Chromatin Remodeling and Transcription Repression

描述（由申请人提供）：ISWI型染色质重塑复合体已被证明参与转录抑制，转录延伸和终止的调节。染色质和染色体区域的重组是细胞发育和分化的基础。我们建议通过(1)ISWI蛋白家族的广泛生化表征和(2)体内突变型与野生型ISWI复合物的分子遗传分析来进一步了解这些过程。我们的目标是通过ISWI型atp依赖性染色质重塑复合体将DNA包装在细胞内的方式，研究调节基因表达和细胞发育的机制。已知这些染色质重塑复合体调节细胞发育和分化的许多不同方面，因此也参与肿瘤的发生。该项目的长期意义在于阐明影响和控制细胞中DNA读取、修复、复制或改变等过程的基本生物学过程。细胞从干细胞发展到最终分化细胞类型的重要关键调控步骤已被证明涉及染色质形式的DNA重组，特别是涉及这种类型的染色质重塑复合体介导的转录抑制。ISWI型复合物也在siRNA介导的沉默和非编码RNA的合成中发挥作用。我们将结合酵母遗传学的优势和我们为研究大型大分子组装的结构和动力学而开发的技术，以缩小ISW2， ISW1a和ISW1b染色质重塑复合体的结构和功能特性，并结合高通量技术，提供细胞中这些特性的基因组视图。所有的缺失和突变都将在体内和体外进行广泛的检查，这样就有可能揭示ISW2、ISW1a和ISW1b染色质重塑的重要机制细节，这些细节在生理学上是重要的，但由于已经完成了潜在的生化分析，因此很容易理解。

DESCRIPTION (provided by applicant): The ISWI type chromatin remodeling complexes have been shown to be involved in transcription repression, and the regulation of transcription elongation and termination. Reorganization of chromatin and regions in chromosomes are fundamental in cellular development and differentiation. We propose to further our understanding of these processes by a two-prong approach of (1) extensive biochemical characterization of the ISWI family of proteins and (2) the molecular genetic analysis of mutant versus wild type ISWI complexes in vivo. PUBLIC HEALTH RELEVANCE Our objective is to study the mechanism of regulating gene expression and cellular development by the manner in which DNA is packaged DNA inside the cell by the ISWI type of ATP-dependent chromatin remodeling complexes. These chromatin remodeling complexes are known to regulate many different aspects of cell development and differentiation, and as such are also involved in oncogenesis. The long term significance of this project is the elucidation of a fundamental biological process that influences and controls those processes in the cell that involves reading, repairing, copying, or changing of DNA. Important key regulatory steps of cells developing from stem cells to terminally differentiated cell types have been shown to involve reorganization of DNA in the form of chromatin and in particular to involve the type of transcription repression mediated by this type of chromatin remodeling complexes. ISWI type complexes also have a role in siRNA mediated silencing and in the synthesis of noncoding RNA. We will incorporate both the strength of yeast genetics and that of the techniques that we have developed for investigating the structure and dynamics of large macromolecular assemblies to narrow in on the structural and functional properties of the ISW2, ISW1a, and ISW1b chromatin remodeling complexes in combination with high through put techniques to afford a genomic view of these properties in the cell. All the deletions and mutations that are made will be examined extensively both in vivo and in vitro such that it will be possible to reveal important mechanistic details of chromatin remodeling by ISW2, ISW1a, and ISW1b that are physiologically important and yet well understood because of the underlying biochemical analysis that has been done.