HETEROCHROMATIN REGULATION BY IMITATION SWITCH COMPLEXES

真核生物的基因表达受位置信息和高阶抑制性染色质结构（异染色质）调控。染色质重塑复合体与这种抑制性染色质的破坏有关，导致转录、DNA复制和DNA修复的调节。这些染色质修饰复合物组分的突变可导致严重的发育缺陷（如ATRx、MeCP2）和癌症发展（如SNF5、CBP）。我们专注于含有人类染色质重塑因子模仿开关（ISWI）蛋白的染色质重塑复合物。我们假设含有iswi的复合物通过改变高阶染色质结构（即异染色质）来促进基因表达。这三个目标的目的是提供一个彻底的生化和功能表征人类ISWI-containing复合物。目的1将确定1-1.5 MDa含ISWI/WSTF复合物的剩余亚基，并描述将开始定义复合物和复合物中单个蛋白质的染色质重塑活性的实验。目的2描述了人类iswi复合物的详细功能分析，重点是它们在异染色质调节中的假定作用。Aim 3将工作扩展到包含人类SNF2L的组织特异性染色质重塑复合物，并使用表达flag标记的SNF2L蛋白的细胞系来鉴定和功能表征SNF2L染色质重塑复合物。

Gene expression in eukaryotes is modulated by positional information and higher order repressive chromatin structure (heterochromatin). Chromatin remodeling complexes have been implicated in the disruption of such repressive chromatin resulting in modulation of transcription, DNA replication, and DNA repair. Mutations in the components of these chromatin modifying complexes leads to severe developmental defects (e.g. ATRx, MeCP2) and cancer development (e.g. SNF5, CBP). We are focused on chromatin remodeling complexes containing the human chromatin-remodeling factor Imitation Switch (ISWI) protein.We hypothesize that ISWI-containing complexes facilitate gene expression by altering the structure of a higher order chromatin structure, known as heterochromatin. The three Aims are designed to provide a thorough biochemical and functional characterization of human ISWI-containing complexes. Aim 1 will identify the remaining subunits of the 1-1.5 MDa ISWI/WSTF- containing complex and describe experiments that will begin to define the chromatin remodeling activity of the complex and the individual proteins in the complex. Aim 2 describes a detailed functional analysis of human ISWI-complexes with emphasis on their putative role in heterochromatin modulation. Aim 3 extends the work to tissue-specific chromatin remodeling complexes containing human SNF2L and use a cell line expressing FLAG-tagged SNF2L protein to identify and functionally characterize the SNF2L chromatin remodeling complex.