Chromatin and Transcriptional Regulation of CHA1 in Yeast

该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。在包括人类在内的高等生物中，DNA被包裹在组蛋白八聚体周围，被包装成核小体。这对DNA转录为RNA所需的蛋白质-DNA相互作用造成了潜在的障碍，因此在许多基因上，这些核小体必须被改造以允许转录发生。对于一些基因，允许染色质重塑的酶和途径已经被确定，但对于许多其他基因，这些途径仍然未知。为了获得染色质调控转录的新见解，本研究将重点研究酿酒酵母的CHA1基因。在培养基中加入丝氨酸可诱导CHA1启动子，激活后染色质发生重构。遗传学和生物化学方法将被用来确定在激活过程中CHA1染色质结构重塑的机制。在缺乏诱导信号的情况下，CHA1的抑制也将被研究。结果表明，在非诱导条件下，组蛋白H3氨基末端的缺失导致CHA1转录强烈，并伴有染色质重塑。导致CHA1抑制的新的组蛋白H3突变已经被分离出来，并将用于抑制筛选，以发现导致组蛋白介导的CHA1抑制的基因。这项工作的意义在于探究了染色质介导的基因调控的基本机制。染色质与发育中的基因调控和激素调控有关，因此具有重要的基础意义。由于CHA1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包括人类在内的高等真核生物之间高度保守，这些研究将揭示在真核生物王国中可能普遍存在的调控方面。

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.