ROLE OF HISTONE H3 AND H1 PHOSPHORYLATION ON CHROMATIN

我们提出的研究的长期目标是确定组蛋白H3和H1的磷酸化如何影响染色质结构，以及这些组蛋白修饰如何可能导致白血病和其他致癌疾病。组蛋白H3和H1的磷酸化是细胞快速增殖的标志，这些修饰组蛋白的水平在细胞转化过程中被诱导，并随着细胞周期和细胞生长条件的变化而变化。H3和H1磷酸化位点位于已知在核小体阵列的高阶折叠中起关键作用的正电荷结构域内；磷酸化已被假设导致染色质的去浓缩，这可能更允许转录。此外，我们提出组蛋白H3和H1的磷酸化可能促进atp依赖性染色质“重塑”因子识别其靶染色体位点并促进核小体破坏的能力。本研究将采用生物化学和细胞生物学相结合的方法，直接测试H3烷H1磷酸化在核小体结构、模型核小体阵列折叠以及atp依赖性染色质重塑酶SWI/SNF家族的募集和活性中的作用。这项建议有三个具体目标。在第一个目的中，我们提出了一种生化方法来研究组蛋白H3和H1磷酸化对染色质结构的作用。这一目标可以通过核酸酶探测单核小体、模型核小体阵列的折叠以及atp依赖性染色质重塑酶的SW/SNF家族的募集和活性来解决。这项建议有三个具体目标。在第一个目的中，我们提出了一种生化方法来研究组蛋白H3和H1磷酸化对染色质结构的作用。这一目标是通过核小体阵列的生物物理特性，以及单核小体或核小体阵列中DNA对限制性酶和转录因子的可及性分析来解决的。目的2的目的是验证组蛋白H3和H1的磷酸化增强SWI/ snf样染色质重塑酶功能的假设。在这个目的中，我们提出了单核小体和核小体阵列分析来定量酵母SWI/SNF、酵母RSC和人类SWI/SNF染色质重塑复合物的重塑活性。本研究还将利用能够诱导表达SWI/SNF显性阴性亚基的新型哺乳动物细胞系来研究SWI/SNF在体内的作用。Aim 33将研究磷酸化组蛋白H3和H1的细胞定位，以及人类SWI/SNF蛋白在哺乳动物细胞中的定位。这一目标将通过完整细胞和核基质制备的免疫荧光来解决；染色质免疫沉淀分析也将被采用。

The long-range objective of our proposed research is to determine how phosphorylation of histones H3 and H1 affects chromatin structure and how these histone modifications might contribute to leukemias and other oncogenic disease. Phosphorylation of histones H3 and H1 is a hallmark of rapidly proliferating cells, and the levels of these modified histones are induced upon cellular transformation and vary as a function of cell cycle and cell growth condition. The sites for H3 and H1 phosphorylation are contained within positive charged domains that are known to play crucial roles in the higher order folding of nucleosomal arrays; phosphorylation has been hypothesized to lead to a decondensation of chromatin that might be more permissive for transcription. In addition, we propose that the phosphorylation of histones H3 and H1 might facilitate the ability of ATP-dependent chromatin "remodeling" factors to recognize their target chromosomal loci and facilitate nucleosome disruption. This proposal will use a combination of biochemical and cell biological approaches to directly test the role of H3 ane H1 phosphorylation in the structure of the nucleosome, the folding of model nucleosomal arrays, and in the recruitment and activity of the SWI/SNF family of ATP-dependent chromatin remodeling enzymes. This proposal has three specific aims. In the first aim we propose a biochemical approach to investigate the role o f histone H3 and H1 phosphorylation on chromatin structure. This aim is addressed by nuclease probing of mononucleosome, the folding of model nucleosomal arrays, and in the recruitment and activity of the SW/SNF family of ATP-dependent chromatin remodeling enzymes. This proposal has three specific aims. In the first aim we propose a biochemical approach to investigate the role of histone H3 and H1 phosphorylation on chromatin structure. This aim is addressed by biophysical properties of nucleosomal arrays, and analysis of the accessibility of DNA in the context of a mononucleosome or nucleosomal arrays to restriction enzymes and transcription factors. The objective of aim 2 is to test the hypothesis that phosphorylation of histones H3 and H1 enhances the functioning of SWI/SNF-like chromatin remodeling enzymes. In this aim we propose mononucleosome and nucleosomal array assays to quantitative the remodeling activities of the yeast SWI/SNF, yeast RSC, and human SWI/SNF chromatin remodeling complexes. This aim will also use novel mammalian cell lines that inducibly express dominant negative SWI/SNF subunits to investigate the role of SWI/SNF in vivo. Aim 33 will investigate the cellular localization of phosphorylated histones H3 and H1, and the localization of human SWI/SNF proteins in mammalians cells. This aim will be addressed by immunofluorescence of intact cells and nuclear matrix preparations; chromatin immunoprecipitation analyses will also be employed.