Transcriptional control in eukaryotic cells can be visualized as involving several levels of regulation. The concentrations and activities of activators and repressors that control transcription of many protein-coding genes are regulated during cellular differentiation and in response to hormones and signals from neighboring cells. These activators and repressors in turn regulate changes in chromatin structure and histone acetylation and deacetylation, thereby influencing the ability of general transcriptions factors to bind to promoters. In addition, activators and repressors directly regulate assembly of transcription-initiation complexes and the rate at which they initiate transcription. In this section, we review current understanding of how activators and repressors control chromatin structure and initiation-complex assembly and how these molecular events work together to regulate gene expression according to the needs of the cell and organism.
As discussed in Chapter 9, the DNA in eukaryotic cells is not free, but is associated with a roughly equal mass of protein in the form of chromatin. In some cases, the ability of transcription factors to interact with long stretches of DNA sequence is regulated by controlling chromatin structure. The basic structural unit of chromatin is the nucleosome, which is composed of ≈146 base pairs of DNA wrapped tightly around a disk-shaped core of histone proteins. In condensed chromatin, the nucleosomes associate with each other into a 30-nm fiber (see Figure 9-31). The amino acid residues at the N-terminus of each histone (≈20–60 residues depending on the histone) extend from the surface of the nucleosome (see Figure 9-30). These histone N-termini are rich in lysine residues, which can be reversibly modified by acetylation, phosphorylation, and methylation, as well as by the addition of a single ubiquitin molecule, a highly conserved 76-residue protein.
Phosphorylation has an important, but as yet poorly understood, function in the condensation of chromosomes during mitosis. The functions of the other modifications are also poorly understood, except in the case of acetylation. Acetylation of the histone N-termini is associated with gene control during interphase. Other proteins less abundant than histones, but nonetheless present in large numbers, also are associated with chromatin. These include HMG proteins, which participate in formation of enhancesomes (see Figure 10-48).
For many years it has been clear that inactive genes in eukaryotic cells are often associated with heterochromatin, regions of chromatin that stain more darkly with DNA dyes than euchromatin where most transcribed genes are located (see Figure 9-39). Heterochromatin stains more darkly than euchromatin because it is more highly condensed. The DNA in heterochromatin is less accessible to externally added proteins than DNA in euchromatin. For instance, in an experiment described in the last chapter, the DNA of inactive genes was found to be far more resistant to digestion by DNase I than the DNA of transcribed genes (see Figure 9-32).
Two haploid cells that differ in mating type, called a and α, can mate to form a diploid a/α cell, which multiplies by budding. Under starvation conditions, diploid cells undergo meiosis, forming haploid ascospores. Rupture of an ascus releases four haploid spores, which can germinate into haploid cells. Once each generation a haploid cell is converted to the opposite mating type.
). When diploid
cells are starved, they sporulate, forming four haploid spores, each of
which can germinate when supplied with nutrients, generating haploid cells.
A normal haploid cell switches its mating type each generation. Genetic and
molecular analyses have traced this remarkable phenomenon to regulated
changes in the DNA sequence of chromosome III.
Silent (unexpressed) mating-type genes (either a or α, depending on the strain) are located at HML. The opposite mating-type genes are present at the silent HMR locus. Once every other cell division, the DNA sequence at HML is transferred to the MAT locus; in the alternate cell divisions, the DNA sequence from HMR is transferred to the MAT locus. When the α or a sequences are present at the MAT locus, they can be transcribed into mRNAs whose encoded proteins are transcription factors that regulate the expression of mating-type specific genes (see Figure 14-1). The silencer sequences near HML and HMR bind proteins that are critical for repression of these silent loci.
). The central
locus is termed MAT, the mating-type locus. Genes at the
MAT locus are actively transcribed into mRNA. The mRNAs
expressed from the MAT locus encode transcription factors
that regulate other genes that give the cell its a or
α phenotype (Section 14.1). Two additional
“silent” (nontranscribed) copies of the genes for these
transcription factors are “stored” at loci termed
HML and HMR, near the left and right
telomere, respectively, of chromosome III. These sequences are transferred
alternately from HMLα or
HMRa into the MAT locus,
once during each cell generation, by a type of recombination called gene conversion (Section 12.5).
When the MAT locus contains the DNA sequence from
HMLα, the cells behave as α cells.
When the MAT locus contains the DNA sequence from
HMRa, the cells behave like a
cells.
). If the silencer is
deleted, the adjacent silent locus is transcribed. Remarkably, any gene
placed near the yeast mating-type silencer by recombinant DNA techniques is
repressed, or “silenced,” even a tRNA gene transcribed
by RNA polymerase III, which uses a different set of general transcription
factors from RNA polymerase II.
Several lines of evidence indicate that repression of the HML and HMR loci results from a condensed chromatin structure that sterically blocks transcription factors from interacting with the DNA. In one telling experiment, the gene encoding a DNA methylase of E. coli was introduced into yeast cells under the control of a yeast promoter so that the enzyme was expressed. This enzyme methylates adenine residues in the sequence GATC. Methylation at this sequence can be assayed easily with restriction enzymes that digest either the methylated or unmethylated sequence. Using these methods, researchers demonstrated that the E. coli methylase expressed in yeast cells was able to methylate GATC sequences within the MAT locus and most other regions of the yeast genome, but not within the HML and HMR loci. These results indicate that the DNA of the silent loci is inaccessible to proteins in general, including transcription factors and RNA polymerase. In similar experiments conducted with various yeast histone mutants, mutations in the N-terminal region of histones H3 and H4 were found to derepress the silent loci, allowing the E. coli DNA methylase to gain access to GATC sequences in HML and HMR. This result suggested that specific interactions involving the H3 and H4 N-termini are required for formation of a fully repressing chromatin structure.
Other studies indicate that the telomeres of every yeast chromosome also behave like silencers. For instance, when a gene is placed within a few kilobases of any of the yeast telomeres, its expression is repressed. In addition, this repression is relieved by the same mutations in the H3 and H4 N-termini that interfere with repression at the silent mating-type loci.
(a) Confocal micrograph 0.3 μm thick through three diploid yeast cells, each containing 34 telomeres. Telomeres were labeled by hybridization to a fluorescent telomere-specific probe (yellow). DNA was stained red to reveal the nuclei. The 34 telomeres coalesce into a much smaller number of regions near the nuclear periphery. (b,c) Confocal micrographs of yeast cells labeled with a telomerespecific hybridization probe (b) and a fluorescent-labeled antibody specific for Sir3 (c). Note that Sir3 is localized in the repressed telomeric heterochromatin. Similar experiments with Rap1, Sir2, and Sir4 have shown that these proteins also co-localize with the repressed telomeric heterochromatin. [From M. Gotta et al., 1996, J. Cell Biol. 134:1349; courtesy of M. Gotta, T. Laroche, and S. M. Gasser.]
Multiple copies of Rap1 bind to a simple repeated sequence at each telomere region, which lacks nucleosomes (top). This nucleates the assembly of a multiprotein complex (bottom) through protein-protein interactions between Rap1, Sir2, Sir3, Sir4, and the hypoacetylated amino-terminal tails of histones H3 and H4 of nearby nucleosomes. Asterisks represent hyperacetylated histone amino-terminal tails. The heterochromatin structure encompasses ≈4 kb of DNA neighboring the Rap1- binding sites, irrespective of its sequence. The actual structure of the higher-order heterochromatin is not yet understood. See text. [Adapted from M. Grunstein, 1997, Curr. Opin. Cell Biol. 9:383.]
). These results have led
to the model for silencing at yeast telomeres depicted in Figure 10-57. An important feature of
this model, which has been experimentally demonstrated, is that the histone
N-termini are hypoacetylated.
In this model, formation of heterochromatin is nucleated by the multiple Rap1 proteins bound to repeated sequences in the nucleosome-free region at the telomere. Rap1 binds Sir3 and Sir4, which then form a network of protein-protein interactions with Sir2, hypoacetylated histones H3 and H4, and additional Sir3 and Sir4 proteins, creating a stable, higher-order nucleoprotein complex in which the DNA is largely inaccessible to external proteins. One additional protein, Sir1, is also required for silencing of the silent mating-type loci. Although the function of Sir1 is not yet understood, it is thought to allow the telomeric silencing mechanism to encompass HML and HMR. The dependence of the silencing mechanism on histone hypoacetylation was shown in experiments in which arginines and glutamines were substituted for lysines in histone N-termini of constructed yeast mutants. Arginine is positively charged like lysine, but cannot be acetylated, and glutamine stimulates lysine acetylation. Substitution to arginine was compatible with silencing, whereas substitution with glutamine was not.
Regulation of transcription through heterochromatin-mediated repression is also important in multicellular eukaryotes. For example, expression of Hox transcription factors, which regulate development of the “body plan” (i. e., normal anatomy) in nearly all animals (Section 14.3), is subject to heterochromatin repression. The mechanism of this repression is still being worked out, but genetic analysis in Drosophila has revealed that multiple proteins nucleate formation of heterochromatin regions at specific sites within the Hox genes. In Drosophila, some of these proteins can be visualized binding to multiple, specific locations in the chromosome by in situ binding of specific-labeled antibodies to salivary gland polytene chromosomes.
The role of histone deacetylation in chromatin-mediated gene repression has been further supported by the discovery of yeast proteins that repress transcription of multiple genes at internal chromosomal positions. These proteins are now known to act in part by causing deacetylation of histone N-termini in nucleosomes that bind to the TATA box of the genes they repress. In vitro studies have shown that when promoter DNA is assembled onto a nucleosome with unacetylated histones, the general transcription factors cannot bind to the TATA box and initiation region. In unacetylated histones, the N-terminal lysines are positively charged and interact strongly with DNA phosphates, increasing the affinity of DNA for the nucleosome surface. This strong interaction may prevent access of general transcription factors to the promoter region. In contrast, binding of general transcription factors is repressed much less by histones with hyperacetylated N-termini in which the positively charged lysines are neutralized and electrostatic interactions with DNA phosphates are eliminated. Moreover, the binding of transcription factors to promoter DNA in this case is greatly stimulated by transcriptional activators.
(a) Repressor-directed deacetylation of histone N-terminal tails. The DNA-binding domain (DBD) of the repressor Ume6 interacts with a specific upstream control element (URS1) of the genes it regulates. The Ume6 repression domain (RD) binds Sin3, a subunit of a multiprotein complex that includes Rpd3, a histone deacetylase. Deacetylation of histone N-terminal tails on nucleosomes in the region of the Ume6-binding site inhibits binding of general transcription factors at the TATA box, thereby repressing gene expression. (b) Activator-directed hyperacetylation of histone N-terminal tails. The DNA-binding domain of Gcn4 interacts with specific upstream-activating sequences (UAS) of the genes it regulates. The Gcn4 activation domain (AD) then interacts with a multiprotein histone acetylase complex that includes the Gcn5 catalytic subunit. Subsequent hyperacetylation of histone N-terminal tails on nucleosomes in the vicinity of the Gcn4-binding site facilitates access of the general transcription factors required for initiation. Repression and activation of some genes in higher eukaryotes occurs by similar mechanisms.
Nucleosomes are lightly cross-linked to DNA in vivo using a cell-permeable, reversible, chemical cross-linking agent. Chromatin is then isolated, sheared to an average length of three nucleosomes, and subjected to immunoprecipitation with an antibody specific for a particular acetylated N-terminal histone sequence. The DNA in the immunoprecipitated chromatin fragment is released by reversing the cross-link and then is quantitated using a sensitive PCR method. [See S. E. Rundlett et al., 1998, Nature 392:831.]
). Additional
experiments, using the technique outlined in Figure 10-59, demonstrated that in wild-type yeast, one or two
nucleosomes in the immediate vicinity of Ume6-binding sites are hypoacetylated.
These DNA regions include the promoters of genes repressed by Ume6. In
sin3 and rpd3 deletion mutants, not only
were these promoters derepressed, but the nucleosomes neighboring the
Ume6-binding sites were hyperacetylated. This finding provides considerable
support for the model shown in Figure
10-58a.
The SIN3 and RPD3 genes are required for complete repression by a number of other yeast repressors, which bind to DNA at different sites than does Ume6. These repressors are thought to function by the same mechanism as the Ume6-Sin3-Rpd3 system. Also, three other histone deacetylase complexes have been identified in yeast extracts. Some of these may also be targeted to specific promoters to repress transcription through deacetylation of histones in specific nucleosomes.
).
These recent findings provide an explanation for earlier observations that in vertebrates transcriptionally inactive DNA regions often contain the modified cytidine residue 5-methylcytidine (mC) followed immediately by a G, whereas transcriptionally active DNA regions lack mC residues. DNA containing 5-methylcytidine has been found to bind a specific protein that in turn interacts specifically with mSin3. This finding suggests that association of mSin3-containing histone deacetylase complexes with methylated sites in DNA leads to deacetylation of histones in neighboring nucleosomes, making these regions inaccessible to general transcription factors and Pol II, and hence transcriptionally inactive.
is
consistent with the observation that nucleosomes near the promoter region of a
gene regulated by GCN5 are specifically hyperacetylated, as
determined by the nucleosome immunoprecipitation method (see Figure 10-59). The activator-directed
hyperacetylation of nucleosomes near a promoter region changes the chromatin
structure so as to facilitate the binding of other proteins required for
transcription initiation.
A similar activation mechanism operates in higher eukaryotes. In mammals, for instance, there is a small family of ≈400-kDa, multidomain CBP proteins. As noted earlier, one domain of CBP binds the phosphorylated CREB transcription factor (see Figure 10-46). Other domains of CRB interact with distinct classes of activation domains in other transcription factors. Interaction between CBP and various activators is required for their maximal activity, reflecting the function of CBP as a co-activator. Yet another domain of CBP has histone acetylase activity, and large multiprotein CBP–histone acetylase complexes, functionally analogous to the yeast Gcn5-containing complexes, have been identified in nuclear extracts from mammalian cells. Activators are thought to function in part by directing a CBP – histone acetylase complex to specific nucleosomes, where it acetylates histone N-terminal tails, facilitating the interaction of general transcription factors with promoter DNA. In addition, the largest TFIID subunit (TAFII145 in yeast and TAFII250 in higher eukaryotes) has been shown to interact with a number of activation domains. This TFIID subunit also has histone acetylase activity and may function by acetylating histone N-terminal tails in the vicinity of the TATA box.
As noted previously, chromosomal DNA in the region of a transcribed gene is more sensitive to digestion by DNase I than DNA in a transcriptionally silent region (see Figure 9-32). Nonetheless, transcriptionally active DNA is more resistant to DNase I digestion than “naked” DNA because most of the DNA is bound to the surface of histone octamers in nucleosomes. However, within transcriptionally active regions of chromatin, some sites are nearly as sensitive to DNase I digestion as naked DNA. These DNase I – hypersensitive sites occur in regions where transcription factors are bound, and probably result from digestion at sites immediately adjacent to the bound factors. DNase I – hypersensitive sites can be mapped by Southern blotting and may be useful in identifying transcription factor – binding sites for a gene of interest.
Genetic analyses in yeast first revealed another type of multiprotein complex, called the Swi/Snf chromatin-remodeling complex, required for activation at some promoters. Several of the largest Swi/Snf subunits have homology to DNA and RNA helicases, enzymes that use energy released by ATP hydrolysis to disrupt interactions between base-paired nucleic acids. Other enzymes with the helicase homology disrupt nucleic acid – protein interactions. Even in transcriptionally active regions of chromatin, which are more susceptible to DNase than inactive regions, bound histone octamers partially protect the DNA from digestion. However, in the presence of the purified Swi/Snf complex, nucleosomal DNA becomes more susceptible to DNase I digestion. This finding suggests that the ability of the Swi/Snf complex to facilitate the in vitro binding of some transcription factors to sites in nucleosomal DNA results from transient dissociation of the DNA from the surface of nucleosomes. Yeast also contains other homologous multiprotein complexes with similar “chromatin-remodeling” activities, raising the possibility that different chromatin-remodeling complexes may be required by distinct families of activators.
Higher eukaryotes also contain multiprotein complexes with homology to the yeast Swi/Snf chromatin-remodeling factors. Drosophila genetic studies have revealed that some of these are required for the normal regulation of Hox genes through chromatin structure. Also, protein complexes isolated from nuclear extracts of mammalian and Drosophila cells have been found to assist binding of transcription factors to their cognate sites in nucleosomal DNA in an ATP-requiring process. Consequently, it seems clear that chromatin- remodeling factors participate in the regulation of genes in higher eukaryotes as well as in yeast.
),
transcriptional activators are thought to stimulate the assembly of an
initiation complex and regulate the frequency at which new Pol II molecules
reinitiate transcription. This function of activators, which provides a second
level of transcriptional control, often can be demonstrated in in vitro
reactions lacking histones. For example, some activators stimulate the binding
of TFIID or the simultaneous binding of TFIID plus TFIIA to the TATA box in
vitro. Other activators interact with other general transcription factors and
with subunits in the multiprotein Mediator complex associated with the CTD of
the largest Pol II subunit. As discussed earlier, many of these general
transcription factors and the Mediator complex may occur in a preassembled
holoenzyme complex that can bind to a
TFIID – promoter DNA complex in a single step.
CBP and other co-activators also participate in the network of protein- protein
and protein-DNA interactions in the large nucleoprotein complexes that assemble
at eukaryotic promoters.
HNF = hepatocyte nuclear factor. [See R. Costa et al., 1989, Mol. Cell Biol. 9:1415; K. Xanthopoulus et al., 1989,Proc. Nat’l. Acad. Sci. USA 86:4117.]
Four activators enriched in hepatocytes plus the ubiquitous AP1 factor bind to sites in the hepatocytespecific enhancer and promoter-proximal region of the TTR gene. The activation domains of the bound activators interact extensively with co-activators, TAF subunits of TFIID, Srb/Mediator proteins, and general transcription factors, resulting in looping of the DNA and formation of a stable activated initiation complex. Because of the highly cooperative nature of complex assembly, an initiation complex does not form on the TTR promoter in intestinal epithelial cells, which contain only two of the four hepatocyte-enriched transcription factors. Many of the general transcription factors, Srb/Mediator proteins, and RNA polymerase II (Pol II) may be pre-assembled into a holoenzyme complex.
):
HNF3, a hepatocyte-specific winged-helix protein
HNF4, a nuclear receptor that also is expressed in intestinal epithelial cells and kidney tubule cells
C/EBP, a basic-zipper heterodimer that also is expressed in intestinal epithelial cells, fat cells, and some neurons
AP1, a small family of basic-zipper heterodimeric proteins that are expressed in virtually all cell types
).
Different genes that encode prominent hepatocytespecific proteins, such as serum albumin or α1-antitrypsin, have different arrangements of protein-binding sites and use overlapping but not identical sets of factors. Thus there is no single arrangement of sites that dictates hepatocytespecific gene expression. Serum albumin is expressed at far higher levels than transthyretin because the serum albumin gene is transcribed much more frequently in hepatocytes than the transthyretin gene. This difference reveals another level of control by transcription factors, regulation of the frequency of transcription initiation for those genes that are transcribed in a specific cell type. Much remains to be learned about the mechanisms that result in differential transcription-initiation frequency within a given cell type.
). Histone deacetylation, in turn, inhibits the interaction of
general transcription factors with their binding sites in nucleosomal DNA,
thereby repressing transcription. However, the finding that a number of
eukaryotic repressor proteins repress in vitro transcription in the absence of
histones indicates that more direct repression mechanisms also operate.
In the three mechanisms shown, the repressor either inhibits activation or directly interferes with formation of the initiation complex. In addition, some repressors interact with “co-repressor” proteins, that are thought to interact in turn with general transcription factors to inhibit initiation.
). Two mechanisms involve competitive binding
between a repressor and activator or general transcription factor. In both
cases, binding of a repressor molecule to a specific DNA site blocks binding of
proteins required to initiate transcription. In many cases, however, eukaryotic
repressors inhibit transcription without interfering with the binding of an
activator or general transcription factors. In such cases, the bound repressor
may interact with a nearby activator, preventing its function, or with general
transcription factors bound at the promoter, preventing their assembly into an
initiation complex. Presumably, repression of the EGR-1 gene by
WT1 protein, discussed earlier, operates by one of the latter two mechanisms,
since WT1 binding does not interfere with activator binding (see Figure 10-49).
We have seen in the preceding discussion that transcription of eukaryotic genes is regulated by combinations of activators and repressors that bind to specific DNA regulatory sequences. Whether or not a specific gene in a multicellular organism is expressed in a particular cell at a particular time is largely a consequence of the binding and activity of the transcription factors that interact with the regulatory sequences of that gene. Clearly, since different proteins are expressed in different cells at different times in development, the activity of transcription factors must be controlled.
An obvious critical control point for cells is transcription of the genes encoding transcription factors themselves. Hepatocyte-specific expression of transthyretin provides an example: The complete set of activators required for transcription of the TTR gene are expressed only in hepatocytes. The transcription factors expressed in a particular cell type, and the amounts produced, are a consequence of multiple regulatory interactions between transcription-factor genes that occur during the development and differentiation of a particular cell type. In Chapters 14, 20, and 23, we present examples of such regulatory interactions during development and discuss the principles of development and differentiation that have emerged from these examples.
Expression of a particular gene is further controlled by regulating the activities of the factors required for its transcription. In the remainder of this section, we discuss two important mechanisms for regulating transcription-factor activity: interaction of transcription factors with small effector molecules (e.g., lipid-soluble hormones) and post-translational modifications (e.g., phosphorylation).
Like other steroid hormones, it is synthesized from cholesterol. Retinoic acid is a metabolic derivative of vitamin A that has powerful effects on limb bud development in embryos and skin renewal in adult mammals. It is the ligand for the retinoic acid A receptor (RAR). Thyroxine is synthesized from tyrosine residues in the protein thyroglobulin in the thyroid gland. It is a ligand for the thyroid hormone receptor (TR).
). As
discussed earlier, these lipid-soluble hormones, including many different
steroid hormones, retinoids, and thyroid hormones, bind to and regulate specific
transcription factors belonging to the nuclear-receptor superfamily.
The centrally located DNA-binding domain exhibits considerable sequence homology among different receptors and has the C4 zinc-finger motif. The C-terminal hormone-binding domain exhibits somewhat less homology. The N-terminal regions in various receptors vary in length, have unique sequences, and may contain one or more activation domains. This general pattern has been found in the estrogen receptor (553 amino acids [aa]), progesterone receptor (946 aa), glucocorticoid receptor (777 aa), thyroid hormone receptor (408 aa), and retinoic acid receptor (432 aa). [See R. M. Evans, 1988, Science 240:889.]
). All the nuclear receptors have a
unique N-terminal region of variable length
(100 – 500 amino acids) containing regions
that function as transcription-activation domains. The DNA-binding domain
maps near the center of the primary sequence and has the C4
zinc-finger motif. The hormone-binding domain lies near the C-terminal end
of these receptors and contains a hormone-dependent activation domain. In
some cases the hormone-binding domain functions as a repression domain in
the absence of ligand.
The inverted repeats in GRE and ERE and direct repeats in VDRE, TRE, and RARE are indicated by red arrows. [See K. Umesono et al., 1991, Cell 65:1255; A. M. Naar et al., 1991,Cell 65:1267.]
). This finding suggested that these steroid
hormone receptors would bind to DNA as symmetrical dimers, as was later
shown from the x-ray crystallographic analysis of the homodimeric
glucocorticoid receptor’s C4 zinc-finger DNA-binding
domain (see Figure 10-41b).
). The receptors that
bind to such direct-repeat response elements do so as heterodimers with a
common nuclear-receptor monomer called RXR. The vitamin D3
response element, for example, is bound by the RXR-VDR heterodimer, and the
retinoic acid response element is bound by RXR-RAR. The monomers composing
these heterodimers interact with each other in such a way that the two
DNA-binding domains lie in the same rather than inverted orientation,
allowing the RXR heterodimers to bind to direct repeats of the binding site
for each monomer. In contrast, the monomers in homodimeric nuclear receptors
(e.g., GRE and ERE) have an inverted orientation.Hormone binding to a nuclear receptor regulates its activity as a transcription factor. This regulation differs in some respects for heterodimeric and homodimeric nuclear receptors.
). As we saw
earlier, in the presence of hormone, the ligand-binding domain undergoes a
dramatic conformational change (see Figure
10-47). In the ligand-bound conformation, these nuclear receptors
can direct hyperacetylation of histones in nearby nucleosomes, thereby
reversing the repressing effects of the free ligand-binding domain. The
N-terminal activation domain in these nuclear receptors then probably
interacts with additional factors, stimulating the cooperative assembly of
an initiation complex, as described earlier.
Cultured animal cells were transfected with expression vectors encoding the proteins diagrammed at the bottom. Immunofluorescence with a labeled antibody specific for β-galactosidase was used to detect the expressed proteins in transfected cells. (a) When cells were transfected with β-galactosidase alone, the expressed enzyme was localized to the cytoplasm in the presence and absence of the glucocorticoid hormone dexamethasone (Dex). (b) When a fusion protein consisting of β-galactosidase and the entire 794-aa rat glucocorticoid receptor (GR) was expressed in the cultured cells, it was present in the cytoplasm in the absence of hormone but was transported to the nucleus in the presence of hormone. (c) A fusion protein composed of a 382-aa region of GR including the ligand-binding domain (light purple) and β-galactosidase also exhibited hormone-dependent transport to the nucleus. [From D. Picard and K. R. Yamamoto, 1987, EMBO J. 6:3333; courtesy of the authors.]
In the absence of hormone, GR is bound in a complex with Hsp90 in the cytoplasm via its ligand-binding domain (light purple). When hormone is present, it diffuses through the plasma membrane and binds to the GR ligand-binding domain, causing a conformational change in the ligand-binding domain that releases the receptor from Hsp90. The receptor with bound ligand is then translocated into the nucleus where its DNA-binding domain (orange) binds to response elements, allowing the activation domain (green) to stimulate transcription of target genes.
. The GR hormone-binding domain alone mediates this
transport. Subsequent studies showed that, in the absence of hormone, the
glucocorticoid receptor is anchored in the cytoplasm as a large protein
aggregate complexed with inhibitor proteins, including Hsp90, a protein
related to Hsp70, the major heat-shock chaperone. In this situation, the
receptor cannot interact with target genes; hence, no transcriptional
activation occurs. Binding of hormone releases the glucocorticoid receptor
from its cytoplasmic anchor, allowing it to enter the nucleus where it can
bind to response elements associated with target genes (Figure 10-67). Once the receptor with bound hormone
interacts with a response element, it activates transcription by directing
histone hyperacetylation and facilitating cooperative assembly of an
initiation complex.
). Most of these
DNA-binding proteins, referred to as orphan receptors, were
discovered by screening cDNA libraries with probes specific for the
nucleotide sequence encoding the highly conserved DNA-binding domain
characteristic of the nuclear receptors. The precise role of orphan
receptors and identification of the unknown hormones that presumably
regulate their activity are important subjects of current research.Although lipid-soluble hormones can diffuse through the plasma membrane and interact directly with transcription factors in the cytoplasm or nucleus, the second major class of hormones, peptide and protein hormones, cannot. Instead, these hormones function by binding to specific cell-surface receptors, which then pass the signal that they have bound hormone to proteins within the cell, a process called signal transduction (Chapter 20).
In many cases, the mechanism by which a hormonal signal is transduced into an activating signal for transcription factors involves phosphorylation. A simple example is provided by γ-interferon (IFNγ), a hormone released by antigen-stimulated T-helper lymphocytes, which are critical in the immune response. When IFNγ binds to a specific receptor protein that is present on the surface of most cells, it induces expression of a number of genes, producing an antiviral state that decreases the susceptibility of the cells to infection by a broad variety of viruses. IFNγ also stimulates the function of other cells that participate in the immune response. To analyze how this hormone causes induction of a specific set of genes, researchers first identified the IFNγ response element and then purified a protein from the nuclei of IFNγ-treated cells that binds to that sequence (see Figure 10-35). The isolated protein is ≈91 kDa and is called Stat1α, for signal transducer and activator of transcription.
After cultured cells are treated with IFNγ, the DNA-binding activity of Stat1α increases rapidly, in parallel with the rapid rise of transcription of inducible genes. This induction of Stat1α DNA-binding activity occurs even in cells treated with an inhibitor of protein synthesis, indicating that some type of post-translational modification of preexisting Stat1α activates its DNA-binding activity. By staining cells with fluorescein-labeled anti-Stat1α antibody, researchers demonstrated that Stat1α translocates from the cytosol to the nucleus following IFNγ treatment, with kinetics similar to that of gene induction. Analysis of Stat1α from IFNγ-treated cells showed that hormone treatment leads to phosphorylation of a specific tyrosine residue in the protein. Furthermore, phosphorylated Stat1α was found to form a homodimer, whereas the unphosphorylated protein is a monomer. When the critical tyrosine was changed to a phenylalanine by site-specific mutagenesis, the mutant Stat1α failed to activate target genes in a transfection experiment, and failed to translocate to the nucleus.
JAK kinase is activated when the IFNγ receptor dimerizes by binding to IFNγ. Activated JAK kinase phosphorylates a specific tyrosine residue in inactive Stat1α monomers in the cytoplasm. Phosphorylated Stat1α dimerizes, and the phosphorylated dimer then translocates to the nucleus where it binds to corresponding response elements, promoting transcription of IFNγ-regulated genes. [See K. Shuai et al., 1992, Science 258:1808.]
. Phosphorylation of specific serine or threonine residues also
regulates the activity of a number of other transcription factors. Various
signal-transduction pathways that regulate transcription factors in this way are
considered in Chapter 20. In some
cases (e.g., Stat1α), phosphorylation of the free transcription factor
modulates its DNA-binding activity. In other cases (e.g., CREB), the inactive,
nonphosphorylated transcription factor binds to its DNA recognition sequence;
phosphorylation then alters the functioning of the activation domain, so that
the protein can stimulate transcription.
Eukaryotic transcriptional control operates at three levels: modulation of the levels and/or activities of activators and repressors; changes in chromatin structure directed by activators and repressors; and direct influence of activators and repressors on assembly of initiation complexes.
Heterochromatin refers to condensed regions of chromatin in which the DNA is relatively inaccessible to transcription factors and other proteins, so that gene expression is repressed.
).
). This
inhibits interaction between the promoter DNA and general transcription
factors, thereby repressing transcription initiation.
). This
facilitates interaction between the promoter DNA and general
transcription factors, thereby stimulating transcription initiation.Chromatin-remodeling factors cause transient dissociation of DNA from histone cores in an ATPdependent reaction. These factors thereby promote binding of other DNA-binding proteins needed for initiation to occur at some promoters.
In vitro, combinations of activators can stimulate the assembly of initiation complexes on a nearby promoter. This direct effect of activators is thought to occur in vivo subsequent to histone acetylation.
). A cell must
produce the specific set of activators required for transcription of a
particular gene in order to express that gene.Some repressors competitively inhibit binding of activators or general transcription factors. Others interact directly with general transcription factors or with activators.
).
Hormone binding to these transcription factors induces conformational
changes that modify their interactions with other proteins.
).
