Latchman. Eukariotic Transciption Factors
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182 EUKARYOTIC TRANSCRIPTION FACTORS
CHAPTER 6
REPRESSION OF GENE EXPRESSION
BY TRANSCRIPTION FACTORS
6.1 REPRESSION OF TRANSCRIPTION
Although the majority of transcription factors that have so far been described
act in a positive manner, a number of cases have now been reported in which
a transcription factor exerts an inhibitory effect on transcription initiation.
This effect can occur by indirect repression, in which the repressor interferes
with the action of an activating factor so preventing it stimulating transcrip-
tion (Fig. 6.1a–d). Alternatively, it can occur via direct repression in which the
factor reduces the activity of the basal transcriptional complex (Fig. 6.1e).
These two mechanisms will be discussed in sections 6.2 and 6.3 respectively.
Other targets for transcriptional repression, such as the alteration of chroma-
tin structure or the inhibition of transcriptional elongation, are discussed in
section 6.4 (for reviews of transcriptional repression see Hanna-Rose and
Hansen, 1996; Latchman, 1996; Maldonado et al., 1999; Courey and Jia, 2001).
6.2 INDIRECT REPRESSION
Several mechanisms exist by which an inhibitor can interfere with the action
of an activator and these will be discussed in turn.
6.2.1 INHIBITION OF ACTIVATOR BINDIN G BY MASKI NG OF ITS
DNA BINDING SITE
One means by which repression can occur is by the masking of the DNA
binding site for the factor so preventing it binding to the DNA and activating
transcription. By preventing the binding of the positively acting factor, the
negatively acting factor effectiv ely inhibit s gene activation. This masking of
the binding site can be achieved simply by the negatively acting factor binding
to the same site as the positively acting factor but failing to activate transcrip-
tion (Fig. 6.1a). This is seen for example in the case of the Sp3 factor, a factor
related to the Sp1 factor . Thus the Sp3 factor binds to the same Sp1 binding
site as Sp1 itself (see Chapter 1, section 1.3.2) but unlike Sp1 it cannot activate
transcription. It therefore blocks the Sp1 binding site, preventing Sp1 binding
and activatin g transcription (for review see Lania et al., 1997).
A similar example is seen in the case of the homeobox proteins, discussed
in Chapter 4 (section 4.2), the proteins engrailed (eng), fushi-tarazu (Ftz),
paired (prd) and zerknult (zen) can all bind to the sequence
TCAATTAAAT (Hoey and Levine, 1988). When plasmid s expressing each
of these genes are co-transfected with a target promoter carrying multiple
copies of this binding site, the Ftz, prd and zen proteins can activate transcrip-
tion of the target promoter (Jaynes and O’Farrell, 1988; Han et al., 1989). In
contrast, the eng protein has no effect on the transcription of such a
184 EUKARYOTIC TRANSCRIPTION FACTORS
Figure 6.1
Potential mechanisms by
which a transcription
factor can repress gene
expression. This can
occur by the repressor
(R) binding to DNA and
preventing an activator
(A) from binding and
activating gene
expression (a), by the
repressor interacting with
the activator in solution
and preventing its DNA
binding (b), by the
repressor binding to DNA
with the activator and
neutralizing its ability to
activate gene expression
(c), by the repressor
promoting degradation of
the activator (d) or by
direct repression by an
inhibitory transcription
factor (e).
promoter. It does, however, interfere with the ability of the activating proteins
to induce transcription, presumably by blocking the binding of the activating
factor. Thus, for example, while Ftz can stimulate the target promoter when co-
transfected with it, it cannot do so in the presence of eng since eng prevents
binding of Ftz to its binding site (Jaynes and O’Farrell, 1988). Hence, the
expression of Ftz alone in a cell would activate particular genes whereas its
expression in a cell also expressing engrailed would not have any effect
(Fig. 6.2).
Hence, both Sp3 and eng act purely as transcriptional repressors by block-
ing binding of activators to their binding sites. Interestingly, the glucocorti-
coid receptor can also repress transcription of specific genes in this way, even
though, as discussed in Chapter 4 (section 4.4), it acts primarily as an activator
of target gene expression. Thus, treatment with glucocorticoid and activation
of the glucocorticoid receptor inhibits, for example, expression of the genes
encoding bovine prolactin and human pro-opiomelanocortin. The inhibitory
effect observed in these cases is mediated by binding to DNA of the identical
receptor/hormone complex which activates glucocorticoid-inducible genes
(see Chapter 4, section 4.4 and Chapter 8, section 8.2.2) and these genes
are therefore repressed by glucocorticoid. However, the DNA sequence ele-
ment to which the complex binds when mediating its negative effect (nGRE) is
distinct from the glucocorticoid response element (GRE) to which it binds
when inducing gene expression, although the two are related (Fig. 6.3).
REPRESSION OF GENE EXPRESSION BY TRANSCRIPTION FACTORS 185
Figure 6.2
Blockage of gene
induction by Ftz in cells
expressing the engrailed
(Eng) protein which binds
to the same sequence as
Ftz but does not activate
transcription.
Figure 6.3
Relationship of the sites
in DNA which mediate
gene activation or
repression by binding the
glucocorticoid receptor.
Note that the sites are
related but distinct.
This has led to the suggestion that the sequence difference causes the
receptor/hormone complex to bind to the nGRE in a configuration in
which its activation domain cannot interact with other transcription factors
to activate transcription as occ urs following binding to the positive element
(Fig. 6.4). In agreement with this idea, the glucocorticoid receptor has been
shown to bind to the nGRE in the POMC gene as a trimer rather than the
dimer form which binds to the GRE and stimulates transcription (for review
see Latchman, 2001). The receptor bound in this configuration to the nega-
tive element apparently acts by preventing binding of a positive acting factor
to this or an adjacent site, thereby preventing gene induction. In agreement
with this idea, the nGRE in the human glycoprotein hormone alpha subunit
gene, which overlap s a cyclic AMP response element (CRE), is only able to
inhibit gene expression when the CRE is left intact. Hence, it is likely that
receptor bound at the negative element prevents binding of a transcriptional
activator to the CRE and thereby inhibits gene expression (Fig. 6.5).
186 EUKARYOTIC TRANSCRIPTION FACTORS
Figure 6.4
Consequences of
glucocorticoid receptor
binding to the DNA
binding sites which
mediate gene activation
(GRE) or repression
(nGRE). Note that the
receptor is likely to bind
in a different
configuration to the two
different sequences
resulting in its ability to
activate transcription
only following binding to
the GRE.
Figure 6.5
Inhibition of gene
expression by
glucocorticoid receptor
binding to an nGRE is
likely to be mediated by
preventing the binding of
a positively acting
activator protein (A) to a
site adjacent to or
overlapping the nGRE.
Hence the inhibition of DNA binding by a specific activator via masking of
its binding site is a major method of transcriptional inhibition. This can
involve either factors such as Sp3 or eng, which function only as transcrip-
tional inhibitors, as well as factors such as the glu cocorticoid receptor which
either repress via this mechanism or activate transcription depending on the
nature of their DNA binding site in a specific target gene.
6.2.2 INHIBITION OF ACTIVATOR BINDIN G BY FORMATION O F A
NON-DNA BINDING COMPLEX
As well as preventing activator binding to DNA via masking its binding site, an
inhibitor can also inhibit transcription via the formation of a non-DNA bind-
ing complex with an activating factor (see Fig. 6.1b). Thus, as discussed in
Chapter 7 (section 7.2.1 ), the MyoD transcription factor is specifically
expressed in skeletal muscle cells and plays a key role in activating skeletal
muscle genes. Its expression can be indu ced in the 10T‰ fibroblast cell line by
treatment with 5-azacytidin e and this converts the fibroblast cells into muscle
cell precursors, known as myoblasts. However, ac tivation of muscle-specific
genes and the production of differentiated myotubes require these cells to be
incubated in the absence of serum (Fig. 6.6). Paradoxically, however, MyoD
levels do not change in this transition from myoblast to myotubes and yet
MyoD-dependent muscle specific genes are activated.
The explanation of this paradox was provided by the identification of the
Id protein (Benezra et al., 1990) which, like MyoD, contains a helix-loop-helix
motif but lacks the basic domain m ediating DNA binding (see Chapter 4,
section 4.5 for a discussion of these motifs). Because the helix-loop-helix
motif mediates dimerization of proteins containing it, Id can dimerize with
other helix-loop-helix proteins such as MyoD and inhibit their DNA binding
since the resulting heterodimer lacks the necessary pair of DNA binding
motifs (Fig. 6.7). When 10T‰-derived myoblasts are induced to form myo-
tubes, Id levels dec line indicating that this second stage of myogenesis is
mediated by a decline in the inhibitory protein rather than an increase in
the activator, MyoD.
The role of inhibitory helix-loop-helix proteins is not confined to myogen-
esis. Thus, the level of Id also declines during differentiation of early embryo-
nic cells and erythroid cells. Moreover, inactivation of Id proteins in knock
out mice results in abnormal development of the brain and the blood vessels
providing direct evidence for the key role of pr oteins of this type in develop-
ment (Lyden et al., 1999; for review see Carmeliet, 1999). Similarly, the pro-
duct of the emc gene, which regulates neurogenesis in Drosophila , also contains
a helix-loop-helix motif and lacks a basic DNA binding domain (for review
REPRESSION OF GENE EXPRESSION BY TRANSCRIPTION FACTORS 187
188 EUKARYOTIC TRANSCRIPTION FACTORS
Figure 6.6
Differentiation of 10T½
cells into myoblasts by 5-
azacytidine and then into
myotubes by removal of
growth factors. Note that
the MyoD-dependent
induction of genes
encoding terminal
differentiation markers
such as creatine kinase
(CK), which occurs in
myotubes, occurs without
an increase in MyoD
concentration.
Figure 6.7
Dimerization of functional
basic helix-loop-helix
proteins (bHLH) with Id.
Note that while Id can
dimerize with other
proteins via the helix-
loop-helix domain, it lacks
the basic DNA domain
and hence the Id-
containing heterodimer
cannot bind to DNA.
see Jones, 1990. Hence, this form of repression is not confined to mammalian
cells.
6.2.3 QUENCHING OF AN ACTIVATOR
The cases of repressi on described so far all involve the inhibition of DNA
binding either by blocking the binding site for a factor (see Fig. 6.1a) or
by forming a non-DNA binding protein–protein complex (see Fig. 6.1b).
Since DNA binding is a necessary prerequisite for gene activation, this con-
stitutes an effective form of repression. In addition, however, inhibition of
transcription can also be achieved by interfering with transcriptional activa-
tion by a DNA bound factor in a phenomenon known as quenching (see
Fig. 6.1c).
A simple exam ple of this type is seen in the case of the negatively acting
yeast factor GAL80 which inhibits gene activation by the positively acting
GAL4 protein . This is achieved by the binding of GAL80 to DNA-bound
GAL4, such binding occ urring via the thirty amino acids located at the
extreme C terminus of the GAL4 molecule. As these amino acids are located
close to the GAL4 activation domain, the binding of GAL80 to GAL4 masks
the GAL4 activation domain and hence inhibits the activation of gene expres-
sion by GAL4. In response to treatment with galactose, GAL80 dissociates
from GAL4 allowing GAL4 to fulfill its function of activating galactose indu-
cible genes. Hence this system provides an elegant means of modulating gene
expression in response to galactose with the activating GAL4 factor being
bound to DNA both prior to and after galactose addition but being able to
activate gene expression only following the galactose-induced dissociation of
the quenching GAL80 factor (Fig. 6.8). Similar cases involving repression
of p53 by MDM2 and E2F by Rb-1 both of which involve, at least in part,
masking of the activation domain are discussed in Chapter 9 (sections 9.4.2
and 9.4.3).
A related example of quenching, in which the inhibitory factor binds to a
DNA sequence adjacent to the quenched factor rather than only to the fact or
itself, is seen in the case of the c-myc promoter. Thus an inhibitory transcrip-
tion factor myc-PRF binds to a site adjacent to that occupied by an activating
factor myc-CF1 and interferes with its ability to activate c-myc gene transcrip-
tion (Kakkis et al., 1989). Hence quenching can occur either by an inhibitory
factor binding to the positively acting factor (Fig. 6.9a) or by the inhibitory
factor binding to DNA adjacent to the positive factor (Fig. 6.9b). In both cases,
however, this effect involves the inhibitor interfering with the ability of the
activator’s activation domain to stimulate transcription.
REPRESSION OF GENE EXPRESSION BY TRANSCRIPTION FACTORS 189
6.2.4 DEGRADATION OF THE ACTIVATOR
As well as interfering functionally with the action of the activator by prevent-
ing its DNA binding or q uenching its activation domain, an indirect repressor
can act by targeting an activator for degradation (see Fig. 6.1d). This is seen in
the case of MDM2, which as well as quenching the activation domain of p53
(see abo ve), also targets it for degradation so using multiple mechanisms to
prevent p53 activating transcription (see Chapter 9, section 9.4.2).
In the case of p53, MDM2 is likely to do this by stimulating the recognition
of p53 by protease enzymes which are pr esent in the cell (Fig. 6.10a).
However, the AEBP1 transcription factor, which regulates adipocyte differen-
190 EUKARYOTIC TRANSCRIPTION FACTORS
Figure 6.8
Galactose activates gene
expression by removing
the GAL80 protein from
DNA-bound GAL4
protein, unmasking the
activation domain on
GAL4.
Figure 6.9
The ability of a bound
activator (A) to stimulate
transcription via its
activation domain
(hatched) can be inhibited
by quenching of the
activation domain by
inhibitory factors (R),
which either bind to the
activator without binding
to DNA (a) or which bind
to a site adjacent to the
activator (b).