Latchman. Eukariotic Transciption Factors
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In view of its metabolic efficiency, it is not surprising therefore that the
regulation of their synthesis is widely used to control the activity of the factors
which mediate cell type-specific gene regulation where differences in the
activity of a given factor in different cell types are maintained for long periods
of time. Similarly, alternative splicing or use of different translational initia-
tion codons is used to produce different forms of the same factor which often
have antagonistic effects on gene expression.
The regulation of factor activity by regulating its synthesis, does suffer,
however, from the defect that a change in the level of activity of a factor
which is controlled purely by a change in its actual amount can take some
time to occur. Thus in response to a signal which induces new transcription of
the gene encod ing a particular factor, it is necessary to go through all the
stages illustrated in Figure 7.21, before the product ion of active factor which
is capable of activating the expression of other genes in response to the
inducing signal. It is not surprising therefore that although some factors
such as GCN4 which mediate inducible gene express ion are regulated by
the regulation of their synthesis, the majority of such factors are regulated
by post-translational mechanisms which activate pre-existing transcription fac-
tor protein in response to the inducing signal. Thus, althoug h mechanisms of
this type are metabolically expensive in that they require the synthesis of the
factor in situations where it is not required, they have the necessary rapid
response time required for the regulation of inducible gene expression.
Moreover, unlike transcriptional regulation, they constitute an independent
method of gene regulation rather than requiring the activation of other tran-
scription factors in order to activate the transcription of the gene encoding
the factor itself. The regulation of transcription factor activity and the manner
in which it is achieved is discussed in the next chapter.
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244 EUKARYOTIC TRANSCRIPTION FACTORS
CHAPTER 8
REGULATION OF TRANSCRIPTION
FACTOR ACTIVITY
8.1 EVIDENCE FOR THE REGULATED ACTIVITY OF
TRANSCRIPTION FACTORS
In a number of cases, it has been shown that a particular transcription factor
pre-exists in an inactive form prior to its activation and the consequent switch-
ing on of the genes that depend on it for their activity. Thus, as discussed in
Chapter 5 (section 5.5.1) and in section 8.3.1 of this chapter, the activation of
heat-inducible genes by elevated temp erature is dependent on the activity of
the heat shock transcription factor (HSF). However, this induction can be
achieved in the presence of the protein synthesis inhibitor cycloheximide
(Zimarino and Wu, 1987; for review see Morimoto, 1998). Hence this process
cannot be dependent on the synthesis of HSF in response to heat but rather
must depend on the heat-induced activation of pre-existing inactive HSF (for
further details see section 8.3.1). Similarly, as discussed in Chapter 6 (section
6.2.3), the yeast GAL4 transcription factor pre-exists in cells prior to galactose
treatment, which activates it by causing the dissociation of the inhibitory
GAL80 protein.
Although for the reasons discussed in Chapter 7 (section 7.4) the activation
of pre-existing transcription factors is predominantly used to modulate tran-
scription factors involved in controlling inducible rather than cell type-specific
gene expression, it has also been reported for factors involved in regulating
cell type-specific gene expression. Thus, the transcription factor NFB (which
is a heterodimer of two subunits p50 and p65) plays an impor tant role in the B
cell-specific expression of the immunoglobulin gene (for reviews see Foo
and Nolan, 1999; Karin and Ben-Neriah, 2000; Perkins, 2000; Dixit and Mak,
2002). However, both subunits of NFB are expressed in a wide variety of cell
types and the factor is present in an inactive form both in pre-B cells and in a
wide variety of other cell types such as T cells and HeLa cells which do not
express the immunoglobulin genes. This pre-existing form of NFBcanbe
activated by treatment of pre-B cells with substances such as lipopolysacchar-
ides. As in the case of HSF, this activation can take place in the presence of
inhibitors of protein synthesis, indicating that it does not require de novo
synthesis of NFB protein. These treatments therefore activate pre-existing
NFB and thus result in the activation of the immunoglobulin gene in pre-B
cells which do not normally express it.
Interestingly, the inactive form of NFB is widely distributed in different
cell types and can be activated in both T cells and HeLa cells by treatment with
phorbol ester. Although in these cases NFB activation does not result in
immunoglobulin light chain gene expression since the gene has not rear-
ranged and is tightly packed within inactive chromatin, it does play a role
in gene regulation. Thus the activation of NFB by agents which activate
T cells, results in the active transcription factor inducing increased expression
of cellular genes such as that encoding the interleukin-2 receptor and is also
responsible for the increased activity of the human immunodeficiency virus
promoter in activated T cells. NFB therefore plays a role not only in B-cell
specific gene activity but also in gene activity specific to activate d T cells.
Indeed further work has suggested an additional role for NFB in bone
development indicating that it plays a key role in a number of different cell
types (for review see Abu-Amer and Tondravi, 1997; Dixit and Mak, 2002).
The process in which pre-existing NFB becomes activated both during B-
cell differentiation and by agents such as phorbol esters which activate T cells
therefore allows NFB to play a dual role both in B-cell specific gene expres-
sion and in the expression of particular genes in response to T-cell activation
by various agents (Fig. 8.1). This effect would otherwise require a complex
pattern of regulation in which NFB was synthesized both in response to B-
cell maturation and to agents which activate T cells.
246 EUKARYOTIC TRANSCRIPTION FACTORS
Figure 8.1
Activation of NFB during
B-cell differentiation or by
agents such as PMA
which activate T cells
allows it to activate
expression of the
immunoglobulin chain
gene in B cells and the
interleukin 2 receptor
gene in activated T cells.
Hence modulating the activity of a transcription factor represents a rapid
and flexible means of activating a particular factor. Moreover, unlike transcrip-
tional control, such mechanisms allow a direct linkage between the inducing
stimulus and the activation of the factor rather than requiring the regulated
activity of other transcription factors which, in turn, activate transcription of
the gene encoding the regulated factor. Hence they represent a highly efficient
means of allowing specific cellular signalling pathways to produce changes in
cellular transcription factor activity and hence affect gene expression (for
reviews see Barolo and Posakony, 2002; Brivanlou and Darnell, 2002).
In the most extreme example of the linkage between signalling pathways
and transcription factors, the signalling molecule and the transcription factor
are identical. Thus, in response to microbial infection, mammalian neutro-
phils secrete the protein lactoferrin into the medium. It has been shown that
the lactoferr in protein can be taken up by other cells of the immune system. It
then enters the nucleus of the cells and binds to specific DNA sequences,
activating genes whose protein products are required for the cells to neutra-
lize the microbial infection (He and Furmanski, 1995). Hence in this case, the
signalling factor and the transcription factor are the same protein (for discus-
sion see Baeuerle, 1995). In most cases, however, the signalling molecule acts
indirectly to produce a change in the activity of a distinct transcription factor
which pre-existed within the cell in an ina ctive form prior to exposure to the
signal. Four basic means by which such mechanisms can regulate factor activ-
ity have been described (Fig. 8.2) and these will be discussed in turn.
8.2 REGULATION BY PROTEIN–LIGAND BINDING
8.2.1 EXAMPLES OF REGULATION BY LIGAND B INDING
As discussed above, one of the principal advantages of regulating the activity
of a factor in response to an inducing stimulus is that it allows a di rect inter-
action between the inducing stimulus and the activation of the factor, ensur-
ing a rapid response. The simplest method for this is for an inducing ligand to
bind to the transcription factor and alter its structure so that it becomes
activated (Fig. 8.2a).
An example of this effect is seen in the case of the ACE1 factor which
mediates the induction of the yeast metall othionein gene in response to cop-
per. In this case, the transcription factor undergoes a major conformational
change in the presence of copper whi ch converts it to an active form that is
able to bind to its appropriate binding sites in the metallothionein gene
promoter and activate transcription (Fig. 8.3; for review see Thiele, 1992).
REGULATION OF TRANSCRIPTION FACTOR ACTIVITY 247
A similar example in mammalian cells involves the DREAM transcription
factor which represses the transcription of the dynorphin gene and thereby
enhances the response to painful stimuli (for review see Costigan and Woolf,
2002). The activity of this factor is directly modulated by the level of calcium,
which binds direc tly to the DREAM protein and reduces its ability to bind to
its binding site in the dynorphin gene. Hence, the repression of the gene by
DREAM is relieved and the dynorphin gene is transcribed (for review see
Mandel and Goodman, 1999) (Fig. 8.4).
8.2.2 THE NUCLEAR RECEPTORS
A more complex example of regulation by ligand binding to a tr anscription
factor is provided by the steroid hormone receptors. These receptors are
members of the nuclear receptor or steroid-thyroid receptor family discussed
248 EUKARYOTIC TRANSCRIPTION FACTORS
Figure 8.2
Methods of activating a
transcription factor in
response to an inducing
stimulus. This can occur
by a ligand-mediated
conformational change
(a), by removal of an
inhibitory protein (b), by a
modification to the protein
such as phosphorylation
(c) or by stabilizing the
factor so that it is not
degraded (d).
in Chapter 4 (section 4.4) and mediate gene activation in response to steroids
such as glucocorticoid or oestrogen (for review see Weatherman et al., 1999).
Following identification of the steroid hormone receptors, it was very
rapidly shown that the receptors were only found associated with DNA after
hormone treatment. These early studies were subsequently confirmed by
using DNAseI footprinting on whole chromatin to show that the receptor
was only bound to the hormone response sequence following hormone treat-
ment (Becker et al., 1986). These studies were therefore consistent with a
model in which the hormone induces a conformational change in the recep-
tor activating its ability to bind to DNA and thereby activate transcription.
Subsequent studies have suggested that the situation is more complex how-
ever. Thus, altho ugh in the intact cell the receptor binds to DNA only in the
presence of the hormone, purified receptor can bind to DNA in vitro in a
REGULATION OF TRANSCRIPTION FACTOR ACTIVITY 249
Figure 8.3
Activation of the ACE1
factor in response to
copper results in
transcription of the
metallothionein gene.
Figure 8.4
In the absence of
calcium, the DREAM
repressor binds to its
response element (DRE)
in the dynorphin gene
and represses its
transcription. When
calcium is present, it
binds to the DREAM
factor and changes its
conformation so that it
does not bind to the
DRE. This relieves the
repression and allows
transcription of the
dynorphin gene.
band shift or footprinting assay regardless of whether hormone is present or
not (Wilmann and Beato, 1986; Figs 8.5 and 8.6).
This discrepancy led to the suggestion that the receptor is inherently cap-
able of binding to DNA but is prevented from doing so in the absence of
steroid because it is anchored to another protein. The hormone acts to release
it from this association and allow it to fulfill its inherent ability to bind to
DNA. In agreement with this possibility, in the absence of hormone, the
glucocorticoid receptor protein is found in the cytoplasm complexed to a
90 000 molecular weight heat-inducible protein (hsp90) in an 8S complex.
250 EUKARYOTIC TRANSCRIPTION FACTORS
Figure 8.5
DNAseI footprint analysis of the binding of the
glucocorticoid receptor to the glucocorticoid-
inducible mouse mammary tumour virus long
terminal repeat promoter (MMTV-LTR). In
tracks I and II the DNAseI digestion has been
carried out without any added receptor. In
tracks 1–3, glucocorticoid receptor has been
added prior to DNAseI digestion either alone
(track 1+), with the glucocorticoid hormone
corticosterone (track 2) or with the anti-
hormone RU486 which inhibits steroid-
induced activation of the receptor (track 3).
Track 1 shows the result of adding receptor to
the DNA in the absence of DNAseI addition in
which some cleavage by endogenous
nucleases (e) occurs, while track G is a
marker track produced by cleaving the same
DNA at each guanine residue. Minus signs
indicate footprinted regions protected by
receptor, plus signs are hypersensitive sites at
which cleavage is increased by the presence
of the receptor. The DNA fragment used and
position of the radioactive label (diamond) are
shown together with the distances upstream
from the initiation site for transcription.
Note that the identical footprint is produced by
the receptor either alone or in the presence of
hormone or anti-hormone. Hence in vitro the
receptor can bind to DNA in the absence of
hormone.