We have seen in the preceding discussion how combinations of transcription factors that bind to specific DNA regulatory sequences control the transcription of eukaryotic genes. 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 nuclear concentrations and activities of the transcription factors that interact with the transcription-control regions of that gene. (Exceptions are due to the “transcriptional memory” that results from the epigenetic mechanisms discussed in the next section.) Which transcription factors are expressed in a particular cell type, and the amounts produced, are determined by multiple regulatory interactions between transcription factors and control regions in genes encoding transcription factors that occur during the development and differentiation of that cell type. Recent advances in the analysis of transcription-factor-binding sites through identification of DNase I hypersensitive sites on a genomic scale have given us the first high-resolution view of how transcription-factor binding changes during the development and differentiation of multiple human cell types.

DNase I Hypersensitive Sites Reflect the Developmental History of Cellular Differentiation

 In Chapter 8, we learned that an expressed gene is far more sensitive to digestion by DNase I (a bovine pancreatic enzyme) than the same gene in a different cell type in which it is not expressed. In addition to this general increase in DNase I sensitivity over long regions, researchers later found that specific short regions of the genome, on the order of a hundred base pairs in length, are extremely sensitive to DNase I digestion and are the first regions cut when isolated nuclei are treated with low levels of DNase I. These sites are known as DNase I hypersensitive sites (DHSs). High-throughput sequencing methods have allowed mapping of DHSs across the genome in multiple differentiated and embryonic cell types. Briefly, after digestion of isolated nuclei with low levels of DNase I, DNA is isolated from the treated chromatin. Oligonucleotide linkers of a known sequence are ligated to the DNA ends generated by DNase I digestion. Then the DNA is sheared into small fragments by sonication, amplified by PCR, and sequenced. Human DNA sequences adjacent to the known sequence of the oligonucleotide linker were thus identified as DHSs.

Figure 1a shows plots of the number of times a DHS was sequenced—a measure of the DNase sensitivity of the site—in samples from the human cell types indicated at the left. A roughly 600-kb region of the genome on chromo some 12, located 96.2–96.8 Mb from the left end of the chromosome, is shown. The height of each vertical bar rep resents the degree of sensitivity of the DNA sequence at that position to digestion in nuclei isolated from each of the cell types.

Fig1. Maps of DNase I hypersensitive sites in embryonic and adult cells reflect their developmental history. (a) DHSs from each of the human cell types shown at the left are mapped in the interval on chromosome 12 between 96.2 and 96.8 Mb from the left end. The height of each vertical bar in the figure represents the number of times a sequence in a 50-bp interval at that position was sequenced after following the protocol described in the text to ligate a linker of known sequence to DNA ends resulting from low-level DNase I digestion of chromatin. The plots are color-coded according to the embryonic tissue from which they developed, as shown in (b). (b) Dendrogram showing the relationships among the DHS maps for each cell type across the entire genome. The embryonic tissue from which each of these cell types develops is shown at the right. Embryonic stem cells form the root of the dendrogram. The DHS maps for all other cell types are derived from those for the embryonic stem cell by loss of some DHSs and the acquisition of other DHSs. The dendrogram, based on how closely DHS maps from two cell types are related, parallels the developmental relationships among the cell types. [Republished with permission of Elsevier, Stergachis, A.B., et al., “Developmental Fate and Cellular Maturity Encoded in Human Regulatory DNA Landscapes,” Cell, 2013, 154: 888-903; permission conveyed through Copyright Clearance Center, Inc.]

Mapping of binding sites for specific transcription fac tors by chromatin immunoprecipitation (see Figure 9-18) has shown that most transcription-factor-binding sites are coincident with DHSs. This may be because the DNA-binding domain of the bound transcription factor exposes DNA flanking the binding site to DNase I digestion, or because the transcription-factor activation domain interacts with chromatin-remodeling complexes that destabilize the interaction of DNA with histone octamers in neighboring nucleosomes, causing the DNA to be more sensitive to DNase I digestion. Because DHSs are coincident with bound transcription factors, the DHS pattern in a region of chromatin represents the positions of bound transcription factors, although the transcription factors bound are not directly identified.

In Figure 1a, the type of tissue from which the DHS data were determined is shown on the left, and the embryonic tissues from which these tissue types developed are color coded as indicated in Figure 1b. It is apparent that more closely related cell types, such as fibroblasts from different regions of the body, or endothelial cells that line the inner surfaces of blood vessels from different organs, have more similar DHSs than more distantly related cell types. With computer methods, it is possible to compare the similarity of the DHS maps for each of these cell types across the entire genome. With these computational methods, a dendrogram can be generated showing how closely the DHS map from one cell type resembles those of other cell types (see Figure 1b). This dendrogram is similar to the dendrograms used to show the relatedness, and hence the evolution, of gene sequences.

Importantly, the DHS pattern of embryonic stem cells is at the root of the DHS dendrogram for all cell types (see Figure 1b). These cells from the inner cell mass of the early mammalian embryo, discussed in Chapter 21, are the progenitors of all cells in the adult organ ism. Embryonic stem cells appear to have the most complex transcriptional control of all cells in that they have the largest number of DHSs: about 257,000 in one study, compared with 90,000–150,000 in differentiated cells. This difference probably reflects the developmental potential of embryonic stem cells. Approximately 30 percent of the DHSs observed in adult differentiated cells are also observed in embryonic stem cells, but a different 30 percent is retained in each adult cell type. An additional 50,000–100,000 new DHSs not found in embryonic stem cells arise during development, but a different set of DHSs arises in each cell type. These DHS patterns reveal the complexity of the combinations of transcription factors that regulate each gene. Approximately a million distinct DHSs were characterized in the cell types shown in Figure 1, suggesting that on average, combinations of four or five enhancers regulate the transcription of each of the roughly 21,000 genes in the human genome. This analysis excluded the central nervous system, probably the most complex organ system of all, so the total number of human enhancers may be much larger. But in the tissues analyzed, the maps of DHSs reveal where binding of early embryonic transcription factors is lost and where new cell-type-specific combinations of transcription factors bind as a cell differentiates from the embryonic stem cell. Even this estimate fails to capture the complexity of transcriptional control, since many transcription-factor- binding sites detected as one DHS are bound by different related transcription factors expressed in different cell types. Often different related transcription fac tors bind to the same transcription-control region in different cell types to regulate the appropriate level of transcription for that cell type.

Nuclear Receptors Are Regulated by Extracellular Signals

In addition to controlling the expression of transcription factors, cells also regulate the activities of many of the transcription factors expressed in a particular cell type. For example, many transcription factors are regulated by intercellular signals. Interactions between the extracellular domains of transmembrane receptor proteins on the surface of the cell and specific protein ligands for these receptors secreted by other cells or expressed on the surfaces of neighboring cells activate the intracellular domains of these transmembrane proteins, transducing the signal received on the outside of the cell to a signal on the inside of the cell. The intracellular signal then regulates the activities of enzymes that modify transcription factors by phosphorylation, acetylation, and other types of post-translational protein modifications. These post-translational modifications activate or inhibit transcription factors in the nucleus. In Chapter 16, we de scribe the major types of cell-surface receptors for protein ligands and the intracellular signaling pathways that regulate transcription-factor activity.

Here we discuss another major group of extracellular signals that regulate the activities of transcription factors: small, lipid-soluble hormones including many different steroid hormones, retinoids, and thyroid hormones. These lipid-soluble hormones can diffuse through the plasma and nuclear membranes and interact directly with the transcription factors they control (Figure 2). As noted earlier, transcription factors regulated by lipid-soluble hormones include the nuclear-receptor superfamily. These transcription factors function as transcription activators only when bound to their ligands.

Fig2. Examples of hormones that bind to nuclear receptors. These and related lipid-soluble hormones diffuse through the plasma and nuclear membranes and bind to receptors located in the cytosol or nucleus. The ligand-receptor complex functions as a transcription activator.

All Nuclear Receptors Share a Common Domain Structure

 Sequencing of cDNAs derived from mRNAs encoding various nuclear receptors has revealed remarkable conservation in their amino acid sequences. It has also revealed that each of these receptors has three functional regions (Figure 3). The first is a unique N-terminal region of variable length (100–500 amino acids). Portions of this variable region function as activation domains in most nuclear receptors. The second is a DNA-binding domain that maps near the center of the primary sequence and contains a repeat of the C4 zinc-finger motif. The third region, the hormone- binding domain, located near the C-terminal end, contains a hormone-dependent activation domain. In some nuclear receptors, the hormone-binding domain functions as a repression domain in the absence of ligand.

Fig3. General design of transcription factors in the nuclear-receptor superfamily. The centrally located DNA-binding domain exhibits considerable sequence homology among different receptors and contains two copies of the C4 zinc-finger motif. The C-terminal hormone-binding domain exhibits somewhat less homology. The N-terminal regions of various receptors vary in length, have unique sequences, and may contain one or more activation domains. See R. M. Evans, 1988, Science 240:889.

Nuclear-Receptor Response Elements Contain Inverted or Direct Repeats

 The DNA sites to which nuclear receptors bind are called response elements. The characteristic nucleotide sequences of several response elements have been determined. The consensus sequences of response elements for two steroid hormone receptors, the glucocorticoid receptor response element (GRE) and the estrogen receptor response element (ERE) are 6-bp inverted repeats separated by any three base pairs (Figure 4a, b). This finding suggested that the cognate steroid hormone receptors would bind to DNA as symmetric dimers (i.e., dimers with twofold rotational symmetry), as was later confirmed by x-ray crystallographic analysis of the homodimeric glucocorticoid receptor’s C4 zinc-finger DNA-binding domain.

Fig4. Consensus sequences of DNA response elements that bind five nuclear receptors. (a, b) The glucocorticoid and estrogen receptors are twofold symmetric dimers that bind, respectively, to the glucocorticoid receptor response element (GRE) and the estrogen receptor response element (ERE). Each of these response elements contains inverted repeats separated by three base pairs. (c–e) The heterodimeric nuclear receptors each contain one RXR subunit associated with another nuclear-receptor subunit that defines the hormone response. RXR-VDR mediates responses to vitamin D3 by binding to a direct repeat separated by three base pairs (a VDRE). RXR-TR mediates responses to thyroid hormone by binding to the same DNA bases in a direct repeat separated by four base pairs (a TRE). Similarly, RXR-RAR mediates a response to retinoic acid by binding to the same direct repeat separated by five base pairs, comprising a RARE. The repeat sequences bound by the reading helices of these receptors are indicated by red arrows. (f) Crystal structures of the glucocorticoid receptor bound to DNA containing a GRE (top) and of the RXR-TR heterodimer bound to DNA containing a TRE (bottom). Red arrows indicate the orientation from N to C of the helices below them. Note that in the twofold symmetric glucocorticoid receptor, the reading helices are inverted relative to each other so that they “read” an AGAACA on the top strand of the left half-site and on the bottom strand of the right half-site, separated by 3 base pairs. Consequently, the binding site for the glucocorticoid receptor and other twofold symmetric homodimers such as the estrogen receptor is an inverted repeat (see a and b). In contrast, the reading helices in the RXR-TR heterodimer are in the same orientation. Consequently, they read an AGGTCA sequence in the same orientation in the two-half sites separated by four base pairs, a direct-repeat binding site. The interface between the RXR subunit and the vitamin D3 receptor (VDR) subunit bound to a VDRE brings the two reading helices closer together so that they bind to the same half-sites separated by three rather than four base pairs. Similarly, the interface between the RXR and RAR subunits bound to a RARE positions the two reading helices in the heterodimer farther apart than in the RXR-TR, so that they bind the same AGGTCA sequences separated by five base pairs. See K. Umesono et al., 1991, Cell 65:1255, and A. M. Naar et al., 1991, Cell 65:1267. [Part (f) top data from B. F. Luisi et al., 1991, Nature 352:497–505, PDB ID 1glu. Part (f) bottom data from F. Rastinejad et al., 1995, Nature 375:203, PDB ID 2nll.]

Some nuclear-receptor response elements, such as those for the receptors that bind nonsteroids such as vitamin D3, thyroid hormone, and retinoic acid, are direct repeats of the same sequence that is recognized by the estrogen receptor, separated by three, four, or five base pairs (Figure 4c–e).

The specificity of these response elements is determined by the spacing between the repeats. The nuclear receptors that bind to these direct-repeat response elements do so as heterodimers, all of which share a monomer called RXR. The vitamin D3 response element (VDRE), for example, is bound by the RXR-VDR heterodimer, and the retinoic acid response element (RARE) is bound by RXR-RAR. The monomers com posing 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 (Figure 4f). 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

The mechanism whereby hormone binding controls the activity of nuclear receptors differs between heterodimeric and homodimeric receptors. Heterodimeric nuclear receptors (e.g., RXR-VDR, RXR-TR, and RXR-RAR) are located exclusively in the nucleus. In the absence of their hormone ligand, they repress transcription when bound to their cognate sites in DNA. They do so by directing histone deacetylation at nearby nucleosomes by associating with his tone deacetylase complexes, as described earlier for other repressors. When heterodimeric nuclear receptors bind their ligand, they undergo a conformational change, and as a consequence, they bind histone acetylase complexes, thereby reversing their own repressing effects. In the presence of ligand, the ligand-bound conformation of the receptor also binds Mediator, stimulating preinitiation complex assembly.

In contrast to heterodimeric nuclear receptors, homodimeric receptors are found in the cytoplasm in the absence of their ligands. Hormone binding to these receptors leads to their translocation to the nucleus. The hormone-dependent translocation of the homodimeric glucocorticoid receptor (GR) was demonstrated in the transfection experiments shown in Figure 5a–c. The GR hormone-binding domain alone mediates this transport. Subsequent studies showed that in the absence of hormone, GR cannot be transported into the nucleus because its ligand-binding domain is partially unfolded by the major cellular chaperone Hsp70. As long as the receptor is confined to the cytoplasm, it cannot interact with target genes and hence cannot activate transcription. Hormone binding promotes a “handoff” of GR from Hsp70 to Hsp90, which, with coupled hydrolysis of ATP, refolds the GR ligand binding domain, increasing the affinity for hormone and re leasing GR from Hsp70 so that it can enter the nucleus. Once in the nucleus in the conformation induced by ligand binding, it can bind to response elements associated with target genes (Figure 5d). Once the receptor with bound hormone binds to a response element, it activates transcription by interacting with chromatin-remodeling and histone acetylase complexes and Mediator.

Fig5. Fusion proteins demonstrate that the hormone-binding domain of the glucocorticoid receptor mediates trans location to the nucleus in the presence of hormone. 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) In cells that expressed β-galactosidase alone, the enzyme was localized to the cytoplasm in the presence and absence of the glucocorticoid hormone dexamethasone (Dex). (b) In cells that expressed a fusion protein consisting of β-galactosidase and the entire glucocorticoid receptor (GR), the fusion protein was present in the cytoplasm in the absence of hormone but was transported to the nucleus in the presence of hormone. (c) Cells that expressed a fusion protein composed of β-galactosidase and only the GR ligand-binding domain (light purple) also exhibited hormone-dependent transport of the fusion protein to the nucleus. (d) Model of hormone-dependent gene activation by a homodimeric nuclear receptor. In the absence of hormone, the receptor is kept in the cytoplasm by interaction between its ligand-binding domain (LBD) and chaperone proteins. When hormone is present, it diffuses through the plasma membrane and binds to the ligand-binding domain, causing a conformational change that releases the receptor from the chaperone proteins. The receptor with bound ligand is then translocated into the nucleus, where its DNA-binding domain (DBD) binds to response elements, allowing the ligand binding domain and an additional activation domain (AD) at the N-terminus to stimulate transcription of target genes. [Parts (a)–(c) from Picard, D. and AD Yamamoto, K. R., “Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor,” EMBO J., 1987, 6(11):3333–3340; courtesy of the authors.]

Metazoans Regulate the RNA Polymerase II Transition from Initiation to Elongation A recent unexpected discovery that resulted from application of the chromatin immunoprecipitation technique is that a large fraction of genes in metazoans have a paused elongating RNA polymerase II within about 100 bp of the transcription start site. Thus expression of the encoded protein is controlled not only by transcription initiation, but also by transcription elongation early in the transcription unit. The first genes discovered to be regulated by control of transcription elongation were heat-shock genes (e.g., hsp70), which encode molecular chaperones that help to refold de natured proteins and other proteins that help the cell to deal with the effects of heat shock. When heat shock occurs, the heat-shock transcription factor (HSTF) is activated. Binding of activated HSTF to specific sites in the promoter-proximal region of heat-shock genes stimulates the paused polymerase to continue chain elongation and promotes rapid reinitiation by additional Pol II molecules, leading to many transcription initiations per minute. This mechanism of transcriptional control permits a rapid response: these genes are always paused in a state of suspended transcription and therefore, when an emergency arises, no time is required to remodel and acetylate chromatin at the promoter and assemble a transcription pre initiation complex.

Another transcription factor shown to regulate transcription by controlling elongation by Pol II paused near the transcription start site is MYC, which functions in the regulation of cell growth and division. MYC is often expressed at high levels in cancer cells and is a key transcription factor in the reprogramming of somatic cells into pluripotent stem cells capable of differentiation into any cell type. The ability to in duce differentiated cells to convert to pluripotent stem cells has elicited enormous research interest because of its potential for the development of therapeutic treatments for traumatic injuries to the nervous system and degenerative diseases.

Termination of Transcription Is Also Regulated

Once Pol II has transcribed about 200 nucleotides from the transcription start site, elongation through most genes is highly processive. Chromatin immunoprecipitation with antibody to Pol II, however, indicates that the amount of Pol II at various positions in a transcription unit in a population of cells varies greatly. This finding indicates that the enzyme can elongate through some regions much more rapidly than others. In most cases, Pol II does not terminate transcription until after a sequence is transcribed that directs cleavage and polyadenylation of the RNA at the sequence that forms the 3′ end of the encoded mRNA. Pol II can then terminate transcription at any of multiple sites located 0.5–2 kb beyond this poly(A) addition site. Experiments with mutant genes show that termination is coupled to the process that cleaves and polyadenylates the 3′ end of a transcript, which is discussed in the next chapter.

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مواضيع ذات صلة

Activators Are Composed of Distinct Functional Domains

DNase I Footprinting and EMSA Detect Protein-DNA Interactions

Most Eukaryotic Genes Are Regulated by Multiple Transcription-Control Elements

DNA-Binding Domains Can Be Classified into Numerous Structural Types

Promoter-Proximal Elements Help Regulate Eukaryotic Genes

Elongation Factors Regulate the Initial Stages of Transcription in the Promoter-Proximal Region

General Transcription Factors Position RNA Polymerase II at Start Sites and Assist in Initiation

Microarrays (DNA Chips)

Analysing Genetic Mutations and Polymorphisms

The Largest Subunit in RNA Polymerase II Has an Essential Carboxy-Terminal Repeat

Three Eukaryotic RNA Polymerases Catalyze Formation of Different RNAs

Regulatory Elements in Eukaryotic DNA Are Found Both Close to and Many Kilobases Away from Transcription Start Sites