分子生物学资料：chapter7 Transcriptional regulation in eukaryotes£±-3

1、Chapter 7 Transcriptional Regulation in EukaryotesConserved Mechanisms of Transcriptional Regulation from Yeast to MammalsRecruitment of Protein Complexes to Genes by Eukaryotic ActivatorsSignal Integration and Combinatorial ControlTranscriptional RepressorsSignal Transduction and the Control of Tra

2、nscriptional RegulatorsGene Silencing by Modification of Histones and DNAEpigenetic Gene RegulationCombinatorial Control of the Mating-Type Genes from S. cerevisiaeThe yeast S. cerevisiae exists in three forms: two haploid cells of different mating typesa and and the diploid formed when an a and an

3、cell mate and fuse. Cells of the two mating types differ because they express different sets of genes: a-specific genes and -specific genes. These genes are controlled by activators and repressors in various combinations.The a cell and the cell each encodes cell-type-specific regulators: a cells mak

4、e the regulatory protein a1, and cells make the proteins 1 and 2. A fourth regulatory protein, called Mcm1, is also involved in regulating the mating-type-specific genes (and many other genes) and is present in both cell types. How do these various regulators work together to ensure that in a cells,

5、 a-specific genes are switched on and -specific genes are off; vice versa in cells; and in diploid cells, both sets are kept off?Figure 19-21Control of cell-type-specific genes in yeastIn a cells, the -specific genes are off because no activators are bound there, whereas the a-specific genes are on

6、because Mcm1 is bound and activates those genes.In cells, the -specific genes are on because Mcm1 is bound upstream and activates them. At these genes, Mcm1 binds to a weak site and does so only when it binds cooperatively with a monomer of the protein 1. This ensures that Mcm1 activates these genes

7、 only in cells. The a-specific genes are kept off in cells by the repressor 2. This repressor binds, as a dimer, cooperatively with Mcm1 at these genes. In diploid cells, both a-specific and -specific genes are off. This is done as follows: the a-specific genes bind Mcm1 and 2, just as they do in ce

8、lls. This keeps those genes off. The -specific genes are off because, as in a cells, no activators bind there.Both the haploid cell types (a and ) express another class of genes called haploid-specific genes. These are switched off in the diploid cell by 2, which binds upstream of them as a heterodi

9、mer with the a1 protein. Only in diploid cells are both of these regulators present. Figure 19-22TRANSCRIPTIONAL REPRESSORSFigure 19-22a(a) By binding to a site on DNA that overlaps the binding site of an activator, a repressor can inhibit binding of the activator to a gene and thus block activation

10、 of that gene. In a variation on this theme, a repressor can be a derivative of the same protein as the activator but lack the activating region. In another variation, an activator that binds to DNA as a dimer can be inhibited from doing so by a derivative that retains the region of the protein requ

11、ired for dimerization but lacks the DNA-binding domain. Such a derivative forms inactive heterodimers with the activator.Figure 19-22b(b) A repressor binds to a site on DNA beside an activator and interacts with that activator, occluding its activating region.Figure 19-22c(c) A repressor binds to a

12、site upstream of a gene and, by interacting with the transcriptional machinery at the promoter in some specific way, inhibits transcription initiation.Figure 19-22d(d) Repression is caused by recruiting histone modifiers that alter nucleosomes in ways that inhibit transcription (e.g., deacetylation,

13、 as shown here, but also methylation in some cases, or even remodeling at some promoters).Figure 19-3Gal4 protein activates transcription of the galactose genes in the yeast Saccharomyces cerevisiae. These genes, like their bacterial counterparts, encode enzymes required for galactose metabolism. On

14、e such gene is GAL1. Gal4 binds to four sites located 275 bp upstream of GAL1 (Fig. 19-3). When bound there, in the presence of galactose, Gal4 activates transcription of the GAL1 gene 1000-fold.Figure 19-23SIGNAL TRANSDUCTION AND THE CONTROL OF TRANSCRIPTIONAL REGULATORSSignals Are Often Communicat

15、ed to Transcriptional Regulators through Signal Transduction PathwaysIn a signal transduction pathway, the initiating ligand is typically detected by a specific cell surface receptor: the ligand binds to an extracellular domain of the receptor, and this binding is communicated to the intracellular d

16、omain. From there, the signal is relayed to the relevant transcriptional regulator, often through a cascade of kinases. How is the binding of ligand to the extracellular domain communicated to the intracellular domain? This can be through an allosteric change in the receptor, whereby binding of liga

17、nd alters the shape (and thus activity) of the intracellular domain. Alternatively, the ligand can act simply to bring together two or more receptor chains, allowing interactions between the intracellular domains of those receptors to activate each other.Figure 19-24Figure 19-24aThe STAT (signal tra

18、nsducer and activator of transcription) pathway. In this example, a kinase is bound to the intracellular domain of a receptor. When the receptor is activated by its ligand (a cytokine), it brings together two receptor chains and triggers the kinase in each chain to phosphorylate a particular sequenc

19、e in the intracellular domain of the opposing receptor. This phosphorylated site is then recognized by a particular STAT protein that, once bound, gets phosphorylated itself. Once phosphorylated, the STAT dimerizes, moves to the nucleus, and binds DNA.Figure 19-24bThe Ras pathway leading into the do

20、wnstream MAPK pathwayThe mitogen-activated protein kinase (MAPK) pathway that controls activators such as Jun, one of the activators that works at the interferon- enhancer. In this case, the activated receptor induces a cascade of signaling events, ending in activation of an MAPK that phosphorylates

21、 Jun (and other transcriptional regulators). The most common way in which information is passed through signal transduction pathways is via phosphorylation, but proteolysis, dephosphorylation, and other modifications are also used.Signals Control the Activities of Eukaryotic Transcriptional Regulato

22、rs in a Variety of WaysIn eukaryotes, transcriptional regulators are not typically controlled at the level of DNA binding (although there are exceptions). Regulators are instead usually controlled in one of the following two basic ways.Unmasking an Activating Region Unmasking an activating region is

23、 done either by a conformational change in the DNA-bound activator, revealing a previously buried activating region, or by release of a masking protein that previously interacted with, and eclipsed, an activating region. The conformational changes required in each case can be triggered either by bin

24、ding ligand directly or through a ligand-dependent phosphorylation.Figure 19-25Gal4 is controlled by a masking protein. In the absence of galactose, Gal4 is bound to its sites upstream of the GAL1 gene, but it does not activate that gene because another protein, Gal80, binds to Gal4 and occludes its

25、 activating region. Galactose triggers the release of Gal80 and activation of the gene.Signals Control the Activities of Eukaryotic TranscriptionalRegulators in a Variety of WaysTransport into and out of the Nucleus When not active, many activators and repressors are held in the cytoplasm. The signa

26、ling ligand causes them to move to the nucleus, where they act. Release and transport into the nucleus in response to a signal can be mediated through proteolysis of an inhibitor or tethering region or by allosteric changes. There, Cactus is an inhibitory protein that binds the transcriptional regul

27、ator Dorsal in the cytoplasm. In response to a specific signal, Cactus is phosphorylated and destroyed, allowing Dorsal to enter the nucleus, bind specific sites within appropriate enhancers, and regulate the transcription of associated genes. This same mechanism is used to control the activity of N

28、F-B, one of the regulators of -interferon. NF-B is held in the cytoplasm by IB; NF-B is related to Dorsal and IB to Cactus.GENE “SILENCING” BY MODIFICATION OF HISTONES AND DNAThe mechanisms of transcriptional silencingSilencing is a position effect: a gene is silenced because of where it is located,

29、 not in response to a specific environmental signal. In addition, silencing can “spread” over large stretches of DNA, switching off multiple genes, even those quite distant from the initiating event.Figure 19-26The most common form of silencing is associated with a dense form of chromatin called het

30、erochromatin. Telomeres and centromeres are typically composed of repetitive sequences and contain few, if any, protein-coding genes. If a gene is experimentally moved into these regions, that gene is typically switched off.Transcription can also be silenced by methylation of DNA by enzymes called D

31、NA methylases. This kind of silencing is not found in yeast but is common in mammalian cells. Methylation of DNA sequences can inhibit binding of proteins, including the transcriptional machinery, and thereby block gene expression. But methylation can also inhibit expression in another way: some DNA

32、 sequences are recognized only when methylated by specific repressors that then switch off nearby genes, often by recruiting histone modifying enzymes.Figure 19-27Silencing in Yeast Is Mediated by Deacetylation and Methylation of HistonesThe chromatin at the telomere is less acetylated than that fou

33、nd in most of the rest of the genomethe so-called euchromatinwhere genes are more readily expressed. Insulator elements can block the spread of histone modifications. Thus, methylation of Lys-9 in the H3 tail (H3K9) is a modification associated with silenced heterochromatin in these organisms. In co

34、ntrast, other sites of methylation (e.g., Lys-4 on that same tailH3K4) are associated with increased transcription.Figure 19-27Silencing in Yeast Is Mediated by Deacetylation and Methylation of HistonesFigure 19-28In Drosophila, HP1 Recognizes Methylated Histones and Condenses ChromatinThe HP1 prote

35、in interacts with modified chromatin containing methylated histone H3. This particular modification is produced by an enzyme encoded by Su(Var)3-9, a suppressor of so-called variegation. Variegation is seen in some cases when a gene is moved into a region of heterochromatin. Instead of being silence

36、d in all cells all the time, the gene switches between the silenced and expressed state apparently at random, being “on” in some cells and “off” in others. Su(Var)3-9Repression by Polycomb Also Uses Histone MethylationHistone methylationtriggered chromosome condensation is also used by Polycomb (Pc)

37、, an important group of repressors in animal cells. Pc repressors are found in two major protein complexes, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). The PRC2 complex is recruited by sequence-specific DNA-binding proteins (the repressive complex PhoRC) that interact with so-called Polyc

38、omb Response Elements (PREs). PRC2 contains a histone methyltransferase (Enhancer of Zeste) that trimethylates lysine-27 (K27) on the tails of histone H3. This methylation leads to the recruitment of the PRC1 complex, which is thought to either condense chromatin or lead to the positioning of a nucl

39、eosome at or near the transcription start site. Figure 19-29DNA Methylation Is Associated with Silenced Genes in Mammalian CellsSome mammalian genes are kept silent by methylation of nearby DNA sequences. In fact, large regions of the mammalian genome are marked by methylation of DNA sequences, and

40、often DNA methylation is seen in regions that are also heterochromatic. This is because methylated sequences are often recognized by DNA-binding proteins (such as MeCP2) that recruit histone deacetylases and histone methylases, which then modify nearby chromatin. Thus, methylation of DNA can mark si

41、tes where heterochromatin subsequently forms.Figure 19-30Switching a gene off through DNA methylation and histone modification.DNA methylation lies at the heart of a phenomenon called imprinting In a diploid cell, there are two copies of most genes: one copy on a chromosome inherited from the father

42、 and the other copy on the equivalent chromosome from the mother. In the majority of cases, the two alleles are expressed at comparable levels. This is hardly surprisingthey carry the same regulatory sequences and are in the presence of the same regulators; they are also located at the same position

43、 along homologous chromosomes. But there are a few cases in which one copy of a gene is expressed while the other is silent.Two well-studied examples are the human H19 and insulin-like growth factor 2 (Igf2) genes. These are located close to each other on human chromosome 11. In a given cell, one co

44、py of H19 (that on the maternal chromosome) is expressed, whereas the other copy (on the paternal chromosome) is switched off; for Igf2 the reverse is truethe paternal copy is on and the maternal copy is off.Figure 19-31Two regulatory sequences are critical for the differential expression of these genes: an enhancer (downstream from the H19 gene) and an insulator (called th