cytosol [37]. Other examples of this group
are the adenovirus E1A243R (12S) repressor protein and
the Mot1 repressor protein. These proteins interact with
TBP and dissociate TBP bound to TATA boxes [38, 39].
Class IIIB repressors post-translationally modify their
targets. Examples are the PHO80/85 kinase cyclin com-
plex from yeast that phosphorylates and inactivates the
activator PHO4 [40] and the mammalian CARM-1 pro-
tein which methylates the co-activator CBP [41].
As pointed out earlier, some repressors fall into more than
one of the categories described above. PHO80, for exam-
ple, can be considered a class IIIA repressor as it binds to
PHO4, a DNA-binding activator protein, and ‘masks’ the
PHO4 activation domain [42]. However, the PHO80/
PHO85 complex can phosphorylate PHO4, and in this
context, PHO80 acts as a class IIIB repressor [40]. Simi-
larly, the homeodomain protein TGIF is a class IA re-
pressor protein that binds to the retinoid X receptor
(RXR) response element [43]. However, TGIF can also
function as a class IIB repressor protein at promoters that
are regulated by the SMAD proteins [44].
Repression mechanisms
There are three major routes through which repressor
proteins can down-regulate specific genes: inhibition of
the basal transcription machinery, ablation of activator
function, and remodelling/compaction of chromatin [45].
The details for each route to transcriptional repression
can vary in a promoter- and repressor-specific fashion.
Below, we describe some of the best-understood repres-
sion mechanisms.
Repression via the basal transcription machinery
Although targeting of the basal transcription machinery
might be expected to result in a global shutdown of tran-
scription, there are a number of repressor proteins that act
in a gene-specific fashion by interacting with one or more
of the GTFs or core subunits. This can result in the inhi-
bition of basal and activated transcription at specific pro-
moters and is often known as ‘active’ or ‘direct’ repres-
sion.
Modifying the RNA polymerase II large subunit
The RNA polymerase II large subunit C-terminal domain
(CTD) is a target for direct repression (fig. 2A). The CTD
of RNA polymerase is glycosylated and dephosphory-
lated during transcriptional initiation, whereas it is degly-
cosylated and phosphorylated during elongation [46, 47].
Altering the extent or the timing of these modifications
on the CTD brings about transcriptional repression. As
mentioned earlier, the RNA polymerase II holoenzyme in
yeast contains several ‘suppressors of RNA polymerase
B/Mediator’ (Srb/Med) components that mediate the re-
sponse of the holoenzyme to many transcriptional activa-
tors and repressors. One protein present in this complex
is the kinase Srb10. Srb10 represses the transcription of a
set of genes involved in cell type specificity, meiosis and
sugar utilisation by phosphorylating the CTD before the
holoenzyme associates with promoter DNA. Phosphory-
lation by Srb10 thus inhibits formation of the PIC and
hence transcription initiation [48]. How the activity of
Srb10 is regulated so that can repress only specific sets of
genes is not yet known, however, one possibility is that
Srb10 activity is regulated by Tup1. The yeast Tup1 pro-
tein is a member of the Groucho family of class II repres-
sors. Genetic and biochemical experiments have demon-
strated that Srb10 and Tup1 interact and that the kinase
activity of Srb10 is important for repression by Tup1 [49,
50]. Furthermore, Tup1 prevents the association of the
RNA polymerase II holoenzyme with promoters [50].
One mechanism through which Tup1 represses transcrip-
tion could be by enhancing the activity of the Srb10 ki-
nase [50].
Recruitment of the enzyme O-GlcNac transferase (OGT)
to promoters by the mammalian class II repressor protein
Sin3a has been suggested to be another mechanism for
the direct repression of transcription. This suggestion is
based on the fact that Sin3a can interact with OGT and
that directly tethering OGT to promoter DNA as a Gal4-
OGT fusion protein results in the repression of transcrip-
tion. Furthermore, proteins bound to Sin3-dependent
promoters are more heavily glycosylated when the pro-
moter is repressed [51]. Since the RNA polymerase CTD
is known to be glycosylated, gene-specific repression
might be brought about by recruiting OGT to Sin3-regu-
lated promoters. Glycosylation or hyperglycosylation of
the CTD might inhibit elongation or the recycling of
RNA polymerase between transcription elongation and
initiation [51].
Inhibiting the binding of TBP to DNA
An important mechanism of gene-specific repression is
interference with the binding of TBP and hence TFIID to
the TATA box (fig. 2B). This can be gene specific for
three reasons. First, repressors that act in this way can be
targeted to individual promoters either through direct or
indirect binding to specific DNA sequences. Second,
many genes transcribed by RNA polymerase II do not
contain TATA box sequences and are thus probably not
sensitive to this type of repression; this is particularly true
CMLS, Cell. Mol. Life Sci. Vol. 60, 2003 Review Article 725
of housekeeping genes. Third, at least some of the genes
transcribed by RNA polymerase II are regulated by a PIC
that does not contain TBP [52]. Several class I repressor
proteins have been shown to bind TBP including the
Drosophila homeodomain protein Even-skipped (Eve)
[53]. In vitro, the Eve repression domain together with
the Eve homeodomain bind directly to TBP and this in-
teraction can block the binding of TBP/TFIID to the
TATA box and thus prevent assembly of the PIC [54]. Ev-
idence to support this mechanism of repression comes
from the fact that phosphorylation of Eve prevents the
Eve-TBP interaction and results in the loss of repression
[55]. The adenovirus E1A243R (12S) protein also re-
presses transcription by making direct contacts with TBP
that interfere with formation of the TBP-TATA complex.
Intriguingly, a pre-formed TBP-TATA box complex can
be dissociated by the E1A N-terminal repression domain
[38]. The N-terminal repression domain of E1A also
binds to the co-activator CBP and a mutational analysis
has demonstrated that the repression domain in E1A is
not separable from the CBP interaction domain. This has
led to a model for repression where E1A243R interacts
with CBP at CBP-regulated promoters and then dis-
sociates/interferes with formation of the TBP-TATA
complex [56].
Mot1 is a repressor protein that can dissociate a TBP-
TATA complex in an ATP-dependent manner. Mot1 is a
member of the SWI/SNF family of ATP-dependent nu-
cleosome-remodelling proteins [see below and ref. 57].
Mot1 removes TBP from DNA by interacting with TBP
[39]. Interestingly, Mot1 competes with TFIIA for bind-
ing to TBP as they bind to the same surface of TBP. Since
TFIIA stabilises the TFIID/DNA complex, Mot1 coun-
teracts the stabilising activity of TFIIA. Although Mot1
was originally thought to function as a general regulator
of basal transcription, it has recently been demonstrated
to repress (and activate) the transcription of specific sets
of genes [58]. One possibility is that Mot1 is recruited to
726 K. Gaston and P. S. Jayaraman Transcriptional repression mechanisms
A B
C D
Figure 2. Repression via the basal machinery. (A) Repressors bind to and/or modify RNA polymerase or GTFs and block binding to the
promoter. (B) Repressors block the binding of TFIID to the TATA element either by competing for the TATA element (R1) or by binding
to TFIID (R2). (C) Repressors block interactions between GTFs. (D) Repressors block activator-dependent interactions between GTFs.
promoters in a gene-specific fashion by interaction with
DNA-binding proteins [59].
The inhibition of TBP-DNA binding does not necessarily
involve protein-protein interactions between the repres-
sor and the TBP. Several repressor proteins can bind to
TATA box sequences and sterically hinder the binding of
TBP. For example, En is a class I repressor that can bind
to A/T-rich sequences and compete with TFIID for bind-
ing to TATA box sequences [60]. Whether En uses this
mechanism to repress transcription in vivo is not known,
but studies in haematopoietic cells suggest that the verte-
brate homeodomain protein PRH (also known as Hex)
might repress transcription in this way. Like En, PRH can
bind to TATA box sequences in vitro; furthermore, muta-
tions in the PRH homeodomain that block binding to
these sequences abolish homeodomain-dependent tran-
scriptional repression [61]. In vitro studies with Eve sug-
gest that when this protein is bound to high-affinity bind-
ing sites upstream of the promoter it can recruit further
Eve proteins to low-affinity sites around the TATA box.
The binding of Eve to these low-affinity sites is thought
to prevent TBP binding and hence inhibit PIC formation.
This has been termed ‘cooperative blocking’ of the pro-
moter [62] and may also occur when the papillomavirus
E2 protein binds cooperatively to its four binding sites
within the papillomavirus genome and represses viral
gene expression [63].
Inhibiting interactions between the GTFs
A number of repressors inhibit GTF-GTF interactions
and thereby repress transcription (fig. 2C). For example,
the NC2 (Dr1-Drap or Bur6-Ydr1) heterodimer [64–66]
binds to TBP in vitro and blocks interactions between
TBP and TFIIA. The NC2- and TFIIB-binding sites on
TBP are adjacent and the binding of NC2 possibly di-
rectly impedes the binding of TFIIB. However, the NC2-
and TFIIA-binding sites on TBP are on opposing sur-
faces, suggesting that the interaction of NC2 with TBP al-
ters the conformation of TBP so that it can no longer in-
teract with TFIIA [65–67, reviewed in ref. 57]. Although
NC2 has been characterised as a general repressor of tran-
scription, evidence suggests that it can also function in a
gene-specific fashion as both an activator and a repressor
of transcription [68]. The class I repressor proteins Krüp-
pel and the unliganded thyroid receptor (TR) are also
thought to repress transcription by altering interactions
between GTFs. Whilst monomeric Krüppel is an activa-
tor that binds to TFIIB, dimeric Krüppel binds to the
TFIIE
b
subunit and inhibits formation of the PIC [69].
Similarly, in the presence of ligand, TR is an activator that
interacts with TFIIB. However, in the absence of ligand,
TR can interact with TBP and inhibit the formation of a
functional PIC. In vitro transcription studies have shown
that TR binds directly to TBP and interferes with the for-
mation of TBP-TFIIA or TBP-TFIIA-TFIIB complexes
[70–72]. The class II repressor proteins MDM2 and N-
CoR also interact directly with GTFs. MDM2 binds to
TFIIE and TBP and directly interferes with basal tran-
scription [73]. Similarly N-CoR, a protein that mediates
repression by unliganded nuclear hormone receptor pro-
teins such as TR, can interact with TAF32, TFIIB and
TAFII70 simultaneously and inhibit the functional inter-
action of TFIIB and TAF32 [74]. This suggests that N-
CoR, like the repressors described above, makes interac-
tions with the GTFs that lock the PIC into a non-func-
tional state or conformation.
In all of the examples described so far, the repressors
block activator-independent GTF-GTF contacts. In con-
trast, RBP/CBF1 is a class I repressor that is able to dis-
rupt activator-dependent GTF-GTF interactions (fig. 2D)
[75]. Sp1-activated transcription can be repressed by
RBP. Sp1 interacts with Drosophila TAF110 (dTAF110)
in vitro and dTAF110 associates with TFIIA resulting in
transcription activation. RBP binds to TFIIA and prevents
the Sp1-induced interaction of TFIIA and dTAF110. RBP
does not repress transcription if the Sp1-dTAF110-TFIIA
complex is preformed and RBP does not prevent the in-
teraction of Sp1 with dTAF110. Thus RBP does not in-
hibit the interaction of the activator with a GTF but,
rather, inhibits activator-dependent interactions between
GTFs [75].
Finally, one should note that many proteins involved in
chromatin remodelling can also repress transcription via
direct contacts with GTFs. The Polycomb group (PcG)
proteins, for example, are chromatin-binding proteins
that are recruited to specific promoters by interaction
with class I and class II repressors. PcG proteins can re-
press transcription by interacting directly with a number
of GTFs [76, 77]. Similarly, MeCP2, a methyl-CpG-bind-
ing protein involved in transcriptional silencing, interacts
with TFIIB to inhibit basal transcription and also can or-
ganise DNA into large nucleoprotein complexes [78].
Repression via the ablation of activator function
Many transcriptional repressor proteins regulate the ac-
tivity or location of a transcription activator/co-activator
(fig. 3). This can be achieved by regulating the turnover
and hence levels of an activator, regulating its intracellu-
lar localisation, inhibiting its DNA-binding activity, or by
inhibiting any of the protein-protein interactions that the
activator makes with the transcription machinery.
Regulating activator/co-activator turnover
Proteins that regulate the stability and turnover of an ac-
tivator can indirectly regulate transcription. The MDM2
oncoprotein is an example of a repressor that ubiquiti-
CMLS, Cell. Mol. Life Sci. Vol. 60, 2003 Review Article 727
nates an activator and thereby promotes its degradation
by the proteasome. Ubiquitination of p53 by MDM2 trig-
gers the nuclear export of p53 and its degradation [79,
80]. Another example of a repressor that promotes the
degradation of an activator is the kinase Srb10. As men-
tioned earlier, Srb10 can repress specific subsets of yeast
genes by phosphorylating the RNA polymerase CTD.
Srb10 can also repress genes activated by the transcrip-
tion factor GCN4 [81]. In this case, however, Srb10 acts
by phosphorylating GCN4. The phosphorylation of
GCN4 by Srb10 results in the recruitment of a complex
that ubiquitinates GCN4 and marks it for degradation by
the proteasome. Thus, Srb10 ultimately represses GCN4-
dependent genes by promoting the degradation of their
activator [82]. Although the promotion of degradation/
turnover of an activator by a repressor is generally medi-
ated by the proteasome, there is at least one case where a
repressor is thought to directly cleave proteins involved in
transcription. The class I repressor protein AEBP1 has
carboxypeptidase activity and this activity is required for
its repressor function [83]. Whether activators or compo-
nents of the PIC are cleaved by this protein remains un-
clear.
Promoting the degradation of a co-activator can also
bring about the repression of transcription. The LIM
homeodomain proteins (LIM-HDs) are involved in cell
lineage determination and the regulation of differentia-
tion [84]. When associated with CLIM proteins (co-fac-
tor of LIM-HD), the LIM-HDs activate transcription.
However, R-LIM proteins inhibit the activity of LIM-
HD-CLIM complexes in several ways. They compete
with CLIM proteins for binding to LIM-HD, they recruit
histone-modifying complexes and, uniquely, they can
also ubiquitinate CLIM factors bound to LIM-HDs whilst
they are bound to DNA [84, 85]. Ubiquitination results in
the degradation of CLIM proteins and thus R-LIM re-
presses LIH-HD activity by regulating the turnover of
CLIM co-activator proteins [84].
Regulating the intracellular localisation
of an activator
A well-known transcription factor that functions by pre-
venting nuclear localisation of the activator is the repres-
sor I
k
B. I
k
B blocks the nuclear import of NF
k
B, a het-
erodimer comprised of p50 and p65, two members of the
Rel family of transcription activators [86]. The interac-
tion of NF
k
B with I
k
B ‘masks’ the nuclear localisation
signal of NF
k
B and thereby prevents its nuclear import
and, hence, the activation of NF
k
B-dependent genes [37].
The interaction of I
k
B with NF
k
B is subject to extensive
regulation by kinases and ubiquitinylating enzymes that
target the repressor for degradation [reviewed in ref. 87].
Post-translational modification of an activator can also
inhibit nuclear localisation. The PHO80 repressor protein
from Saccharomyces cerevisiae is a kinase that interacts
with a cyclin-like partner protein PHO85. Under condi-
tions where phosphate is in excess, the PHO80/85 het-
erodimer phosphorylates the activator protein PHO4
[40]. Phosphorylated PHO4 becomes localised to the cy-
tosol resulting in the repression of genes required for
phosphate uptake [88].
Inhibiting activator-DNA interactions
One of the first mechanisms of repression to be studied
in detail was that arising as a consequence of competi-
tion between two different transcription factors for a
728 K. Gaston and P. S. Jayaraman Transcriptional repression mechanisms
Figure 3. Repression by the ablation of activator function. Repressors often target activators or co-activators for degradation (R1) or hold
activators in non-productive complexes (R2 and R3). Some repressors block activator-PIC contacts (R4). Other repressors post-transla-
tionally modify activators (R5). Note that R1 and R5 need not necessarily act only in the cytoplasm and nucleus, respectively.
common binding site. This type of repression is analo-
gous to that observed when proteins compete with TBP
for binding to the TATA box. Engrailed competes with
the activator Fushi-taratzu (Ftz) for a common binding
site and can thereby repress transcription [89]. Similarly,
the GC-rich sequence-binding factor is able to repress
transcription of several genes by competing with the
transcription activator Sp1 for binding sites [90]. This re-
pression mechanism allows transcription factors that
normally activate transcription to down-regulate gene
expression. The transcription factor AP1 provides an ex-
ample of this situation. AP1 is usually an activator, how-
ever, it represses basal and retinoic acid-induced tran-
scription of the osteocalcin gene by competing with the
retinoic acid receptor protein for overlapping DNA-
binding sites [91]. Some repressors which bind to single-
stranded DNA sequences have also been proposed to re-
press transcription by competing with activator proteins
that bind to the same sequences when they are in double-
stranded DNA [92, 93].
Post-translational modification of many activators can in-
hibit their ability to bind DNA (fig. 3). For example, dur-
ing mitosis, phosphorylation of the Oct-1 homeodomain
by protein kinase A inhibits Oct-1 DNA-binding activity
and represses the transcription of Oct-1-dependent genes
[94]. Similarly, acetylation of the C-terminal zinc-finger
domain of YY1 decreases the DNA-binding activity of
this bifunctional activator/repressor protein [95]. Many
other repressors regulate transcription by preventing acti-
vators from binding DNA but do not post-translationally
modify the activator. This type of repression usually in-
volves protein-protein interactions between the repressor
and the activator. Repressor proteins belonging to the he-
lix-loop-helix (HLH) family and the basic-leucine zipper
(B-LZ) family function by forming non-DNA-binding
heterodimers with other family members that activate
transcription. The class IIIA repressor protein, Id, for ex-
ample is an HLH protein that lacks DNA-binding activity
but which heterodimerises with the activators MyoD, E12
and E47 and inhibits their binding to DNA [96]. The class
I repressor protein Mad is a member of the basic-helix-
loop-helix-leucine-zipper (B-HLH-LZ) family of tran-
scription factors and forms DNA-binding heterodimers
with the B-HLH-LZ proteins Myc and Max [97]. Myc ac-
tivates transcription but only as a heterodimer with Max.
Heterodimerisation of Mad with Max competes with the
formation of the Myc/Max complex and can thereby
block transcription activation. In addition, the Mad/Max
heterodimer can compete with Myc/Max for DNA se-
quences known as E-boxes. Thus, Mad competes with
Myc for an interacting partner and the Mad/Max het-
erodimer competes with Myc/Max for a common binding
site on DNA. In addition, the Mad/Max complex recruits
proteins that alter chromatin structure [see below and
ref. 98].
Inhibiting activator-target interactions
Many class II repressors bind to activators and prevent
them from interacting with their targets. When the re-
pressor binds to the activation domain, this type of re-
pression is sometimes referred to as masking. Masking
was first characterised in detail for the yeast repressor
protein GAL80 which binds directly to the activator
GAL4. The GAL4 activation domain interacts with the
Srb4 subunit of the RNA polymerase II holoenzyme [99].
Since the binding site for the GAL80 protein on GAL4
partially overlaps with the GAL4 activation domain, the
interaction of GAL80 with GAL4 can inhibit the interac-
tion of GAL4 with the transcription machinery [33, 100].
Presumably, at least one of the interactions that is masked
by GAL80 is the Srb4-GAL4 interaction. Interestingly,
repression is augmented if there is more than one GAL4-
binding site at the promoter and protein-protein interac-
tions between GAL80 repressor molecules that are si-
multaneously bound to GAL4 activator proteins have
been suggested to enhance masking of the GAL4 activa-
tion domain [101]. Masking of an activation domain has
been suggested as a mechanism of repression for at least
three other class II repressor proteins. The Rb protein
‘masks’ the activation domain of the activator E2F [1, 2].
Similarly, MDM2 ‘masks’ the p53 activation domain
[102, 103]. Masking of p53 by MDM2 involves an am-
phipathic
a
helix in p53 that is involved both in the bind-
ing of MDM2 and in transcription activation [104]. Fi-
nally, the yeast PHO80 repressor protein has been sug-
gested to ‘mask’ the activation domain of PHO4.
However, in this case, the PHO80 interaction region is ad-
jacent rather than co-incident with the PHO4 activation
domain [42].
Some repressors that inhibit activator-target interactions
do not bind to activation domains. Fuse binding protein
(FBP) binds to the c-myc promoter and activates tran-
scription. The FBP-interacting repressor protein (FIR)
binds to FBP and to its target GTF. FBP contacts TFIIH
and stimulates the 3¢-5¢ helicase activity of the p89/XPB
TFIIH subunit. The 3¢-5¢ helicase activity of this GTF is
required for promoter melting, transcription initiation
and promoter escape. FIR inhibits activated transcription
by simultaneously interacting with FBP and p89/XPB
and decreasing the 3¢-5¢ helicase activity of TFIIH [105,
106]. Repressors can also act by inhibiting co-activator-
polymerase II/GTF contacts. The yeast Tup1 protein was
recently proposed to repress transcription by inhibiting
the interaction of Srb7, an essential component of the
RNA polymerase II holoenzyme, with the co-activator
Med6. Tup1 can compete with Med6 for binding to Srb7
and since the interaction of Srb7 with Med6 is essential
for full activation by several activators, this leads to the
repression of a subset of yeast genes [107].
When class I repressor proteins block interactions be-
tween a DNA-bound activator and one or more compo-
CMLS, Cell. Mol. Life Sci. Vol. 60, 2003 Review Article 729
nents of the PIC, this is sometimes known as ‘quenching’.
YY1, is a mammalian Krüppel-like transcription factor
[108, 109] that can quench transcription activated by
AP-1 [110]. In this case, the repression mechanism is
known: YY1 binds to the co-activator CBP and prevents
the interaction of CBP and AP-1 [110]. However, quench-
ing does not refer to a particular repression mechanism
and other class I repressors can ‘quench’ in other ways. A
novel mechanism of transcriptional repression has re-
cently been described where activator-co-activator inter-
actions are disrupted but where the disruption does not
require direct interactions between the repressor and ei-
ther the activator or the co-activator. The IRF-2 oncopro-
tein is a class I repressor that represses virus-induced
transcription of the interferon (IFN)-
b
gene. IRF2 re-
presses transcription by binding to the IFN-
b
enhancer
and inhibiting the recruitment of the co-activator CBP by
the activator IRF-1 and/or by destabilising the CBP-IRF-
1 interaction. Remarkably, repression by IRF-2 does not
rely on direct protein-protein interactions between the re-
pressor and CBP. Instead, there is a small basic domain in
IRF-2 that has been proposed to ‘repel’incoming CBP (or
alternatively to destabilise the interaction of CBP and ac-
tivator proteins such as NF
k
B, IRF-1 and ATF-2/c-Jun
present at the IFN-
b
enhancer) [111].
Post-translational modification of an activator
or co-activator
We have already described how phosphorylation and
acetylation can regulate the DNA-binding activity of ac-
tivators. However, in many cases, repressors post-transla-
tionally modify activators and thereby prevent their inter-
action with other proteins. These modifications act as
molecular switches that can rapidly regulate the tran-
scription of a set of genes. An example of a repressor that
acts in this fashion is the calcium/calmodulin-dependent
kinase II (CCK-II). This class III B repressor phosphory-
lates the activator protein CREB. Phosphorylation of
CREB inhibits the interaction of CREB with the co-acti-
vator CBP and results in the down-regulation of tran-
scription [112]. CBP itself has been shown to acetylate
the Drosophila activator protein TCF and in vitro this re-
duces the affinity of TCF for the co-activator
b-
catenin
[113]. The glycosylation and deacetylation of transcrip-
tion factors can also block activation. For example, the
glycosylation of Sp1 by OGT inhibits the interaction be-
tween Sp1 and the co-activator dTAF110 as well as in-
hibiting the self-association of Sp1 [114]. Deacetylation
of MyoD by HDAC1 has been proposed as a mechanism
for regulating MyoD activity in undifferentiated cells.
MyoD is a B-HLH protein that activates the expression of
muscle-specific genes. However, MyoD can interact with
HDAC-1 to repress transcription of these genes in undif-
ferentiated cells. HDAC1 interacts directly with MyoD
and can deacetylate MyoD in vitro [115]. Which of the in-
teractions of MyoD with the transcription machinery is
disrupted by its deacetylation is not yet known.
Post-translational modification of a co-activator protein
can also act as a molecular switch that regulates a specific
group of genes. For example, CARM1 (co-activator-as-
sociated arginine methyltransferase) represses transcrip-
tion activated by CREB by methylating CBP and block-
ing the interaction of CBP and CREB. Methylated CBP
is, however, still able to interact with transcription factors
other than CREB and to co-activate transcription in other
contexts [41]. Similarly, the co-activator protein ACTR
functions together with a number of nuclear hormone re-
ceptor proteins, including the estrogen receptor (ER) to
activate transcription, and the acetylation of ACTR by
CBP results in the repression of ER-activated transcrip-
tion [116].
Repression by the recruitment of chromatin-
remodelling factors
The eukaryotic genome is packaged into a complex pro-
tein-DNA fibre known as chromatin (fig. 4). The DNA is
first wrapped around histones H2A, H2B, H3 and H4 to
form nucleosomes. The nucleosomes are then built into
higher-order structures, the exact nature of which is still
not completely understood. At a gross level, chromatin ap-
pears to be of two types: euchromatin and heterochro-
matin. Euchromatin contains most genes that are ex-
pressed in the cell and is sometimes called decompacted
chromatin. In contrast, heterochromatin consists of re-
gions of darkly staining, highly compacted chromatin that
contains very few active genes. Heterochromatin is repli-
cated late in S phase and is commonly located at telomeres
and centromeres and at the silent (HM) mating loci in
S. cerevisiae. The repression of transcription in or by het-
erochromatin is known as gene silencing. Chromatin con-
densation patterns in general are said to be epigenetic be-
cause they are stably inherited after mitosis but do not rely
on specific DNA sequences. This means that a silenced al-
lele and its transcriptionally active counterpart may be of
identical sequence and yet their different transcriptional
states are maintained over many cell divisions [117].
The alteration of chromatin structure into a repressive
state can involve changes in nucleosome-DNA contacts
and/or inter-nucleosomal contacts. One way in which
these changes can be effected is by modifying histones.
There are several different kinds of histone-modifying
enzymes including histone acetyltransferases (HATs), hi-
stone deacetylases (HDACs), histone methyltransferases
(HMTs) and histone kinases, as well as enzymes that
ubiquitinate histones [118]. Transcriptional activation is
generally associated with the acetylation of histones,
whereas transcriptional repression is generally associated
730 K. Gaston and P. S. Jayaraman Transcriptional repression mechanisms
with their deacetylation [119]. Thus, inhibitors of
HDACs derepress many genes and silenced heterochro-
matin is generally deacetylated [120]. Histone modifica-
tions such as acetylation may directly change chromatin
structure by altering the amount of charge on the N-ter-
minal tails of histone proteins. Alternatively, the effects of
histone modifications on chromatin structure may be
mediated by chromatin-binding proteins (see below).
Changes in chromatin structure can also be effected by
proteins that alter nucleosome-DNA contacts or inter-nu-
cleosomal contacts without modifying histones. For ex-
ample, the SWI/SNF ATP-dependent remodelling com-
plex [121, 122], originally isolated from S. cerevisiae but
subsequently found to be conserved across all eukary-
otes, is involved in the repression and activation of tran-
scription [123] and there are many such ATP-dependent
remodelling complexes in yeast and higher eukaryotes
[reviewed in ref. 9]. Evidence from restriction site ac-
cessibility studies suggests that the ATP-dependent
SWI/SNF remodelling subunits from the SWI/SNF com-
plexes continuously generate multiple alternative DNA
conformations. This provides multiple opportunities for
the binding of regulatory factors to DNA to activate or
repress transcription [124].
In higher eukaryotes, cytosine bases within the dinu-
cleotide sequence CpG are often methylated and this also
CMLS, Cell. Mol. Life Sci. Vol. 60, 2003 Review Article 731
A
B
Figure 4. Repression by the recruitment of chromatin-remodelling factors. (A) A model that shows how repressors (R) might recruit chro-
matin-binding proteins (grey ovals) that oligomerise and spread along the chromatin fibre. (B) A model that shows how class I repressors
(RI) can recruit class II repressors (RII) that are components of chromatin-remodelling complexes (CRCs). CRCs bring about histone
deacetylation and histone methylation (blue dots) using HDAC and HMT activity, respectively. Histone methylation brings about the bind-
ing of chromodomain-containing proteins (CHR). CRCs can also remodel chromatin via their ATP-dependent chromatin-remodelling
activity (ACRF). CRCs might bring about CpG methylation (black dots) allowing the binding of methyl-CpG-binding proteins (MeBP),
although this is speculative.
plays an important role in the regulation of chromatin
structure and the control of gene expression. Whilst hete-
rochromatin is generally heavily methylated, CpG methy-
lation patterns in euchromatin are complex, with areas of
heavy methylation and areas of unmethylated CpG se-
quences, sometimes referred to as unmethylated CpG is-
lands. DNA methylation is carried out by DNA methyl-
transferases (DMTs) and CpG sequences are methylated
on both strands. DNA replication results in hemi-methy-
lated DNA and this is a substrate for maintenance DMTs.
Consequently, CpG methylation patterns are stably inher-
ited and, indeed, these patterns play a key role in epige-
netic phenomena such as dosage compensation and im-
printing [reviewed in ref. 125]. Many observations link
DNA methylation, histone modification and the repres-
sion of transcription. CpG methylation can often directly
block the binding of transcription activators and thereby
bring about repression [126]. In addition, CpG methyla-
tion can also block the binding of transcription factors in-
directly by changing the conformation of chromatin
and/or by recruiting methyl-CpG-binding proteins. Sev-
eral methyl-CpG-binding proteins have been described
and shown to compete with transcription factors for bind-
ing to CpG-methylated DNA. In addition, these methyl-
CpG-binding proteins can bring about changes in chro-
matin structure in part at least by recruiting HDACs
[127–129]. More recently, in the filamentous fungus
Neurospora crassa, HMT activity has been shown to be
required for CpG methylation, and thus in this organism,
DNA methylation appears to be dependent upon histone
methylation [130]. Furthermore, several repressor proteins
recruit DMTs [28, 29, 131]. The class I repressor protein
RP58, for example, binds to Dnmt3. However, the enzy-
matic activity of Dnmt3 does not appear to be required for
RP58-mediated repression. Instead, Dnmt3 interacts with
HDAC1 to bring about repression [28]. Similarly, Rb
forms a complex with Dnmt1 and HDAC1 that can re-
press E2F responsive promoters and, again, the DMT ac-
tivity of Dnmt1 does not appear to be required for repres-
sion [131]. Thus, although DMT proteins function as
co-repressors, their ability to methylate DNA does not ap-
pear to be required, at least for short-term repression. Of
course, long-term transcriptional repression might be a
different matter and the ability to methylate DNA might
be important in the maintenance of the repressed state
through multiple rounds of cell division [reviewed in
ref. 132].
Lessons from gene silencing
Three groups of chromatin-binding proteins that nega-
tively regulate transcription have been characterised in
detail: the Sir proteins, the HP1 family and the PcG pro-
teins [133, 134]. The Sir proteins are required for telom-
eric silencing and the silencing of mating type in S. cere-
visiae. These proteins can homo- and heterodimerise and
they are thought to form a highly stable transcriptionally
repressed state that spreads along the chromatin fibre. At
telomeres, the DNA-binding protein Rap1p [135] recruits
Sir3p and Sir4p [136] and the yeast telomere end-binding
complex Ku70/80 helps in this process [137, 138]. At the
mating type loci, the DNA-binding proteins Rap1p, ORC
and Abf1 are together responsible for the recruitment of
Sir proteins. Sir1p binds to ORC and can recruit Sir4p
[139]. Both Sir4p and Sir3p bind directly to the N-termi-
nal tails of histones [140] and Sir3p has been found to
propagate along nucleosomes [141]. Sir4p is thought to
recruit Sir2, an NAD-dependent histone deacetylase
[142] and histone deacetylation is in turn thought to
favour the assembly of Sir complexes.
The mechanisms that bring about pericentric centromeric
silencing in the fission yeast Schizosaccharomyces
pombe have been elucidated in some detail. Chromatin
immunoprecipitation assays have identified a number of
proteins that are localised to different regions of the S.
pombe centromeres including Mis6, Chp1 and Swi6.
Swi6 is a member of the HP1 family of chromatin-bind-
ing proteins [143] that also includes the Drosophila pro-
tein SU(VAR)2-5 [144] and several mammalian HP1 pro-
teins including HP1
a
, Hp1
b
and Hp1
g
[145] (reviewed in
[146]). These proteins are essential for the silencing of
pericentric heterochromatin in Drosophila and mam-
malian cells. HP1 proteins are involved in a phenomenon
known as position effect variegation (PEV). This refers to
the silencing of genes that are placed adjacent to regions
of pericentric heterochromatin (reviewed in [147]).
Swi6/HP1 can coat and mediate the silencing of large
noncentromeric DNA inserts within centromeric DNA
[148]. The HP1 proteins bind to histones [149] and bind
with high affinity to methylated histones using a domain
known as a chromodomain [150, 151]. They can also self-
associate using a domain known as a chromoshadow do-
main [149]. In S. pombe, the HDACs Clr6 and Clr3 are
thought to remove acetyl groups from the N-terminal tail
of histone H3 at lysine 9 and lysine 14, respectively. The
HMT Clr4 can then methylate histone H3 on lysine 9 cre-
ating a high-affinity binding site for Swi6/HP1 [152].
Since Swi6/HP1 proteins can self-associate, bind to his-
tones, and silence genes placed within centromeric DNA
and adjacent to pericentric chromatin, it seems likely that
these proteins silence by ‘spreading’ along the chromatin
fibre in a manner analogous to the Sir proteins. Recent
experiments suggest that RNA interference is involved in
gene silencing in S. pombe [153–155]. In cells, double-
stranded RNAs are processed into small interfering
RNAs (siRNAs) that target mRNA transcripts with the
same sequence for degradation. S. pombe mutants defec-
tive in siRNA processing also show a loss of methylation
at histone H3 lysine 9 and loss of binding of Swi6 [155].
Proteins involved in siRNA pathways will likely also play
732 K. Gaston and P. S. Jayaraman Transcriptional repression mechanisms
an important role in the establishment and maintenance
of heterochromatin in other species.
Like the HP1 proteins, PcG proteins contain chromod-
omains suggesting that these proteins also bind to methy-
lated histones [146, 156]. PcG proteins are found as
homo- and hetero-oligomers in complexes that are asso-
ciated with the maintenance of transcriptionally inactive
homeotic gene complexes in Drosophila [157–159] and
implicated in the control of the cell cycle and oncogene-
sis in humans [160, 161]. The PcG complexes appear to
retain a molecular memory of the early state of activity of
a gene complex such that once a gene has been silenced,
it remains silent even after many rounds of cell division.
PcG proteins use several mechanisms to repress tran-
scription [reviewed in refs 162, 163]. In vitro studies with
purified hPRC1 demonstrate that this human PcG com-
plex inhibits chromatin remodelling. hPRC1 somehow
alters the regulated locus such that it excludes SWI/SNF
remodelling complexes. However, the DNA is still open
to attack by micrococcal nuclease, suggesting that al-
though PcG proteins exclude remodelling factors, the
regulated locus is still accessible to other proteins [158,
164]. This finding is broadly in agreement with the fact
that PcG complexes and GTFs can co-occupy promoters
and that PcG complexes can also repress transcription by
interacting with the basal transcription machinery [76,
77]. PcG complexes could also bring about the modifica-
tion of histones, as some PcG proteins can recruit HDACs
[165]. Alternatively, or in addition, the ability of PcG pro-
teins to homo- and hetero-oligomerise suggests that PcG
proteins could form ‘spreading’ complexes [149].
In the examples given above, chromatin-binding proteins
from the SIR and PcG complexes act at specific sites be-
cause they are able to associate with DNA-binding pro-
teins. The Sir proteins are recruited to DNA by DNA-
binding complexes that include the Rap proteins. Several
gene-specific repressors are thought to repress transcrip-
tion by recruiting the PcG chromatin-binding proteins
(fig. 4A). Repressors that recruit PcG complexes include
RYBP, a class I repressor, and CtBP, a class II repressor.
Repression by PcG proteins involves interactions be-
tween distal control elements (PREs) and promoters
[166, 167]. RYBP binds to both YY1 and PcG proteins
and the interaction between RYBP and YY1 is thought to
be instrumental in targeting PcG complexes formed at
distal PREs to RYBP-dependent promoters to bring about
promoter-specific repression [168]. Like RYBP, the class
II repressor CtBP can recruit PcG proteins. CtBP recruits
HPC2 at some promoters [169] and is found in PcG com-
plexes with the Rb protein [8]. In contrast to these gene-
specific repressor proteins that recruit chromatin-binding
proteins, some repressor proteins are themselves chro-
matin-binding proteins. For example, the class II repres-
sor Tup1 can position nucleosomes at the STE6 gene
[170]. Tup1 is recruited to promoters by several class I re-
pressor proteins. The interaction between Tup1 and the
class I repressor proteins is not direct, but is mediated by
the Tup1-interacting protein Ssn6 [reviewed in ref. 171].
Tup1 and Groucho can bind directly to deacetylated his-
tones H3 and H4 [172–174]. Tup1 has been suggested to
repress transcription by interacting with hypoacetylated
histones located close to the promoter [172]. Several ob-
servations have led to the proposal that Tup1/Groucho
proteins bring about long-range repression by spreading
along the chromatin fibre and recruiting HDACs in a
manner similar to that observed for the Sir silencing com-
plexes [reviewed in refs 18, 19]. First, members of the
Groucho family of proteins allow the repression of pro-
moters in a distance- and orientation-independent manner
(long-range repression) [16, 19]. Second, chromatin im-
munoprecipitation assays with Tup1 at the yeast STE6 lo-
cus have shown that Tup1 is associated with the entire ge-
nomic STE6 coding region [175]. Third, Groucho and
human TLE/Groucho proteins assemble into large
oligomeric structures [173, 176, 177]. Finally, both Tup1
and Groucho proteins interact with histone deacetylases
[178–180] (see below). The formation of spreading com-
plexes by Tup1 is, however, still the subject of some con-
troversy [180].
Chromatin-remodelling complexes
Many proteins involved in chromatin remodelling appear
to be components of large multi-functional complexes,
such as the NuRD and SIN complexes. Recruitment of any
of the components of these complexes by a repressor is
likely to result in the recruitment of all the proteins in ei-
ther complex to the promoter (fig. 4B). The mammalian
SIN complex contains chromatin-binding proteins, his-
tone-modifying enzymes, and the Sin3 class II repressor
protein. Similarly, the mammalian NuRD chromatin-re-
modelling complex contains chromatin-binding proteins,
histone-modifying enzymes and ATP-dependent remodel-
ling activities, and MBD3, a protein that contains a
methyl-CpG-binding domain [129]. MBD3 does not bind
directly to CpG-methylated DNA but instead associates
with two components of NuRD, HDAC-1 and MTA2
[181] and also interacts with the methyl-CpG-binding pro-
tein MBD2 [29]. Recently, the MeCP1 complex was puri-
fied and found to contain both MBD2 and NuRD; this
complex preferentially binds, remodels and deacetylates
methylated nucleosomes [182]. Thus the NuRD complex
also has the potential to bind methylated DNA.
A number of class I repressor proteins recruit NuRD and
SIN to promoters by binding to class II repressors associ-
ated with these complexes. Examples of repressor pro-
teins that recruit the SIN complex are the Mad/Max het-
erodimer, which requires the class II repressor proteins
PML and c-ski for recruitment of the complex [183], and
the unliganded nuclear hormone receptors, which require
CMLS, Cell. Mol. Life Sci. Vol. 60, 2003 Review Article 733
the presence of the class II repressor protein NcoR/SMRT
[reviewed in ref. 184]. Similarly, NuRD can be recruited
by the class I repressors Hunchback, Ikaros, Aiolos and
Tramtrack 69 (Tk69). These repressors interact directly
with Mi-2, the ATP-dependent remodelling subunit of
NuRD, which also contains chromatin-binding motifs
[185–187].
As might be expected from our knowledge of the forma-
tion of silenced heterochromatin, repressor proteins that
function within euchromatin also appear to recruit both
HMTs and HDACs to bring about repression. For exam-
ple, Rb interacts directly with both HDAC1 and the HMT
Su(Var)39H1 and both histone deacetylation and histone
methylation contribute to Rb-mediated repression [3, 4,
6]. Histone methylation by Su(Var)39H1 results in the for-
mation of a high-affinity binding site for HP1. As yet, the
precise function of HP1 in repression by Rb in euchro-
matin is not known [5]; however, it is likely to be involved
in the initiation and propagation of inactive chromatin. In
contrast, E2F-6 is a class I repressor protein that represses
transcription, independently of Rb, by recruiting a com-
plex that contains HMT activity but which does not appear
to contain HDAC activity [188]. This could be an example
of a repressive complex where the formation of inactive
chromatin occurs in the absence of histone deacetylation.
E2F-6 is a member of the E2F family of transcription fac-
tors but lacks the Rb-interacting domain and transcription
activation regions present in the other family members. A
complex known as E2F-6.com is present at the promoters
of E2F-dependent genes when there is no requirement for
transcription, for example, when cells are in the quiescent
state. E2F-6.com contains chromatin-binding proteins and
HMT activity, as well as DP-1 (the heterodimeric binding
partner for E2F) and two other DNA-binding proteins,
Mga and Max. Presumably, the local methylation of his-
tones by the HMT in E2F6.com allows the binding of
chromodomain proteins such as HP1 and PcG proteins,
and this nucleates the formation of complexes that propa-
gate inactive chromatin [188].
Somewhat surprisingly, relatively few class I repressors
appear to interact directly with HDACs. Two class I re-
pressors that do are YY1 and MeCP2. YY1 can both acti-
vate and repress transcription and this protein interacts
directly with HDAC1 [189]. Interestingly, YY1 is related
to Pleiohomeotic, a PcG protein from Drosophila [190].
MeCP2 is a methyl-CpG-binding protein that can interact
directly with HDAC-1 [127, 128]. In contrast with class I
repressors, most, if not all class II repressor proteins in-
teract with HDACs directly and recruit them to the pro-
moter. Rb [3, 4], TGIF [44], CtBP [191, 192] and
Tup1/Groucho [178, 179], for example, all interact with
HDAC s from the Rpd3 (HDAB/class I) group of
HDACs. Both Tup1/Groucho and CtBP also interact with
HDACs from the HDAA/class II group. Tup1 interacts
with HDA1 [180] and CtBP with HDAC5 [193]. Not yet
clear is what differences with regard to transcriptional re-
pression result from recruiting members of these differ-
ent groups of deacetylases. However, recruitment of
HDA1 (HDAA group) by Tup1 has been demonstrated to
lead to the specific deacetylation of histones H3 and
H2AB within the ENA1 promoter [180]. In contrast, re-
cruitment of Rpd3 (HDAB group) by Tup1 at the ENA1
locus leads to deacetylation of all four histones located
within the ENA1 coding sequence. This suggests that
Rpd3 could be important for a more global deacetylation
of a chromatin domain [180].
There are a number of examples where gene-specific
transcriptional repression is brought about by the interac-
tion of activator proteins with chromatin-remodelling
factors. For example, the direct interaction of NF
k
B with
HDAC1 and the indirect interaction of NF
k
B with
HDAC2 through HDAC1 can bring about the repression
of NF
k
B-regulated genes. This is believed to be impor-
tant because although most NF
k
B is held in the cyto-
plasm by I
k
B, some NF
k
B is present in the nucleus even
when the transcription of NF
k
B-activated genes is not re-
quired. Inhibition of HDAC enzymatic activity using the
inhibitor Trichostatin A (TSA) prevents repression by
NF
k
B and leads to the acetylation of histones near the
regulated promoter. Repression in this case is proposed to
have two components. First, the interactions between the
activator and the HDACs cause a local chromatin organi-
sation that decreases basal transcription. Second, the in-
teraction of deacetylases with the activator ‘passively’
prevents protein-protein interactions between the activa-
tor and chromatin re-organising proteins that have a pos-
itive role in transcription [194]. Similarly, under some
conditions, repression of promoters activated by the
SMAD2/SMAD4/SMAD3 complex and the Fast activa-
tor can be brought about by recruitment of HDAC1 to the
promoter. In this case, the interaction of HDAC-1 with
the activator is indirect and occurs via the class II repres-
sor TGIF [35, 44].
Finally, and as mentioned earlier, some repressors recruit
ATP-dependent chromatin-remodelling proteins. Rb, for
example, interacts with hBrm/BRG1, the ATP-dependent
remodelling subunit from the human SWI/SNF complex.
Although the human SWI/SNF complex is generally as-
sociated with the activation of transcription, Rb can in-
teract with hBrm and simultaneously with HDAC1, to re-
press transcription of the G1 phase-transcribed cyclin E
gene. The Rb-hBrm complex is also able to repress tran-
scription of the S phase-transcribed cyclin A and cdc2
genes apparently in the absence of HDAC1, suggesting
that in this case, ATP-dependent chromatin remodelling
may be sufficient for repression [7]. Recently, CtBP and
the PcG proteins HPC2 and Ring1 were also demon-
strated to be present in the complex containing Rb at the
cyclin A promoter. This complex, in association with
hSWI/SNF activity, is required for the repression of cy-
734 K. Gaston and P. S. Jayaraman Transcriptional repression mechanisms
clin A promoter activity [8]. Thus, this appears to be a
case where ATP-dependent nucleosome-remodelling pro-
teins and PcG proteins co-operate to repress transcrip-
tion, possibly in the absence of histone-modifying en-
zymes. The transcription factor Ikaros [195] co-localises
with HP1 in heterochromatin [196] and can also interact
with Mi-2, the ATP-dependent remodelling subunit of
NuRD, HDACs [186], CtBP [192] and with other pro-
teins in the Ikaros family [197]. Interestingly, in cells,
Ikaros-repressed genes are physically located with cen-
tromeres in a heterochromatin environment. However,
when these genes are transcribed they are located within
euchromatin [196, 198]. Ikaros-binding sites are found
both at the promoters of repressed genes and within peri-
centric heterochromatin. Thus Ikaros is thought to bring
about repression by moving genes that are to be silenced
near to regions of heterochromatin and/or by recruiting
silencing complexes to the promoter [198]. Thus, in this
case, nuclear organisation appears to be important for the
regulation of transcription [199].
Nuclear compartmentalisation
Nuclear organisation or compartmentalisation appears to
regulate gene expression and hence transcription [re-
viewed in ref. 200]. The nuclear periphery of budding
yeast is a compartment associated with gene silencing.
The silenced telomeres of yeast are often localised to the
nuclear periphery and are clustered into foci [201]. More-
over, tethering of genes to the nuclear membrane leads to
silencing [202]. However, there are also compartments
present in the nuclear periphery which are associated
with inhibiting the spread of repressive heterochromatin.
These compartments contain proteins that bind a central
protein of the nuclear pore complex, Nup2p, and that can
also bind DNA elements within boundary/barrier regions
(regions of DNA that block the spread of heterochro-
matin). When these proteins are tethered to specific DNA
elements within the boundary region and to the nuclear
pore, the spread of repressive heterochromatin is blocked
[203]. Another nuclear compartment associated with the
regulation of transcription is the scaffold/matrix attach-
ment region (S/MARs). MARs are chromatin regions that
bind the nuclear matrix. The attachment of DNA to the
nuclear matrix via MARs and interaction with MAR-
binding proteins have been shown to alter chromatin con-
formation, promote an extended domain of histone acety-
lation, and thus influence transcription [204, 205]. One
way that MARs may be able to direct the acetylation of an
extended chromatin domain is by the recruitment of the
co-activator CBP/p300. CBP/p300 binds to the MAR-
binding protein scaffold attachment factor A (SAF-A), a
major constituent of the nuclear matrix. Both p300 and
SAF-A bind to MAR elements in the transiently silent
topoisomerase I gene before its activation at G1 during
the cell cycle [206]. This binding is accompanied by the
acetylation of nucleosomes. Thus, the binding of MAR
sequences by SAF-A and p300 likely results in activation
via the initiation of acetylation within a chromatin do-
main [206]. Proteins that interfere in this process would
be expected to bring about repression. The Bright tran-
scription activator and nuclear matrix protein binds to
MARs flanking the immunoglobulin heavy chain intronic
enhancer (Emu). The Cux/CDP homeodomain protein is
a repressor that competes with Bright for binding to these
MAR sequences. In this way, Cux/CDP can eliminate the
activatory effect of the MAR on transcription [207]. In
summary, nuclear compartmentalisation is an important
factor influencing gene regulation. The proteins that alter
gene activity in this context ultimately appear to exert
their effects on transcription by influencing chromatin or-
ganisation.
Conclusions
The rich variety of repression mechanisms that have been
documented suggest that the proteins that repress tran-
scription may be structurally equally diverse. Indeed,
identifying a single feature common to all repressor pro-
teins is impossible. Rather, similar repression motifs will
more likely be found amongst proteins that share a com-
mon mechanism. For example, many repressors that in-
hibit basal transcription by interaction with the GTFs ap-
pear to contain alanine/proline-rich motifs, and hy-
drophobicity is thought to be an important characteristic
for this type of repressor. Similarly, repressors that func-
tion by binding to class II repressor proteins such as
Groucho contain specific protein-protein interaction mo-
tifs. As more repressors are characterised in detail, these
motifs should become increasingly obvious and the mode
of action of each repressor should be more easily pre-
dicted.
In this review we have considered enzymes that post-
translationally modify DNA-binding proteins, chro-
matin-binding proteins or components of the PIC as re-
pressors. Clearly, many of these proteins are also the end
players of signal transduction pathways. The post-transla-
tional modification of proteins involved in gene regula-
tion allows the rapid switching of transcriptional states.
This is particularly important during short-term changes
in gene expression such as those seen in response to
growth factor stimulation. The modification of histones
and other proteins involved in gene regulation may be
ideally suited to this type of gene regulation. However,
long-term gene regulation, such as the repression of
genes during cell differentiation, may call for a different
approach that includes histone and DNA modifications
and the association of chromatin-binding proteins. The
association of Rb, CtBP and the PcG proteins at some
CMLS, Cell. Mol. Life Sci. Vol. 60, 2003 Review Article 735
E2F-dependent promoters has been suggested as a link
between the long-term inhibition of cell proliferation and
the differentiation events that lead to embryonic pattern
formation [8]. Moreover, complexes containing both HP1
and PcG proteins are found at E2F-dependent promoters
in cells that are quiescent.
There are many future challenges in understanding gene
regulation and transcriptional repression. However, un-
derstanding the biochemical nature of chromatin fibres
and their role in the control of gene expression must rep-
resent a key objective. The role that DNA methylation
plays in the structure of heterochromatin and a detailed
understanding of the biochemical nature of heterochro-
matin and euchromatin is still lacking. Furthermore, al-
though short-range repression is relatively well under-
stood, for example when a repressor makes direct contact
with a specific activator or its target, how long-range re-
pression mediated by large assemblies of chromatin-
binding proteins is brought about is still unclear. For ex-
ample, we do not yet know why the recruitment of Grou-
cho tends to bring about long-range repression whereas
the recruitment of CtBP tends to bring about short-range
repression, even though both proteins recruit HDACs.
One possibility is that Groucho oligomerises and spreads
along the chromatin fibre causing the expansion of a re-
pressive domain similar to that seen in silenced hete-
rochromatin (fig. 4B). Although CtBP recruits PcG pro-
teins, perhaps these proteins form complexes that do not
so closely resemble silenced heterochromatin, thus re-
sulting in the repression of smaller domains or in repres-
sion that is more easily reversed. Or perhaps PcG com-
plexes repress transcription in these situations predomi-
nantly by contacting the basal machinery. Lessons
learned from the study of gene silencing and position ef-
fect variegation, areas of research once regarded by some
as esoteric, are sure to continue to throw light upon these
questions.
A question that is related to an understanding of the nature
of chromatin is whether genes travel to specific areas in
the nucleus to be repressed or whether they need to be ex-
cluded from some areas in order to remain active. Recent
work has shown that in yeast cells, the tethering of genes
to the nuclear pore complex prevents the spreading of het-
erochromatin and the consequent gene silencing [203].
Conversely, genes repressed by Ikaros appear to be lo-
calised near to heterochromatin. Repression factories,
analogous to the transcription factories that are believed to
contain groups of transcriptionally active genes, may exist
or most genes may be repressed in situ. The main advan-
tage of a repression factory would be that repressors, and
perhaps more importantly co-repressors, could stay with
the RNA polymerase and repress incoming genes as re-
quired. The advantage of repressing genes individually
could be that stray RNA polymerases would be unable to
transcribe genes that should be repressed. A deeper un-
derstanding of the nature of transcription factories and the
ultrastructure of the nucleus itself should help to establish
the relative importance of these modes of repression.
Acknowledgements. We would like to thank to Dr. S. Roberts for
helpful comments on the manuscript. P.-S. J. is grateful to the MRC
for a Career Development Award.
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