Chromatin Remodeling and Gene Activation

本摘要分为两个部分，反映了两个不同具体目标的工作： 1.转录激活和SWI/SNF依赖的核小体动员 我们选择发芽酵母作为模式生物，是因为染色质结构的生化研究可以与分子遗传学相结合。目前关于SWI/SNFATP依赖的染色质重塑复合体在基因调控中作用的模型主要集中在启动子上，其中染色质结构发生最明显的变化。然而，使用我们的质粒模型系统与HIS3，一个SWI/SNF调节的基因，我们发现转录激活创建了一个重塑的染色质结构域，远远超出启动子，包括整个基因。我们通过定位核小体在基础表达和转录激活染色质中的精确位置，研究了转录激活对HIS3染色质结构的影响。在缺乏Gcn4p激活子的情况下，HIS3基因被组织成一个显性的核小体阵列。在野生型染色质中，这种排列被打乱，形成几个交替的、重叠的核小体排列。优势阵列的破坏还需要SWI/SNF重塑机器，这表明SWI/SNF复合体在核小体动员中发挥着重要作用。Isw1重塑复合体在决定核小体在HIS3上的位置方面起着更微妙的作用，有利于与SWI/SNF复合体不同的位置。我们认为Gcn4p通过SWI/SNF复合体刺激整个HIS3基因的核小体动员。我们认为，在HIS3上，重塑机器之间的相互作用的净影响是创造一个高度动态的染色质结构(Kim等人，2006年)。我们对HIS3的研究和我们早期对CUP1的研究表明，至少对于这两个基因来说，重塑复合体的目标是一个结构域，而不仅仅是启动子。这是一个重要的发现，因为它表明重塑复合体作用于染色质结构域。结构域重构的作用是什么？我们推测，重塑整个基因可能有助于RNA聚合酶II通过核小体延长。我们目前的工作旨在阐明重塑的核小体的结构。至少有两种可能性：不稳定的核小体(改造后很容易解体)和构象发生显著变化的核小体。 首页--期刊主要分类--期刊细介绍--期刊题录与文摘--期刊详细文摘内容酿酒酵母HIS3的激活导致Gcn4p依赖、SWI/SNF依赖的核小体在整个基因上的动员。摩尔。牢房。比奥尔。2006；26：8607-8622。 2.酵母Spt10蛋白含有一个DNA结合域，与一个可能的组蛋白乙酰化酶结构域融合 我们已经表明，铜诱导CUP1导致CUP1启动子上核小体的靶向性乙酰化(沈等人，2002；Clark和Shenin，2006)。这种乙酰化依赖于SPT10，它编码一个可能的组蛋白乙酰基酶(HAT)。SPT10被认为是核心启动子活性的全球调节因子。我们通过表达芯片分析证实了这一点，并探讨了全局调控的机制。使用染色质免疫沉淀(CHIP)分析(包括CUP1)，我们无法在体内任何受影响最强的基因上检测到Spt10p，但我们证实了它存在于核心组蛋白基因启动子处，它激活了组蛋白基因启动子。我们提出的证据表明，在没有Spt10p的情况下，形成了一个有缺陷的染色质结构，从而激活了基本启动子。此外，我们发现Spt10p与核心组蛋白启动子中的一对上游激活序列(UAS元件)特异性地和高度协同地结合(共识：(G/A)TTCC N6 TTCNC)，这与组蛋白基因调控中的直接作用一致。在酵母基因组中没有预测到其他高亲和力的位点。我们的观察结果与以下观点是一致的，即spt10缺失细胞中基因表达的全球变化实际上是核心组蛋白基因调控缺陷的间接影响。因此，Spt10p是组蛋白基因的序列特异性激活子，具有与可能的HAT结构域融合的DNA结合域。我们已经确定了Spt10p的DNA结合域：它含有一个不寻常的锌指(His2-Cys2)，它与泡沫逆转录病毒的DNA整合酶具有同源性。我们认为这个整合酶也可能是一种序列特异性的DNA结合蛋白(Mendiratta等人，2006年)。我们还表明Spt10p是一种二聚体，N-末端结构域是形成二聚体所必需的(Mendiratta等人，2007年)。我们目前的工作有以下几个目的：(1)确证Spt10p的组蛋白/蛋白乙酰基酶活性。(2)鉴定与Spt10p相互作用的蛋白质。(3)确定核心组蛋白基因调控细胞周期的分子机制。 我们还启动了一个新的项目，以确定人类泡沫病毒(HFV)整合酶是否确实是一种序列特异性DNA结合蛋白。 孟迪拉塔G，埃里克森公关，克拉克DJ。酵母Spt10P激活剂与组蛋白UAS的协同结合 元件是通过N-末端二聚结构域来调节的。核子。《酸度研究》2007；35：812-821。 合作者 华盛顿州西雅图弗雷德·哈钦森癌症研究中心博士Toshio Tsukiyama。

This summary is divided into two sections, reflecting work on two different specific aims: 1. Transcriptional activation and SWI/SNF-dependent nucleosome mobilization 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 HIS3, a SWI/SNF-regulated gene, we discovered that transcriptional activation creates a domain of remodeled chromatin structure that extends far beyond the promoter, to include the entire gene. We addressed the effects of transcriptional activation on the chromatin structure of HIS3 by mapping the precise positions of nucleosomes in basal expressing and transcriptionally activated 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. 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 et al., 2006). Our work on HIS3 and our earlier work on CUP1 indicate 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. 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. Kim Y, McLaughlin N, Lindstrom, K, Tsukiyama T, Clark DJ. Activation of Saccharomyces cerevisiae HIS3 results in Gcn4p-dependent, SWI/SNF-dependent mobilisation of nucleosomes over the entire gene. Mol. Cell. Biol. 2006;26:8607-8622. 2. The yeast Spt10 protein contains a DNA-binding domain fused to a putative histone acetylase domain We have shown that induction of CUP1 by copper results in targeted acetylation of nucleosomes at the CUP1 promoter (Shen et al., 2002; Clark and Shen, 2006). 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. We were unable to detect Spt10p at any of the most strongly affected genes in vivo using the chromatin immunoprecipitation (ChIP) assay (including CUP1), but we confirmed its presence at the core histone gene promoters, which it activates. We presented evidence that a defective chromatin structure is formed in the absence of Spt10p, with consequent activation of basal promoters. Furthermore, we find that Spt10p binds specifically and highly cooperatively to pairs of upstream activating sequences (UAS elements) in the core histone promoters (consensus: (G/A)TTCC N6 TTCNC), 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-null 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 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 propose that this integrase might also be a sequence-specific DNA-binding protein (Mendiratta et al., 2006). We have also shown that Spt10p is a dimer and that the N-terminal domain is required for dimer formation (Mendiratta et al., 2007). 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) Identifying the molecular mechanisms underpinning the cell cycle regulation of the core histone genes. We have also initiated a new project to determine whether human foamy virus (HFV) integrase is indeed a sequence-specific DNA-binding protein. Mendiratta G, Eriksson PR, Clark DJ. Cooperative binding of the yeast Spt10p activator to the histone UAS elements is mediated through an N-terminal dimerisation domain. Nucl. Acids Res. 2007;35:812-821. COLLABORATORS Toshio Tsukiyama, PhD, Fred Hutchinson Cancer Research Center, Seattle, WA.