Dynamics and Function of Signal-Induced Nucleosome Disassembly

描述（申请人提供）：转录是基因表达的第一步，也是生物调控的关键点，需要染色质结构和化学发生特异性变化。这项计划的长期目标是了解真核基因调控中染色质动力学的结构基础、功能和机制。最近对酿酒酵母（Saccharomycescerevisiae）的诱导型PHO 5启动子的研究提供了信号诱导启动子核小体解体的第一个例子，并导致了以下关于染色质动力学在基因调控中的性质和功能的工作假设：首先，核小体解体是启动子激活的一般机制;第二，激活的启动子状态的特征在于核小体解体和重组的动态平衡（动态平衡假说）;第三，PHO 5的核小体解体是一个由缓慢的随机速率常数控制的快速反应（核小体解体的随机模型），和第四，PHO 5的表达是由启动子核小体解体的速率控制。为了验证动态平衡假说，我们将使用染色质免疫沉淀来确定新合成的组蛋白是否在激活条件下与启动子DNA结合。核小体解体的随机模型将通过分析启动子染色质结构的多样性进行测试，在从低到高水平的表达的过渡，通过凝胶色谱分离的小启动子环在体内形成。为了测试核小体解体作为启动子激活机制的普遍性，我们将研究核小体解体是否发生在其他启动子上的问题。研究将采用体内形成的染色质环、限制性消化和DNA拓扑分析、沉降和体内交联研究。催化核小体解体的因素尚不清楚。我们将使用遗传和生化工具来鉴定核小体解体因子。转录调控是许多生物学现象的基础，从发育中的模式形成和细胞增殖的调节到长期记忆和动物行为。研究转录控制的分子机制，如这里所建议的，将最终有助于理解和治疗疾病。毫不奇怪，调节染色质结构和化学的因子与肿瘤发生有关。

DESCRIPTION (provided by applicant): Transcription is the first step of gene expression and a key point of biological regulation, which requires specific changes in chromatin structure and chemistry. The long-term objective of this proposal is to understand the structural underpinnings, functions and mechanisms of chromatin dynamics in the regulation of eukaryotic genes. Recent work on the inducible PHO5 promoter of Saccharomyces cerevisiae provided the first example of signal-induced promoter nucleosome disassembly, and has led to the following working hypotheses concerning the nature and function of chromatin dynamics in gene regulation: first, nucleosome disassembly is a general mechanism of promoter activation; second, the activated promoter state is characterized by a dynamic equilibrium of nucleosome dis- and reassembly (dynamic equilibrium hypothesis); third, nucleosome disassembly at PHO5 is a fast reaction governed by a slow stochastic rate constant (stochastic model of nucleosome disassembly), and fourth, PHO5 expression is controlled by the rate of promoter nucleosome disassembly. To test the dynamic equilibrium hypothesis, we will use chromatin immunoprecipitation to determine if newly-synthesized histones associate with promoter DNA under activating conditions. The stochastic model of nucleosome disassembly will be tested by analyzing the diversity of promoter chromatin structure in the transition from low to high levels of expression by gel chromatographic fractionation of small promoter circles formed in vivo. To test the generality of nucleosome disassembly as a mechanism of promoter activation, we will investigate the question of whether nucleosome disassembly occurs at other promoters. The investigation will employ chromatin circles formed in vivo, limit digestion and DNA topology analyses, sedimentation and in vivo cross-linking studies. The factors catalyzing nucleosome disassembly are unknown. We will use genetic and biochemical tools to identify nucleosome disassembly factors. Transcriptional control underlies many biological phenomena from pattern formation in development and the regulation of cell proliferation to long term memory and animal behavior. Investigations of the molecular mechanisms of transcriptional control, as suggested here, will thus eventually contribute the understanding and treatment of disease. Not surprisingly, factors regulating chromatin structure and chemisty have been implicated in oncogenesis.