Molecular Mechanisms in Transcriptional Regulation

描述(由申请人提供)： 对酵母HO基因的研究已经确定了许多重要的转录调节因子，也确定了在后生动物中保守的调节范例。HO的激活涉及转录因子的顺序招募，包括序列特异的DMA结合蛋白、染色质重塑因子、组蛋白乙酰转移酶复合体、建筑转录因子和介体复合体。尽管人们对这些转录激活剂了解很多，但对它们如何机械地激活转录却知之甚少。这一建议结合了遗传和生化方法来理解复杂启动子的调控。 HO的染色质结构是高度抑制的，许多转录因子在复杂的关系中发挥作用来克服这种抑制。我们的实验表明，TATA结合蛋白(TBP)是HO启动子招募的最后一个因子。因此，将因子逐步招募到HO启动子的最终目标是在TATA元件附近组装各种激活剂，以便它们能够在关键时刻协同促进TBP结合。HO-TATA元件被核小体遮挡，我们认为必须移动这个核小体才能与TBP结合和激活HO。我们提议进行实验，以研究包括Swi/Snf和Nhp6结构因子在内的激活剂如何克服核小体抑制，促进TBP与HO的结合。 Swi5是HO表达所必需的，它首先与HO启动子结合，招募染色质修饰因子结合，然后Swi5被降解。值得注意的是，在HO基因转录时，没有Swi5蛋白与HO启动子结合。因此，细胞必须有Swi5存在的“记忆”，并且这种记忆会持续相当长的时间。我们将确定染色质结构的持续变化是否代表这个记忆标记，以及这样的染色质标记是如何产生和保持的。

DESCRIPTION (provided by applicant): Studies of the yeast HO gene have identified many important transcriptional regulators and also identified regulatory paradigms that are conserved in metazoans. Activation at HO involves the sequential recruitment of transcription factors, including sequence specific DMA-binding proteins, chromatin remodeling factors, histone acetyl transferase complexes, architectural transcription factors, and the Mediator complex. Although a great deal is known about these transcriptional activators, relatively little is understood about how mechanistically they activate transcription. This proposal combines genetic and biochemical approaches to understand the regulation of a complex promoter. Chromatin structure at HO is highly repressive, and that numerous transcription factors function in a complex relationship to overcome this repression. Our experiments suggest that TATA-Binding Protein (TBP) is the last factor recruited to the HO promoter. Thus the ultimate goal of the stepwise recruitment of factors to the HO promoter is to assemble various activators in the vicinity of the TATA element, so that they are poised to work in concert to promote TBP binding at the critical time. The HO TATA element is obscured by a nucleosome, and we suggest that this nucleosome must be moved for TBP to bind and for HO activation. Experiments are proposed to examine how activators, including Swi/Snf and the Nhp6 architectural factor, overcome nucleosomal repression to facilitate TBP binding at HO. Swi5, absolutely required for HO expression, binds first to the HO promoter, recruits chromatin modifying factors to bind, and then Swi5 is degraded. Remarkably, there is no Swi5 protein bound to the HO promoter at the time the gene is transcribed. Thus cells must have a "memory" that Swi5 was present, and this memory persists for a remarkably long time. We will determine whether persistent changes in chromatin structure represent this memory mark, and how such a chromatin mark is generated and maintained.