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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.

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Molecular Cell Biology. 4th edition.

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Section 10.7Molecular Mechanisms of Eukaryotic Transcriptional Control

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.

N-Termini of Histones in Chromatin Can Be Modified

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).

Formation of Heterochromatin Silences Gene Expression at Telomeres and Other Regions

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).

Silencing of Yeast Silent Mating-Type Loci and Telomeric Regions

Study of DNA regions in S. cerevisiae that behave like the heterochromatin of higher eukaryotes have provided insight about chromatin-mediated repression of transcription. This yeast can grow either as haploid or diploid cells. Haploid cells exhibit one of two possible mating types, called a and α. Cells of different mating type can “mate,” or fuse, to generate a diploid cell (Figure 10-54). 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.

Figure 10-54. Life cycle of S. cerevisiae.

Figure 10-54

Life cycle of S. cerevisiae. 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. (more...)

Three genetic loci directly involved in mating-type switching have been located (Figure 10-55). 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.

Figure 10-55. Genes on chromosome III involved in mating- type control in the yeast S. cerevisiae.

Figure 10-55

Genes on chromosome III involved in mating- type control in the yeast S. cerevisiae. 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 (more...)

Repression of the silent mating-type loci is critical to haploid cells. If the silent loci are expressed, as they are in yeast mutants with defects in the repressing mechanism, both a and α transcription factors are expressed, causing the cells to behave like diploid cells, which cannot mate. The promoters and UASs controlling transcription of the a and α genes lie near the center of the DNA sequence that is transferred and are identical whether the sequences are at the MAT locus or one of the silent loci. Consequently, the function of the transcription factors that interact with these sequences is somehow blocked at HML and HMR. This repression of the silent loci depends on silencer sequences located next to the region of transferred DNA at HML and HMR (see Figure 10-55). 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.

Additional genetic studies have revealed several genes — RAP1 and three SIR (silent information regulator) genes — that are required for repression of the silent mating-type loci and the telomeres in yeast. RAP1 encodes a protein that binds within the silencer DNA sequences associated with HML and HMR and to a sequence that is repeated multiple times at each yeast chromosome telomere. Further biochemical studies of these proteins have shown that they bind to each other and that two bind to the N-termini of H3 and H4. Immunofluorescence confocal microscopy of yeast cells stained with antibody to the Sir and Rap proteins and hybridized to a labeled telomeric DNA probe revealed that these proteins form large telomeric nucleoprotein structures resembling the heterochromatin found in higher eukaryotes (Figure 10-56). 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.

Figure 10-56. Co-localization of Sir3 protein with telomeric heterochromatin in yeast nuclei.

Figure 10-56

Co-localization of Sir3 protein with telomeric heterochromatin in yeast nuclei. (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 (more...)

Figure 10-57. Schematic model of silencing mechanism at yeast telomeres.

Figure 10-57

Schematic model of silencing mechanism at yeast telomeres. 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 (more...)

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.

Silencing in Higher Eukaryotes

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.

Repressors Can Direct Histone Deacetylation at Specific Genes

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.

The connection between histone deacetylation and repression of transcription at nearby yeast promoters became clearer when the first histone deacetylase was purified (from human cells), and the cDNA encoding it was cloned based on amino acid microsequencing. The cDNA sequence showed high homology to the yeast RPD3 gene, known to be required for the normal repression of a number of yeast genes. Further work showed that the function of the Rpd3 protein at a number of promoters depends on two other proteins: Ume6, a repressor that binds to a specific upstream regulatory sequence (URS1), and Sin3, which is part of a large, multiprotein complex that also contains Rpd3. Sin3 also interacts with the repressor domain of Ume6, thus positioning the Rpd3 histone deacetylase in the complex so it can interact with nearby nucleosomes and remove acetyl groups from specific N-terminal lysines (Figure 10-58a). 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.

Figure 10-58. Role of deacetylation and hyperacetylation of histone N-terminal tails in yeast transcription control.

Figure 10-58

Role of deacetylation and hyperacetylation of histone N-terminal tails in yeast transcription control. (a) Repressor-directed deacetylation of histone N-terminal tails. The DNA-binding domain (DBD) of the repressor Ume6 interacts with a specific upstream (more...)

Figure 10-59. Experimental method for analyzing the acetylation state of histones in chromatin associated with a specific region of the genome.

Figure 10-59

Experimental method for analyzing the acetylation state of histones in chromatin associated with a specific region of the genome. Nucleosomes are lightly cross-linked to DNA in vivo using a cell-permeable, reversible, chemical cross-linking agent. Chromatin is (more...)

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.

Histone deacetylases also have been found associated with repressors from higher eukaryotes. These include two heterodimeric repressors that participate in regulation of the cell cycle in mammals and a group of nuclear receptors that are regulated by lipid-soluble hormones. These sequence-specific DNA-binding repressors interact with mammalian homologs of Sin3 (mSin3), which are found in large, multiprotein histone deacetylases complexes, and are thought to direct the specific hypoacetylation of nucleosomes in the vicinity of their binding sites, similar to the proposed yeast mechanism (see Figure 10-58a).

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.

Activators Can Direct Histone Acetylation at Specific Genes

The yeast gene GCN5 is known from genetic studies to be required for maximal activation by the yeast activator Gcn4 and several other activators with acidic activation domains. As in the case of histone deacetylases, purification, microsequencing, and cloning of the gene encoding a histone acetylase from another source (Tetrahymena, a rich source), which has a strong homology to yeast GCN5, suggested how the Gcn5 protein functions. Subsequent studies showed that Gcn5 is present in two large multiprotein complexes that have histone acetylase activity. Another subunit of these histone acetylase complexes binds to acidic activation domains. The model shown in Figure 10-58b 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.

Chromatin-Remodeling Factors Participate in Activation at Some Promoters

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.

Activators Stimulate the Highly Cooperative Assembly of Initiation Complexes

After participating in the hyperacetylation of chromatin in the vicinity of a promoter region (see Figure 10-58b), 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.

Assembly of the multiprotein initiation complex on promoter DNA is thought to result from multiple cooperative interactions such as those illustrated for the binding of E. coli CAP-cAMP and RNA polymerase to the lac promoter region (see Figure 10-17). In higher organisms, the strong cooperativity of initiation-complex assembly is in part responsible for cell type – specific gene expression. The TTR gene, which encodes transthyretin in mammals, is a good example of this. As noted earlier, transthyretin is expressed in hepatocytes and in choroid plexus cells. Transcription of the TTR gene in hepatocytes is controlled by at least five different transcriptional activators (Figure 10-60):

  • HNF1, a hepatocyte-specific homeobox protein
  • 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
Even though three of these activators are expressed in other cell types, the TTR gene is not transcribed in these cells. Thus hepatocyte-specific transcription of TTR occurs because the complete set of activators is expressed only in hepatocytes. All of the activators must be present to contribute to the highly cooperative assembly of an initiation complex at the TTR promoter (Figure 10-61).

Figure 10-60. Binding sites for activators that control transcription of the mouse transthyretin (TTR) promoter in hepatocytes.

Figure 10-60

Binding sites for activators that control transcription of the mouse transthyretin (TTR) promoter in hepatocytes. HNF = hepatocyte nuclear factor. [See R. Costa et al., 1989, Mol. Cell Biol. 9:1415; K. Xanthopoulus et al., 1989,Proc. Nat’l. (more...)

Figure 10-61. Model for cooperative assembly of an activated transcription-initiation complex at the TTR promoter in hepatocytes.

Figure 10-61

Model for cooperative assembly of an activated transcription-initiation complex at the TTR promoter in hepatocytes. Four activators enriched in hepatocytes plus the ubiquitous AP1 factor bind to sites in the hepatocytespecific enhancer and promoter-proximal (more...)

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.

Repressors Interfere Directly with Transcription Initiation in Several Ways

A repressor is any protein that interferes with transcription initiation when it is bound to a specific site on DNA. As discussed above, some eukaryotic repressors can direct deacetylation of histones in nucleosomes near their cognate binding sites (see Figure 10-58a). 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.

Although repression mechanisms are not well understood, different repressor proteins probably exert their effects in different ways (Figure 10-62). 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).

Figure 10-62. Various eukaryotic repressors can inhibit transcription by mechanisms that do not involve histone deacetylation.

Figure 10-62

Various eukaryotic repressors can inhibit transcription by mechanisms that do not involve histone deacetylation. In the three mechanisms shown, the repressor either inhibits activation or directly interferes with formation of the initiation complex. In (more...)

Regulation of Transcription-Factor Expression Contributes to Gene Control

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).

Lipid-Soluble Hormones Control the Activities of Nuclear Receptors

The activities of many transcription factors are regulated by hormones, which function as extracellular signals in multicellular organisms (Chapter 20). Hormones are secreted from one cell type and travel through extracellular fluids to affect the function of cells at a different location in the organism. One class of hormones comprises small, lipid-soluble molecules, which can diffuse through plasma and nuclear membranes (Figure 10-63). 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.

Figure 10-63. Examples of lipid-soluble hormones that bind to members of the nuclear-receptor superfamily of transcription factors. Cortisol is a steroid hormone that binds to the glucocorticoid receptor (GR).

Figure 10-63

Examples of lipid-soluble hormones that bind to members of the nuclear-receptor superfamily of transcription factors. Cortisol is a steroid hormone that binds to the glucocorticoid receptor (GR). Like other steroid hormones, it is synthesized from cholesterol. Retinoic (more...)

Domain Structure of Nuclear Receptors

Cloning and sequencing of the genes encoding several nuclear receptors permitted comparison of their amino acid sequences. Such studies revealed a remarkable conservation in both the amino acid sequences and different functional regions of various nuclear receptors (Figure 10-64). 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.

Figure 10-64. General design of transcription factors in nuclear-receptor superfamily.

Figure 10-64

General design of transcription factors in 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 (more...)

Nuclear-Receptor Response Elements

The characteristic nucleotide sequences of the DNA sites, called response elements, that bind several major nuclear receptors have been determined. The sequences of the consensus response elements for the glucocorticoid and estrogen receptors are 6-bp inverted repeats separated by any three base pairs (Figure 10-65a, b). 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).

Figure 10-65. Consensus sequences of DNA sites, called response elements, that bind the glucocorticoid receptor (GRE), estrogen receptor (ERE), vitamin D3 receptor (VDRE), thyroid hormone receptor (TRE), and retinoic acid receptor (RARE).

Figure 10-65

Consensus sequences of DNA sites, called response elements, that bind the glucocorticoid receptor (GRE), estrogen receptor (ERE), vitamin D3 receptor (VDRE), thyroid hormone receptor (TRE), and retinoic acid receptor (RARE). The inverted repeats in GRE (more...)

Some nuclear-receptor response elements, such as those for the vitamin D3, thyroid hormone, and retinoic acid receptors, are direct repeats of the same sequence recognized by the estrogen receptor, separated by three to five base pairs (Figure 10-65c – e). 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.

Mechanisms of Hormonal Control of Nuclear-Receptor Activity

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.

When heterodimeric nuclear receptors (e.g., RXR-VDR, RXR-TR, and RXR-RAR) are bound to their cognate sites in DNA, they act as repressors or activators of transcription depending on whether hormone occupies the ligand-binding site. In the absence of hormone, these nuclear receptors direct histone deacetylation at nearby nucleosomes by the mechanism described earlier (see Figure 10-58a). 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.

In contrast to heterodimeric nuclear receptors, which are located exclusively in the nucleus, homodimeric receptors are found both in the cytoplasm and nucleus, and their activity is regulated by controlling their transport from the cytoplasm to the nucleus. The hormone-dependent translocation of the homodimeric glucocorticoid receptor (GR) was demonstrated in the transfection experiments shown in Figure 10-66. 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.

Figure 10-66. Experimental demonstration that hormone-binding domain of the glucocorticoid receptor (GR) mediates translocation to the nucleus in the presence of hormone.

Figure 10-66

Experimental demonstration that hormone-binding domain of the glucocorticoid receptor (GR) mediates translocation to the nucleus in the presence of hormone. Cultured animal cells were transfected with expression vectors encoding the proteins diagrammed (more...)

Figure 10-67. Model of hormone-dependent gene activation by the glucocorticoid receptor (GR).

Figure 10-67

Model of hormone-dependent gene activation by the glucocorticoid receptor (GR). 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 (more...)

Orphan Receptors

The ligands for the hormone-binding domains in many members of the nuclear-receptor superfamily are as-yet unknown. An example is HNF4, which participates in hepatocyte-specific expression of the transthyretin gene (see Figure 10-60). 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.

Polypeptide Hormones Signal Phosphorylation of Some Transcription Factors

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.

The model for IFNγ-mediated activation of Stat1α suggested by these results is illustrated in Figure 10-68. 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.

Figure 10-68. Model of IFNγ-mediated gene activation by phosphorylation and dimerization of Stat1α.

Figure 10-68

Model of IFNγ-mediated gene activation by phosphorylation and dimerization of Stat1α. JAK kinase is activated when the IFNγ receptor dimerizes by binding to IFNγ. Activated JAK kinase phosphorylates a specific tyrosine (more...)

SUMMARY

  •  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.
  •  Heterochromatin-mediated repression occurs in the telomeres and the silent mating-type loci in S. cerevisiae. The interactions of several proteins with each other and the hypoacetylated N-termini of histones H3 and H4 are responsible for the repressing chromatin structure in these regions (see Figure 10-57).
  •  Some repressors function in part by interacting with histone deacetylase complexes, resulting in the deacetylation of histones in nucleosomes near the repressor-binding site (see Figure 10-58a). This inhibits interaction between the promoter DNA and general transcription factors, thereby repressing transcription initiation.
  •  Some activators function in part by interacting with histone acetylase complexes, resulting in the hyperacetylation of histones in nucleosomes near the activator-binding site (see Figure 10-58b). 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.
  •  The highly cooperative assembly of initiation complexes in vivo generally requires several activators (see Figure 10-61). 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.
  •  The activities of the nuclear-receptor superfamily of transcription factors are regulated by lipid-soluble hormones (see Figure 10-67). Hormone binding to these transcription factors induces conformational changes that modify their interactions with other proteins.
  •  The activities of some transcription factors are regulated by phosphorylation induced by binding of polypeptide hormones to their cell-surface receptors (see Figure 10-68).
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Copyright © 2000, W. H. Freeman and Company.
Bookshelf ID: NBK21677