Function of Activation Domains in Gene-Specific Transcription Factors

基因特异性转录因子激活结构域的功能

• 批准号: 0215758
• 负责人: Alexandre Erkine
• 金额: $ 33万
• 依托单位: LSU Health Sciences Center -Shreveport
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2003-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0215758&HistoricalAwards=false
• 关键词: Function; Activation; Domains; Gene; Specific

Despite major advances in characterizing components of eukaryotic transcription machinery, the mechanisms of the recruitment of these components to gene promoters are poorly understood. Activation domains (ADs) of gene-specific transcription factors are critical for these recruitment steps. Amazingly, there are little requirements for the sequences and structure of ADs, and they are easily interchangeable with preservation of functionality not only between different gene specific activators but even between activators belonging to different eukaryotic phyla. The excess of hydrophobic and acidic amino acid residues in natural and synthetic ADs suggests that their interacting targets may be hydrophobic and basic. The nucleosomal histones are the most abundant proteins in the nucleus with such properties. The PI hypothesizes that histones are among the targets of some ADs and that distortion of promoter nucleosomes by ADs triggers a chain of chromatin remodeling events involving different coactivators. In this project, yeast HSF will be used as a model system. The chromatin remodeling mediated by HSF ADs at heat shock promoters will be tested in yeast strains bearing different AD deletions employing Chromatin ImmunoPrecipitation (ChIP) technique. The involvement of known histone-modifying and nucleosome-remodeling activities will be tested in the strains where these activities will be inactivated. The above hypothesis of AD-nucleosome interactions will be addressed in vivo by an attempt to create a synthetic activator with AD substituted by known histone-binding protein and in vitro by DNase I footprinting experiments using a reconstituted nucleosomal template and recombinant HSF. This project potentially can have an impact on the dynamically developing area of gene transcription regulation by characterizing the general chromatin remodeling mechanisms taking place in any eukaryotic cell. Understanding of general mechanisms of natural and especially synthetic ADs function potentially will have an impact on pharmaceutical directions related to the design of synthetic activators and repressors targeting disease-related genes.

尽管真核生物转录机制的组成部分的特征方面取得了重大进展，这些组成部分的基因启动子的招聘机制知之甚少。基因特异性转录因子的激活结构域（AD）对于这些募集步骤至关重要。令人惊讶的是，对AD的序列和结构几乎没有要求，并且它们不仅在不同的基因特异性激活剂之间，而且甚至在属于不同真核生物门的激活剂之间都可以容易地互换，保留功能。在天然和合成的AD中的疏水性和酸性氨基酸残基的过量表明，它们的相互作用靶标可能是疏水性和碱性的。核小体组蛋白是细胞核中最丰富的具有这种性质的蛋白质。PI假设组蛋白是一些AD的靶点，并且AD对启动子核小体的扭曲触发了一系列涉及不同辅激活因子的染色质重塑事件。本研究以酵母HSF为模型系统。将采用染色质免疫沉淀（ChIP）技术在携带不同AD缺失的酵母菌株中测试由热休克启动子处的HSF AD介导的染色质重塑。将在这些活性将被灭活的菌株中检测已知组蛋白修饰和核小体重塑活性的参与。 AD-核小体相互作用的上述假设将在体内通过尝试用已知的组蛋白结合蛋白取代AD来创建合成激活剂来解决，并且在体外通过使用重构的核小体模板和重组HSF的DNA酶I足迹实验来解决。该项目可能会对基因转录调控的动态发展领域产生影响，通过表征任何真核细胞中发生的一般染色质重塑机制。了解天然的，特别是合成的AD功能的一般机制可能会产生影响的药物方向相关的设计合成的激活剂和抑制剂靶向疾病相关基因。