Function of Activation Domains in Gene-Specific Transcription Factors

尽管在鉴定真核转录机制的组件方面取得了重大进展，但这些组件被招募到基因启动子的机制却鲜为人知。基因特异性转录因子的激活结构域(ADS)对这些招募步骤至关重要。令人惊讶的是，ADS对序列和结构的要求很低，而且它们很容易互换，不仅可以在不同的基因特异性激活子之间，甚至在不同的真核生物门的激活子之间保持功能。天然和合成ADS中过量的疏水和酸性氨基酸残基表明，它们的相互作用靶标可能是疏水的和碱性的。核小体组蛋白是细胞核中含量最丰富的蛋白质，具有这种特性。PI假设组蛋白是一些ADS的靶标之一，ADS对启动子核小体的扭曲触发了一系列涉及不同辅活化子的染色质重塑事件。本项目将以酵母菌HSF作为模型系统。利用染色质免疫沉淀(CHIP)技术，将在具有不同AD缺失的酵母菌株中测试HSF ADS在热休克启动子上介导的染色质重塑。已知的组蛋白修饰和核小体重塑活动的参与将在这些活动将被灭活的菌株中进行测试。上述AD-核小体相互作用的假说将在体内通过尝试用已知组蛋白结合蛋白取代AD的合成激活剂来解决，并在体外通过使用重组核小体模板和重组HSF的DNase I足迹实验来解决。这个项目可能通过描述任何真核细胞中发生的一般染色质重塑机制，对基因转录调控的动态发展领域产生影响。了解自然的，特别是合成的ADS功能的一般机制，可能会对与设计针对疾病相关基因的合成激活剂和抑制物相关的药物方向产生影响。

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.