TRANSCRIPTION FACTOR INTERACTIONS WITH NUCLEOSOMES

描述：建立和维护适当的转录 监管对适当的发展和差异化至关重要，以及 在建立适当的转录模式方面的错误与 疾病状态，如癌症。非活性基因启动子区域的研究 真核生物被组装成核小体(通常是更高级的 结构)；核小体的存在抑制了许多步骤 转录过程。广泛的遗传、结构和生化 证据表明，核小体结构的动态重塑 转录激活过程中的启动子可能是一个关键成分 基因调控。蛋白质复合体已经在真核生物中得到了表征 重塑了核小体结构，生化和遗传数据表明 它们可能是转录激活的必要组成部分 染色质。这些复合体都含有一种与酵母有关的蛋白质 SW12/SNF2蛋白。SW12/SNF2蛋白是一种多蛋白 一种能够改变核小体结构并促进 转录因子与核小体结合，两者都依赖于ATP 举止。类似的依赖于ATP的核小体重塑活动已经被 在人SWI/SNF(hSWI/SNF)复合体和果蝇中发现 称为NURF的活动，包括称为ISWI的SW12/SNF2相关基因。 研究人员建议将SW12/SNF2相关的络合物表征在 人类细胞，并检验这些复合体可能起关键作用的假设 在转录激活中的作用。有三个基因被认为是 迄今在与酵母高度相似的人类中发现 SWI2/SNF2：BRG1和hBRM在整个蛋白质中具有广泛的相似性， HSNF2L在ATPase结构域上具有相似性，并具有广泛的相似性 自始至终都有果蝇基因iswi。他提议继续 纯化含有这些蛋白质的复合体，并对其进行表征 这些复合体是否与RNA聚合酶II有关。他将比较 这些复合体改变核小体结构的能力，有助于 转录因子的加载，并调节转录起始和 核小体模板上的伸长。他会表现出显性的否定 在哺乳动物细胞中以有条件的方式将这些蛋白质的版本 确定这些突变体是否改变了肌肉细胞的分化或 热休克基因座的表达。本文件中描述的实验 建议书将提供SWI/SNF相关的生化特征 人类体内的复合体，并将识别这些复合体的自然目标。

DESCRIPTION: Establishment and maintenance of appropriate transcriptional regulation is essential to proper development and differentiation, and mistakes in establishing appropriate transcription patterns are linked to disease states such as cancer. Promoter regions of inactive genes in eukaryotes are assembled into nucleosomes (and frequently higher order structures); the presence of nucleosomes inhibits many steps in the transcription process. Extensive genetic, structural and biochemical evidence suggests that the dynamic remodeling of nucleosome structure over a promoter during transcriptional activation is likely to be a key component of gene regulation. Protein complexes have been characterized in eukaryotes that remodel nucleosome structure, and biochemical and genetic data suggest that they might be a necessary component of transcriptional activation in chromatin. These complexes all contain a protein related to the yeast SW12/SNF2 protein. The SW12/SNF2 protein is a member of a multiprotein complex which is capable of altering nucleosome structure and facilitating transcription factor binding to nucleosomes, both in an ATP-dependent manner. Similar ATP-dependent nucleosome remodeling activities have been found with the human SWI/SNF (hSWI/SNF) complexes and with a Drosophila activity termed NURF that includes a SW12/SNF2 related gene called ISWI. The investigator proposes to characterize the SW12/SNF2-related complexes in human cells, and to test the hypothesis that these complexes might play key roles in transcriptional activation. There are three genes that have been identified to date in humans with high degrees of similarity to yeast SWI2/SNF2: BRG1 and hBRM have extensive similarity throughout the protein, and hSNF2L has similarity in the ATPase domain and has extensive similarity throughout with the Drosophila gene ISWI. He proposes to continue the purification of complexes that contain these proteins, and to characterize whether these complexes associate with RNA polymerase II. He will compare the ability of these complexes to alter nucleosome structure, to facilitate transcription factor loading, and to modulate transcription initiation and elongation on nucleosomal templates. He will express dominant negative versions of these proteins in a conditional manner in mammalian cells to determine whether these mutants alter differentiation of muscle cells or expression of the heat shock loci. The experiments described in this proposal will provide a biochemical characterization of SWI/SNF related complexes in humans, and will identify natural targets of those complexes.