▲ FIGURE 11-33 Examples of the histone code. Specific post-translational modifications of the N-terminal tails in histones H3 and H4 are found in euchromatin, which is accessible to proteins and transcriptionally active. Different modifications are found in heterochromatin, which is condensed and thus largely inaccessible to proteins and transcriptionally inactive. Histone tail sequences are shown in the one-letter amino acid code. CENP-A is a variant form for H3 found in nucleosomes associated with the centromeres of mammalian chromosomes. [Adapted from T Jenuwein and C. D. Allis, 2001, Science 293:1074.]

▲ EXPERIMENTAL FIGURE 11-34 Expression of fusion proteins demonstrates chromatin decondensation in response to an activation domain. A cultured hamster cell line was engineered to contain multiple copies of a tandem array of E. coli lac operator sequences integrated into a chromosome in a region of heterochromatin. (a) When an expression vector for the lac repressor was transfected into these cells, lac repressors bound to the lac operator sites could be visualized in a region of condensed chromatin using an antibody against the lac repressor (red). DNA was visualized by staining with DAPI (blue), revealing the nucleus. (b) When an expression vector for the lac repressor fused to an activation domain was transfected into these cells, staining as in (a) revealed that the activation domain causes this region of chromatin to decondense into a thinner chromatin fiber that fills a much larger volume of the nucleus. Bar = 1 |_im. [Courtesy of Andrew S. Belmont, 1999, J. Cell Biol. 145:1341.]

higher-order chromatin structures. The net result of such chromatin remodeling is to facilitate the binding of transcription factors to DNA in chromatin. Some activation domains have been shown to bind to the SWI/SNF complex, and this binding stimulates in vitro transcription from chro-matin templates (DNA bound to nucleosomes). Thus the SWI/SNF complex represents another type of co-activator complex. Other multi-protein complexes with similar chromatin-remodeling activities have been identified in yeast, raising the possibility that different chromatin-remodeling complexes may be required by distinct families of activators (see chapter opening figure).

Higher eukaryotes also contain multiprotein complexes with homology to the yeast SWI/SNF complex. These 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. The experiment shown in Figure 11-34 dramatically demonstrates how an activation domain can cause decondensation of a region of chromatin. This is thought to result from the interaction of the activation domain with chromatin remodeling and histone acetylase complexes.

Surprisingly, SWI/SNF complexes are also required for the repression of some genes, perhaps because they help expose histone tails to deacetylases or because they assist in the folding of chromatin into condensed, higher-order structures. Much remains to be learned about how this important class of co-activators and co-repressors alters chromatin structure to influence gene expression.

The Mediator Complex Forms a Molecular Bridge Between Activation Domains and Pol II

Still another type of co-activator, the multiprotein mediator complex, assists more directly in assembly of Pol II preiniti-ation complexes (Figure 11-35). Some of the «20 mediator subunits binds to RNA polymerase II, and other mediator subunits bind to activation domains in various activator proteins. Thus mediator can form a molecular bridge between

▲ FIGURE 11-35 Structure of yeast and human mediator complexes. (a) Reconstructed image of mediator from S. cerevislae bound to Pol II. Multiple electron microscopy images were aligned and computer-processed to produce this average image in which the three-dimensional Pol II structure (light blue) is shown associated with the yeast mediator complex (dark blue). (b) Diagrammatic representation of mediator subunits from human cells. Subunits shown in the same color are thought to form a module. Subunits in orange, yellow, and green are homologous with subunits in the yeast mediator complex. Genetic studies in yeast show that mutations in one of the subunits in a module inhibit the association of other subunits in the same module with the rest of the complex. [Part (a) courtesy of Francisco J. Asturias, 2002, Mol. Cell 10:409. Part (b) adapted from S. Malik and R. G. Roeder, 2000, Trends Biochem. Sci. 25:277.]

(a) Yeast mediator-Pol II complex


an activator bound to its cognate site in DNA and Pol II at a promoter. In addition, one of the mediator subunits has hi-stone acetylase activity and may function to maintain a promoter region in a hyperacetylated state.

Experiments with temperature-sensitive yeast mutants indicate that some mediator subunits are required for transcription of virtually all yeast genes. These subunits most likely help maintain the overall structure of the mediator complex or bind to Pol II and therefore are required for activation by all activators. In contrast, other mediator sub-units are required for activation of specific subsets of genes. DNA microarray analysis of gene expression in mutants with defects in these mediator subunits indicates that each such subunit influences transcription of «3-10 percent of all genes (see Figure 9-35 for DNA microarray tech-nique).These mediator subunits are thought to interact with specific activation domains; thus when one subunit is defective, transcription of genes regulated by activators that bind to that subunit is severely depressed but transcription of other genes is unaffected. Consistent with this explanation are binding studies showing that some activation domains do indeed interact with specific mediator subunits.

Large mediator complexes, isolated from cultured mammalian cells, are required for mammalian activators to stimulate transcription by Pol II in vitro. Since genes encoding homologs of the mammalian mediator subunits have been identified in the genome sequences of C. elegans and Drosophila, it appears that most multicellular animals (metazoans) have homologous mediator complexes. About one third of the metazoan mediator subunits are clearly homologous to yeast mediator subunits (see Figure 11-35b). But the remaining subunits, which appear to be distinct from any yeast proteins, may interact with activation domains that

▲ FIGURE 11-36 Model of several DNA-bound activators interacting with a single mediator complex. The ability of different mediator subunits to interact with specific activation domains may contribute to the integration of signals from several activators at a single promoter. See the text for discussion.

are not found in yeast. As with yeast mediator, some of the mammalian mediator subunits have been shown to interact with specific activation domains. For example, the Sur2 subunit of mammalian mediator binds to the activation domain of a TCF transcription factor that controls expression of the EGR-1 gene (see Figure 11-19). The function of this TCF activator in vivo normally is regulated in response to specific protein hormones present in serum. Mouse embryonic stem cells with a knockout of the sur2 gene fail to induce expression of EGR-1 protein in response to serum, whereas multiple other activators function normally in the mutant cells. This finding implicates the mediator Sur2 subunit in the activating function of TCF.

The various experimental results indicating that individual mediator subunits bind to specific activation domains suggest that multiple activators influence transcription from a single promoter by interacting with a mediator complex simultaneously (Figure 11-36). Activators bound at enhancers or promoter-proximal elements can interact with mediator associated with a promoter because DNA is flexible and can form a loop bringing the regulatory regions and the promoter close together. Such loops have been observed in experiments with the E. coli NtrC activator and ct54-RNA polymerase (see Figure 4-17). The multiprotein nucleopro-tein complexes that form on eukaryotic promoters may comprise as many as 100 polypeptides with a total mass of «3 megadaltons (MDa), as large as a ribosome.

Transcription of Many Genes Requires Ordered Binding of Activators and Action of Co-Activators

We can now extend the model of Pol II transcription initiation in Figure 11-27 to take into account the role of activators and co-activators. These accessory proteins function not only to make genes within nucleosomal DNA accessible to general transcription factors and Pol II but also directly recruit Pol II to promoter regions.

Recent studies have analyzed the order in which activators bind to a transcription-control region and interact with co-activators as a gene is induced. Such studies show that assembly of preinitiation complexes depends on multiple protein-DNA and protein-protein interactions, as illustrated in Figure 11-37 depicting activation of the yeast HO gene. This gene encodes a sequence-specific nuclease that initiates mating-type switching in haploid yeast cells (see Figure 11-28). Activation of the HO gene begins with binding of the SWI5 activator to an upstream enhancer. Bound SWI5 then interacts with the SWI/SNF chromatin-remodeling complex and GCN5-containing histone acetylase complex. Once the chromatin in the HO control region is decondensed and hyperacetylated, a second activator, SBF, can bind to several sites in the promoter-proximal region. Subsequent binding of the mediator complex by SBF then leads to assembly of the transcription preinitiation complex containing Pol II and the general transcription factors shown in Figure 11-36.

We can now see that the assembly of a preinitiation complex and stimulation of transcription at a promoter results from the interaction of several activators with various multiprotein co-activator complexes. These include chromatin-remodeling complexes, histone acetylase complexes, and a mediator complex. While much remains to be learned about these processes, it is clear that the net result of these multiple molecular events is that activation of transcription at a promoter depends on highly cooperative interactions initi-

M FIGURE 11-37 Ordered binding and interaction of activators and co-activators leading to transcription of the yeast HO gene. Step 1: Initially, the HO gene Is packaged into condensed chromatin. Activation begins when the SWI5 activator binds to enhancer sites 1200-1400 base pairs upstream of the start site and interacts with the SWI/SNF chromatin-remodeling complex. Step 2|: The SWI/SNF complex acts to decondense the chromatin, thereby exposing histone tails. Step 3: A GCN5-containing histone acetylase complex associates with bound SWI5 and acetylates histone tails in the HO locus as SWI/SNF continues to decondense adjacent chromatin. Step 4|: SWI5 is released from the DNA, but the SWI/SNF and GCN5 complexes remain associated with the HO control region (in the case of GCN5, by poorly understood interactions). Their action allows the SBF activator to bind several sites in the promoter-proximal region. Step 5: SBF then binds the mediator complex. Step 6: Subsequent binding of Pol II and general transcription factors results in assembly of a transcription preinitiation complex whose components are detailed in Figure 11-37. [Adapted from C. J. Fry and C. L. Peterson, 2001, Curr. Biol. 11:R185. See also M. P Cosma et al., 1999, Cell 97:299, and M. P Cosma et al., 2001, Mol. Cell 7:1213.]

ated by several activators. This allows genes to be regulated in a cell-type-specific manner by specific combinations of transcription factors. 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 he-patocytes is controlled by at least five different transcrip-tional activators (Figure 11-38). Even though three of these activators—HNF4, C/EBP, and API— are also expressed in cells of the intestine and kidney, TTR transcription does not occur in these cells, because all five activators are required but HNF1 and HNF3 are missing. Other hepatocyte-


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