TRANSCRIPTION FACTOR INTERACTIONS WITH NUCLEOSOMES

描述：建立和维持适当的转录 监管对于适当的发展和差异化至关重要， 建立适当转录模式的错误与 疾病状态，如癌症。 非活性基因的启动子区 真核生物组装成核小体（并且通常是更高级的 结构）;核小体的存在抑制了许多步骤， 转录过程。 广泛的遗传、结构和生物化学 有证据表明，核小体结构的动态重塑超过一个 启动子在转录激活过程中可能是一个关键组成部分 基因调控。 蛋白质复合物已在真核生物中得到表征 重塑核小体结构，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 这些复合物都含有一种与酵母相关的蛋白质 SW12/SNF2 protein. SW 12/SNF 2蛋白是一种多蛋白， 复合物，其能够改变核小体结构并促进 转录因子结合核小体，无论是在一个ATP依赖的 方式 类似的ATP依赖性核小体重塑活动已经被证实。 在人SWI/SNF（hSWI/SNF）复合物和果蝇中发现 一种称为SNF 2的活性，包括称为ISWI的SW 12/SNF 2相关基因。 研究者建议在以下研究中表征SW 12/SNF 2相关复合物： 人类细胞，并测试假设，这些复合物可能发挥关键作用， 在转录激活中的作用。 有三种基因 迄今为止在人类中发现的与酵母高度相似的 SWI 2/SNF 2：BRG 1和hBRM在整个蛋白质中具有广泛的相似性， 与hSNF 2L在ATP酶结构域上具有相似性， 与果蝇基因ISWI紧密相连。 他建议继续 纯化含有这些蛋白质的复合物，并表征 这些复合物是否与RNA聚合酶II相关。 他会比较 这些复合物改变核小体结构、促进 转录因子加载，并调节转录起始和 在核小体模板上的延伸。 他会表达显性否定 在哺乳动物细胞中以有条件的方式表达这些蛋白质， 确定这些突变体是否改变了肌肉细胞的分化， 热休克位点的表达。 本文中描述的实验 提案将提供SWI/SNF相关的生物化学表征 复合物，并将确定这些复合物的天然目标。

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.