Chromatin Remodeling and Gene Activation

基因激活涉及对适当信号的调控，因子招募到启动子，最终导致RNA聚合酶II和转录延伸形成起始复合物。这些事件必须在核小体存在的情况下发生，核小体是一种紧凑的结构，能够在每一步阻断转录。为了规避染色质阻滞，真核细胞具有一套染色质重塑和核小体修饰复合物。前者（如SWI/SNF复合物）利用ATP驱动核小体的构象变化，并沿着DNA滑动核小体。后者含有酶活性（例如，组蛋白乙酰化酶），其在翻译后修饰组蛋白以标记它们以供其他复合物识别。遗传学家已经描述了染色质组分和转录之间的许多有趣的联系，但是研究这些联系的结构基础的系统一直缺乏。我们已经开发了这样一个模型系统，包括从酵母中纯化的天然质粒染色质，用于对活性和非活性基因的染色质结构进行高分辨率研究。值得注意的是，他们揭示了激活与核小体的大规模运动和整个基因核小体内的构象变化相关。我们目前的工作主要集中在染色质重塑和组蛋白乙酰化在基因调控中的作用：

Gene activation involves the regulated recruitment of factors to a promoter in response to appropriate signals, ultimately resulting in the formation of an initiation complex by RNA polymerase II and transcript elongation. These events must occur in the presence of nucleosomes, which are compact structures capable of blocking transcription at every step. To circumvent the chromatin block, eukaryotic cells possess a set of chromatin remodeling and nucleosome modifying complexes. The former (e.g. the SWI/SNF complex) use ATP to drive conformational changes in nucleosomes and to slide nucleosomes along DNA. The latter contain enzymatic activities (e.g., histone acetylases) which modify the histones post-translationally to mark them for recognition by other complexes. Geneticists have described many interesting connections between chromatin components and transcription, but a system to investigate the structural basis of these has been lacking. We have developed such a model system, involving native plasmid chromatin purified from the yeast Saccharomyces cerevisiae, to perform high resolution studies of the chromatin structures of active and inactive genes. Remarkably, they reveal that activation correlates with large scale movements of nucleosomes and conformational changes within nucleosomes over entire genes. Our current work is focused on the roles of chromatin remodeling and histone acetylation in gene regulation: Activation of yeast HIS3 results in Gcn4p-dependent, SWI/SNF-dependent mobilization of nucleosomes over the entire gene Kim, McLaughlin, in collaboration with Tsukiyama We chose budding yeast as a model organism because biochemical studies of chromatin structure could be combined with molecular genetics. Current models for the role of the SWI/SNF ATP-dependent chromatin remodeling complex in gene regulation are focused on promoters, where the most obvious changes in chromatin structure occur. However, using our plasmid model system with yeast HIS3, a SWI/SNF-regulated gene, we discovered that induction of HIS3 creates a domain of remodeled chromatin structure that extends far beyond the promoter, to include the entire gene (Kim & Clark, 2002). We addressed the effects of transcriptional activation on the chromatin structure of the yeast HIS3 gene by mapping the precise positions of nucleosomes in uninduced and induced chromatin. In the absence of the Gcn4p activator, the HIS3 gene is organized into a dominant nucleosomal array. In wild type chromatin, this array is disrupted, and several alternative, overlapping nucleosomal arrays are formed. Disruption of the dominant array also requires the SWI/SNF remodeling machine, indicating that the SWI/SNF complex plays an important role in nucleosome mobilization over the entire HIS3 gene. The Isw1 remodeling complex plays a more subtle role in determining nucleosome positions on HIS3, favoring different positions from those preferred by the SWI/SNF complex. We propose that Gcn4p stimulates nucleosome mobilization over the entire HIS3 gene by the SWI/SNF complex. We suggest that the net effect of interplay among remodeling machines at HIS3 is to create a highly dynamic chromatin structure (Kim, McLaughlin, Lindstrom, Tsukiyama & Clark, in revision). Our current work is aimed at elucidating the structure of the remodeled nucleosome. There are at least two possibilities: unstable nucleosomes (remodeled such that they fall apart easily) and nucleosomes with a dramatically altered conformation. Our work on CUP1 and HIS3 indicates that, at least for these two genes, the target of remodeling complexes is a domain rather than just the promoter. This is an important finding, because it suggests that remodeling complexes act on chromatin domains. What is the function of domain remodeling? We speculate that remodeling entire genes might facilitate elongation through nucleosomes by RNA polymerase II. In a collaboration with Dr. Len Lutter, we have compared the structures of yeast and mammalian chromatin, leading to the suggestion that yeast chromatin is composed of relatively ordered arrays of closely spaced nucleosomes that are separated by substantial gaps, possibly corresponding to regulatory regions (Tong et al., 2006). Kim Y, Clark DJ. SWI/SNF-dependent formation of a domain of labile chromatin structure at the yeast HIS3 gene. Proc. Nat. Acad. Sci. USA 2002;99:15381-15386. Tong W, Kulaeva OI, Clark DJ, Lutter LC. Topological analysis of plasmid chromatin from yeast and mammalian cells. J. Mol. Biol. 2006;361:813-822. The yeast Spt10 protein is a sequence-specific activator of the histone genes, possessing a DNA-binding domain fused to a putative histone acetylase domain. Eriksson, Mendiratta, McLaughlin, in collaboration with Landsman and Shen Our early studies showed that induction of CUP1 by copper results in targeted acetylation of nucleosomes at the CUP1 promoter. This acetylation is dependent on SPT10, which encodes a putative histone acetylase (HAT). SPT10 has been implicated as a global regulator of core promoter activity. We confirmed this by expression microarray analysis and then addressed the mechanism of global regulation (Eriksson et al., 2005). We were unable to detect Spt10p at any of the most strongly affected genes in vivo using the chromatin immunoprecipitation (ChIP) assay, but we confirmed its presence at the core histone gene promoters, which it activates. We proposed that in the absence of Spt10p, a shortage of histones might occur, resulting in defective chromatin structure and consequent activation of basal promoters. Consistent with this hypothesis, the spt10? phenotype can be rescued by extra copies of the histone genes and chromatin is poorly assembled in spt10? cells, as shown by irregular nucleosome spacing. Furthermore, we find that Spt10p binds specifically and highly cooperatively to pairs of UAS elements in the core histone promoters (consensus: (G/A)TTCCN6TTCNC), consistent with a direct role in histone gene regulation. No other high affinity sites are predicted in the yeast genome. Our observations are consistent with the idea that the global changes in gene expression in spt10? cells are actually the indirect effects of defective regulation of the core histone genes. Thus, Spt10p is a sequence-specific activator of the histone genes, possessing a DNA-binding domain fused to a likely HAT domain. We have now identified the DNA-binding domain of Spt10p: it contains an unusual zinc finger (His2-Cys2) which has homology to the DNA integrase of foamy retroviruses. We proposed that this integrase might also be a sequence-specific DNA-binding protein (Mendiratta et al., 2006). Our current work has the following aims: (1) Demonstration of the putative histone/protein acetylase activity of Spt10p. (2) Identification of proteins which interact with Spt10p. (3) To determine whether human foamy virus (HFV) integrase is indeed a sequence-specific DNA-binding protein. Eriksson PR, Mendiratta G, McLaughlin NB, Wolfsberg TG, Mari?o-Ramirez L, Pompa TA, Jainerin M, Landsman D, Shen CH, Clark DJ. Global regulation by the yeast Spt10 protein is mediated through chromatin structure and the histone UAS elements. Mol. Cell. Biol. 2005;25:9127-9137. Mendiratta G, Eriksson PR, Shen, CH, Clark DJ. The DNA-binding domain of the yeast Spt10p activator includes a zinc finger that is homologous to foamy virus integrase. J. Biol. Chem. 2006;281:7040-7048. COLLABORATORS Chang-Hui Shen, PhD, CUNY Staten Island, NY. David Landsman, PhD, NCBI, NLM, NIH. Toshio Tsukiyama, PhD, Fred Hutchinson Institute for Cancer Research, Seattle, WA. Len Lutter, PhD, Henry Ford Hospital, Detroit,