Molecular Mechanisms of Transcription Activation and Repression

The activators and repressors that bind to specific sites in DNA and regulate expression of the associated protein-coding genes do so by two general mechanisms. First, these regulatory proteins act in concert with other proteins to modulate chromatin structure, thereby influencing the ability of general transcription factors to bind to promoters. Recall from Chapter 10 that the DNA in eukaryotic cells is not free, but is associated with a roughly equal mass of protein in the form of chromatin. The basic structural unit of chromatin is the nucleosome, which is composed of «147 base pairs of DNA wrapped tightly around a disk-shaped core of histone proteins. Residues within the N-terminal region of each histone, and the C-terminal region of histone H2A, called histone tails, extend from the surface of the nucleosome, and can be reversibly modified (see Figure 10-20). Such modifications, especially the acetylation of histone H3 and H4 tails, influence the relative condensation of chro-matin and thus its accessibility to proteins required for transcription initiation. In addition to their role in such chromatin-mediated transcriptional control, activators and repressors interact with a large multiprotein complex called the mediator of transcription complex, or simply mediator.

This complex in turn binds to Pol II and directly regulates assembly of transcription preinitiation complexes.

In this section, we review current understanding of how activators and repressors control chromatin structure and preinitiation complex assembly. In the next section of the chapter, we discuss how the concentrations and activities of activators and repressors themselves are controlled, so that gene expression is precisely attuned to the needs of the cell and organism.

Formation of Heterochromatin Silences Gene Expression at Telomeres, near Centromeres, and in Other Regions

For many years it has been clear that inactive genes in eu-karyotic cells are often associated with heterochromatin, regions of chromatin that are more highly condensed and stain more darkly with DNA dyes than euchromatin, where most transcribed genes are located (see Figure 5-25). Regions of chromosomes near the centromeres and telomeres and additional specific regions that vary in different cell types are organized into heterochromatin. The DNA in heterochromatin is less accessible to externally added proteins than DNA in euchromatin, and consequently often referred to as "closed" chromatin. 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 10-22).

Study of DNA regions in ,S. cerevisiae that behave like the heterochromatin of higher eukaryotes provided early insight into the chromatin-mediated repression of transcription. This yeast can grow either as haploid or diploid cells. Hap-loid cells exhibit one of two possible mating types, called a and a. Cells of different mating type can "mate," or fuse, to generate a diploid cell (see Figure 1-5). When a haploid cell divides by budding, the larger "mother" cell switches its mating type (see Figure 22-21). Genetic and molecular analyses have revealed that three genetic loci on yeast chromosome III control the mating type of yeast cells (Figure 11-28). Only the central mating-type locus, termed MAT, is actively transcribed. How the proteins encoded at the MAT locus determine whether a cell has the a or a phenotype is explained in Chapter 22. The two additional loci, termed HML and HMR, near the left and right telomere, respectively, contain "silent" (nontranscribed) copies of the a or a genes. These sequences are transferred alternately from HMLa or HMRa into the MAT locus by a type of nonreciprocal recombination between sister chromatids during cell division. When the MAT locus contains the DNA sequence from HMLa, the cells behave as a cells. When the MAT locus contains the DNA sequence from HMRa, the cells behave like a cells.

Our interest here is how transcription of the silent mating-type loci at HML and HMR are repressed. If the genes at these loci are expressed, as they are in yeast mutants with defects in the repressing mechanism, both a and a proteins are expressed, causing the cells to behave like diploid cells,

Telomere

Yeast chromosome III

Silencer

Centromere

Silencer

Yeast chromosome III

Silencer

Centromere

Silencer

Telomere

Telomere a sequences at MAT locus a sequences at MAT locus

Telomere a sequences at MAT locus

a sequences at MAT locus a1

▲ FIGURE 11-28 Arrangement of mating-type loci on chromosome III in the yeast S. cerevisiae. Silent (unexpressed) mating-type genes (either a or a, depending on the strain) are located at the HML locus. The opposite mating-type genes are present at the silent HMR locus. When the a or a sequences are present at the MAT locus, they can be transcribed into mRNAs whose encoded proteins specify the mating-type phenotype of the cell. The silencer sequences near HML and HMR bind proteins that are critical for repression of these silent loci. Haploid cells can switch mating types in a process that transfers the DNA sequence from HML or HMR to the transcriptionally active MAT locus.

which cannot mate. The promoters and UASs controlling transcription of the a and a genes lie near the center of the DNA sequence that is transferred and are identical whether the sequences are at the MAT locus or at 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 11-28). If the silencer is deleted, the adjacent silent locus is transcribed. Remarkably, any gene placed near the yeast mating-type silencer sequence 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 than RNA polymerase II uses.

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 an E. coli enzyme that methylates adenine residues in GATC sequences was introduced into yeast cells under the control of a yeast promoter so that the enzyme was expressed. Researchers found that GATC sequences within the MAT locus and most other regions of the genome in these cells were methylated, but not those within the HML and HMR loci. These results indicate that the DNA of the silent loci is inaccessible to the E. coli methylase and presumably to proteins in general, including transcription factors and RNA polymerase. Similar experiments conducted with various yeast histone mutants indicated that specific interactions involving the histone tails of H3 and H4 are required for formation of a fully repressing chromatin structure. Other studies have shown that the telomeres of every yeast chromosome also behave like silencer sequences. For instance, when a gene is placed within a few kilobases of any yeast telomere, its expression is repressed. In addition, this repression is relieved by the same mutations in the H3 and H4 histone tails that interfere with repression at the silent mating-type loci.

Genetic studies led to identification of several proteins, RAP1 and three SIR proteins, that are required for repression of the silent mating-type loci and the telomeres in yeast. RAP1 was found to bind within the DNA silencer sequences associated with HML and HMR and to a sequence that is repeated multiple times at each yeast chromosome telomere. Further biochemical studies showed that the SIR proteins bind to one another and that two bind to the N-terminal tails of histones H3 and H4 that are maintained in a largely unacetylated state by the deacetylase activity of SIR2. Several experiments using fluorescence confocal microscopy of yeast cells either stained with fluorescent-labeled antibody to any one of the SIR proteins or RAP1 or hybridized to a labeled telomere-specific DNA probe revealed that these proteins form large, condensed telomeric nucleoprotein structures resembling the heterochromatin found in higher eukaryotes (Figure 11-29). Figure 11-30 depicts a model for the chromatin-mediated silencing at yeast telomeres based on these and other studies. Formation of heterochromatin at telomeres is nucleated by multiple RAP1 proteins bound to repeated sequences in a nucleosome-free region at the extreme end of a telomere. A network of protein-protein interactions involving telomere- bound RAP1, three SIR proteins (2, 3, and 4), and hypo-acetylated histones H3 and H4 creates a stable, higher-order nucleoprotein complex that includes several telomeres and 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 well understood, it is known

▲ EXPERIMENTAL FIGURE 11-29 Antibody and DNA probes colocalize SIR3 protein with telomeric heterochromatin in yeast nuclei. (a) Confocal micrograph 0.3 ^m thick through three diploid yeast cells, each containing 68 telomeres. Telomeres were labeled by hybridization to a fluorescent telomere-specific probe (yellow). DNA was stained red to reveal the nuclei. The 68 telomeres coalesce into a much smaller number of regions near the nuclear periphery. (b, c)

Confocal micrographs of yeast cells labeled with a telomere-specific 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 colocalize 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.]

to associate with the silencer region, where it is thought to cause further assembly of the multiprotein telomeric silencing complex so that it spreads farther from the end of the chromosome, encompassing HML and HMR.

An important feature of this model is the dependence of silencing on hypoacetylation of the histone tails. This was shown in experiments with yeast mutants expressing his-tones in which lysines in histone N-termini were substituted with arginines or glutamines. Arginine is positively charged like lysine, but cannot be acetylated. It is thought to function in histone N-terminal tails like an unacetylated lysine. Glut-amine on the other hand simulates an acetylated lysine. Repression at telomeres and at the silent mating-type loci was defective in the mutants with glutamine substitutions, but not in mutants with arginine substitutions. Hyperacetylation of the H3 and H4 tails subsequently was found to interfere with binding by SIR3 and SIR4.

Although chromatin-mediated repression of transcription is also important in multicellular eukaryotes, the mechanism of this repression is still being worked out. Genetic and

M FIGURE 11-30 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 interactions between RAP1, SIR2, SIR3, SIR4, and the hypoacetylated N-terminal tails of histones H3 and H4 of nearby nucleosomes. SIR2 deacetylates the histone tails. The heterochromatin structure at each telomere encompasses ~4 kb of DNA neighboring the RAP1-binding sites, irrespective of its sequence. Association of several condensed telomeres forms higher-order heterochromatin complexes, such as those shown in Figure 11-29, that sterically block other proteins from interacting with the DNA. See the text for more details. [Adapted from M. Grunstein, 1997, Curr. Opin. Cell Biol. 9:383.]

biochemical analyses in Drosophila have revealed that multiple proteins associated in large multiprotein complexes participate in the process. These Polycomb complexes are discussed further in Chapter 15. As in the case of SIR proteins binding to yeast telomeres (see Figure 11-29), these Drosophila Polycomb proteins can be visualized binding to the genes they repress at multiple, specific locations in the genome by in situ binding of specific-labeled antibodies to salivary gland polytene chromosomes.

Repressors Can Direct Histone Deacetylation at Specific Genes

The importance of histone deacetylation in chromatin-mediated gene repression has been further supported by studies of eukaryotic repressors that regulate genes at internal chromosomal positions. These proteins are now known to act in part by causing deacetylation of histone tails in nucleo-somes that bind to the TATA box and promoter-proximal region 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 un-

acetylated histones, the N-terminal lysines are positively charged and interact strongly with DNA phosphates. The unacetylated histone tails also interact with neighboring his-tone octamers, favoring the folding of chromatin into condensed, higher-order structures whose precise conformation is not well understood. The net effect is that general transcription factors cannot assemble into a preinitiation complex on a promoter associated with hypoacetylated histones. In contrast, binding of general transcription factors is repressed much less by histones with hyperacetylated tails in which the positively charged lysines are neutralized and electrostatic interactions with DNA phosphates are eliminated.

The connection between histone deacetylation and repression of transcription at nearby yeast promoters became clearer when the cDNA encoding a human histone deacety-lase was found to have 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 RPD3 protein has histone deacetylase activity. The ability of RPD3 to deacetylate histones 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

► EXPERIMENTAL FIGURE 11-31

The chromatin immunoprecipitation method can reveal the acetylation state of histones in chromatin. Histones are lightly cross-linked to DNA in vivo using a cell-permeable, reversible, chemical cross-linking agent. Nucleosomes with acetylated histone tails are shown in green. Step 1 : Cross-linked chromatin is then isolated and sheared to an average length of two to three nucleosomes. Step 2| : An antibody against a particular acetylated histone tail sequence is added, and (step 3) bound nucleosomes are immunoprecipitated. Step 4 : DNA in the immunoprecipitated chromatin fragments is released by reversing the cross-link and then is quantitated using a sensitive PCR method. The method can be used to analyze the in vivo association of any protein with a specific sequence of DNA by using an antibody against the protein of interest in step 2. [See S. E. Rundlett et al., 1998, Nature 392:831.]

Cross-linked chromatin

Cross-linked chromatin

Isolate and shear chromatin mechanically

Isolate and shear chromatin mechanically

Add antibody specific for acetylated N-terminal histone tail

Antibody against acetylated histone N-terminal tail

Nucleosome with acetylated histone tails

Add antibody specific for acetylated N-terminal histone tail

Release immunoprecipitated DNA and assay by PCR

(a) Repressor-directed histone deacetylation

Deacetylation of histone Rpd3 \ N-terminal tails

(a) Repressor-directed histone deacetylation

Deacetylation of histone Rpd3 \ N-terminal tails

Histone

N-terminal tail

(b) Activator-directed histone hyperacetylation

(b) Activator-directed histone hyperacetylation

Hyperacetylation of histone Gcn5 ^ N-terminal tails

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