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，是生物调控的关键环节，需要染色质结构和化学发生特定的变化。本研究的长期目标是了解真核生物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PHO5启动子的研究首次提供了信号诱导启动子核小体拆卸的例子，并对染色质动力学在基因调控中的性质和功能提出了以下工作假设：首先，核小体拆卸是启动子激活的一般机制；第二，激活启动子状态的特征是核小体分裂和重组的动态平衡（动态平衡假说）；第三，PHO5的核小体拆卸是一个由缓慢的随机速率常数（核小体拆卸的随机模型）控制的快速反应；第四，PHO5的表达受启动子核小体拆卸的速率控制。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在激活条件下新合成的组蛋白是否与启动子DNA相关。通过凝胶色谱分离体内形成的小启动子环，分析从低水平到高水平表达转变过程中启动子染色质结构的多样性，从而测试核小体拆卸的随机模型。为了测试核小体拆卸作为启动子激活机制的普遍性，我们将研究核小体拆卸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，限制消化和DNA拓扑分析，沉积和体内交联研究。催化核小体分解的因素尚不清楚。我们将使用遗传和生化工具来识别核小体拆卸因子。转录控制是许多生物现象的基础，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，如本文所建议的，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素与肿瘤的发生有关。

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.