Chromatin and Transcriptional Regulation of CHA1 in Yeast

本项目的长期目标是对染色质介导的基因调控机制有新的认识。包括人类在内的高等生物体中的DNA通过包裹组蛋白八聚体而包装成核小体。 这对DNA转录成RNA所需的蛋白质-DNA相互作用产生了潜在的障碍，因此在许多基因中，这些核小体必须被重塑以允许转录发生。 允许染色质重塑的酶和途径已被确定为一些基因，但对于许多其他基因，这些途径仍然未知。 为了获得新的见解染色质的转录调控，这项工作将集中在酿酒酵母CHA 1基因。 通过向生长培养基中添加丝氨酸，CHA 1启动子易于诱导，并且其染色质在激活后重塑。 遗传和生物化学的方法将被用来确定的机制，其中CHA 1染色质结构是在激活过程中重塑。还将研究在不存在诱导信号的情况下对CHA 1的抑制。 研究表明，组蛋白H3氨基末端的缺失在未诱导的条件下引起强的CHA 1转录，伴随着染色质重塑。 新的组蛋白H3突变，导致CHA 1去阻遏已被分离出来，并将用于抑制筛选，以揭示基因组蛋白介导的CHA 1阻遏。 这项工作的意义在于它的染色质介导的基因调控的基本机制的调查。 染色质与发育中的基因调节和激素调节有关，因此具有根本的重要性。 由于CHA 1基因的重塑不使用已知的染色质重塑机制，这些研究有望对基因调控的这一方面产生根本性的新见解。 此外，由于参与基因调控和染色质的蛋白质在酵母和高等真核生物（包括人类）之间高度保守，这些研究将揭示在真核生物界可能常见的调控方面。

The long term goal of this project is to gain new understanding into mechanisms of chromatin-mediated gene regulation. DNA in higher organisms, including humans, is packaged into nucleosomes by wrapping around an octamer of histone proteins. This creates a potential impediment to protein-DNA interactions needed for transcription of DNA into RNA, and so at many genes these nucleosomes must be remodeled to allow transcription to occur. Enzymes and pathways that allow chromatin remodeling have been identified for some genes, but for many others these pathways remain unknown. To gain new insight into regulation of transcription by chromatin, this work will focus on the Saccharomyces cerevisiae CHA1 gene. The CHA1 promoter is readily inducible by addition of serine to growth medium, and its chromatin is remodeled upon activation. Genetic and biochemical approaches will be used to ascertain the mechanism by which CHA1 chromatin structure is remodeled during activation. Repression of CHA1 in the absence of inducing signal will also be investigated. It was shown that loss of the histone H3 amino terminus causes strong CHA1 transcription under uninduced conditions, accompanied by chromatin remodeling. Novel histone H3 mutations that cause CHA1 derepression have been isolated, and will be used in a suppressor screen to uncover genes contributing to histone-mediated CHA1 repression. The significance of this work lies in its investigation of basic mechanisms of chromatin-mediated gene regulation. Chromatin has been implicated in gene regulation in development and in hormone regulation, and so is of fundamental importance. Since remodeling of the CHA1 gene does not use known mechanisms of chromatin remodeling, these studies are expected to yield fundamental new insight into this aspect of gene regulation. Furthermore, since the proteins involved in gene regulation and in chromatin are highly conserved between yeast and higher eukaryotes, including humans, these studies will reveal aspects of regulation that are likely to be common across the eukaryotic kingdom.