TRANSCRIPTION FACTOR FUNCTION IN CHROMATIN

了解正常细胞生长发育的调控 以及未能适当地监管这些过程是如何导致 异常或疾病状态，有必要确定 细胞转录机器识别的机制 基因启动子和启动转录。POL II的监管 基因表达需要转录激活剂和聚合酶- 相关的通用转录因子(GTF)与 控制基因表达的DNA调控序列。在体内， DNA与组蛋白和非组蛋白形成络合物和缩合物 染色质形成染色质的蛋白质，其基本单位是核小体 大量研究表明，总的来说，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序列可供转录因子访问 功能是基因调控中的一个核心问题，而 细胞生长、发育和分化的调节。 遗传和生化研究表明，组蛋白超乙酰化 和人类SWI/SNF(hSWI/SNF)复合体，它改变了 核小体结构以一种ATP依赖的方式，可以促进一些 染色质基因激活过程中的步骤。SWI/SNF 复合体似乎从酵母到人类都是保守的，而且 组成亚基和相关的同源物已经被牵连到 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的相关性 活跃的转录延伸到所有的真核生物。目的 这项提议是为了表征染色质结构的变化 由hSWI/SNF复合体或组蛋白超乙酰化介导 促进染色质中转录因子的作用。离体 研究将结合纯化的转录激活剂和染色质 利用体外重建技术对成分进行表征 GTF和激活剂与定义的核小体模板的结合 HSWI/SNF和/或过度乙酰化的存在和不存在 组蛋白。GTF和激活剂的能力，单独和在 结合，以进一步改变核小体的结构/稳定性， 从改变后的模板开始转录也将是 检查过了。检测hSWI/SNF对小鼠脑内转录的影响 活体，编码hSWI/SNF显性阴性版本的基因 成分将在哺乳动物细胞中有条件地表达，以 确定核激素受体是否激活了基因 受影响。

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.