TRANSCRIPTION FACTOR FUNCTION IN CHROMATIN

了解正常细胞生长发育的调控 以及未能适当调节这些过程如何导致 异常或疾病状态，有必要确定 细胞转录机制识别 基因启动子并启动转录。 第二阶段的政策 基因表达需要转录激活因子和聚合酶- 相关的一般转录因子（GTF），以与 控制基因表达的DNA调节序列。 在体内， DNA与组蛋白和非组蛋白复合和凝聚 蛋白质形成染色质，其基本单位是核小体。 许多研究表明，一般来说，核小体和 染色质成分抑制转录因子与DNA的结合， 转录起始和转录延伸。 理解 细胞如何使DNA序列可被转录因子 功能是基因调控中的一个重要问题， 调节细胞生长、发育和分化。 遗传和生化研究表明，组蛋白的超乙酰化 和人SWI/SNF（hSWI/SNF）复合物，其改变 核小体结构以ATP依赖的方式，可以促进一些 染色质中基因激活过程中的步骤。 SWI/SNF 复合物似乎是保守的酵母到人类， 组成亚基和相关同系物已经被牵连在 细胞生长、胚胎发育、分化和逆转录病毒 整合，除了转录激活。 类似地， 高乙酰化组蛋白的存在与基因 所有真核生物都有活跃的转录。的目的 这个建议是为了描述染色质结构的改变 由hSWI/SNF复合物或组蛋白超乙酰化介导 促进染色质中转录因子的功能。体外 研究将结合联合收割机纯化的转录激活因子和染色质 采用体外复溶技术表征组分 GTF和激活剂与确定的核小体模板的结合， hSWI/SNF和/或超乙酰化 组蛋白 GTF和激活剂单独和联合应用的能力 组合，以进一步改变核小体的结构/稳定性， 并且从改变的模板开始转录也将是 考察 为了检测hSWI/SNF对转录的影响， 体内，编码hSWI/SNF的显性阴性形式的基因 组分将在哺乳动物细胞中有条件地表达， 确定核激素受体的基因激活是否 影响。

To understand the regulation of normal cell growth and development and how failure to appropriately regulate these processes leads to abnormal or diseased states, it is necessary to determine the mechanisms by which the cellular transcription machinery recognizes gene promoters and initiates transcription. The regulation of pol II gene expression requires transcriptional activators and the polymerase- associated general transcription factors (GTFs) to associate with the DNA regulatory sequences that control gene expression. In vivo, DNA is complexed and condensed with histones and nonhistone proteins to form chromatin, the basic unit of which is the nucleosome. Numerous studies have indicated that, in general, nucleosomes and chromatin components inhibit transcription factor binding to DNA, transcription initiation, and transcription elongation. Understanding how the cell makes DNA sequences accessible for transcription factor function is an issue of central importance in gene regulation and the regulation of cell growth, development, and differentiation. Genetic and biochemical studies indicate that histone hyperacetylation and the human SWI/SNF (hSWI/SNF) comples, which alters nucleosome structure in an ATP dependent manner, can facilitate some steps in the process of gene activation in chromatin. The SWI/SNF complex appears to be conserved from yeast to humans, and constituent subunits and related homologues have been implicated in cell growth, embryonic development, differentiation, and retroviral integration, in addition to transcriptional activation. Similarly, the correlation between the presence of hyperacetylated histones and genes being actively transcribed extends to all eukaryotes. The purpose of this proposal is to characterize how alterations in chromatin structure mediated by the hSWI/SNF complex or histone hyperacetylation facilitate the function of transcriptional factors in chromatin. In vitro studies will combine purified transcriptional activators and chroatin components with in vitro reconstitution techniques to characterize binding of GTFs and activators to defined nucleosomal templates in the presence and absence of hSWI/SNF and/or hyperacetylated histones. The ability of GTFs and activators, alone and in combination, to further alter the structure/stability of the nucleosome, and to initiate transcription from the altered template will also be examined. To examine the effects of hSWI/SNF on transcription in vivo, genes encoding dominant negative versions of hSWI/SNF components will be conditionally expressed in mammalian cells to determine whether gene activation by nuclear hormone receptors is affected.