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，它需要染色质结构和化学的特定变化。这一建议的长期目标是了解染色质动力学在真核基因调控中的结构基础、功能和机制。最近对酿酒酵母可诱导的PHO5启动子的研究提供了第一个信号诱导的启动子核小体分解的例子，并导致了关于染色质动力学在基因调控中的性质和功能的工作假说：第一，核小体分解是启动子激活的一般机制；第二，激活的启动子状态的特征是核小体分解和重组的动态平衡(动态平衡假说)；第三，在PHO5处的核小体分解是一个受慢速随机速率常数(核小体分解的随机模型)控制的快速反应；第四，PHO5的表达受启动子的核小体分解速度控制。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结合。通过对体内形成的小启动子环进行凝胶层析，分析启动子染色质结构在从低水平表达向高水平表达转变过程中的多样性，来检验核小体拆解的随机模型。为了测试核小体拆解作为启动子激活机制的普遍性，我们将调查其他启动子是否发生核小体拆解的问题。这项研究将采用在体内形成的染色质环、极限消化和DNA拓扑分析、沉淀和体内交联性研究。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确定核小体拆解因素。转录调控是许多生物现象的基础，从发育过程中的模式形成和细胞增殖的调节，到长期记忆和动物行为。因此，对转录调控分子机制的研究将最终有助于对疾病的理解和治疗。毫不奇怪，调节染色质结构和化学性质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.