Structure and Function of Gene Regulatory Complexes

描述(申请人提供)：人体中的每个体细胞都含有相同的遗传信息，但分化细胞的不同功能是通过精确控制基因活动来实现的。适当的基因调控对细胞的正常功能至关重要，而人类疾病可以也确实是由于正常基因调控的缺失或规避造成的。因此，我们的长期目标是了解真核细胞中基因表达是如何调控的。这项拨款提案侧重于转录共激活因子，使TATA结合蛋白(TBP)能够与其TATA盒DMA结合位点结合，这是启动基因转录的关键步骤。大量数据表明，SAGA组蛋白乙酰转移酶(HAT)复合体通过将TBP运送到启动子和乙酰化启动子区域的核小体而发挥辅助激活作用。尽管在过去十年中取得了重大进展，但这两种活动的分子机制还不是很清楚。例如，我们不确切地知道佐贺是如何招募TBP到核心推动者的。同样，虽然我们了解HAT亚单位如何识别组蛋白尾部底物，但我们对HAT或其他染色质酶如何识别其生理核小体底物没有同等的了解。我们的具体目标是解决这些不足之处。特别是，我们建议：1.确定SagA的Spt8亚基如何与TBP结合。我们将进行诱变和标记转移实验，以确定相互作用的TBP和Spt8表面。2.确定SAGA如何将TBP招募到推动者TATA盒中。我们将开发和利用体外实验来研究SAGA如何将TBP运送到启动子的分子机制。3.确定Ada2/Ada3/Gcn5和Piccolo/NuA4 HAT酶如何与核小体相互作用。我们将使用光亲和标记来确定与核小体密切相关的HAT复合体亚基，并通过缺失和突变研究来定义溴和染色域在核小体结合和乙酰化中的新功能。我们使用已定义成分的生化研究将通过解决其他方法不容易解决的机械性问题来补充大量的体内可用数据。

DESCRIPTION (provided by applicant): Each somatic cell in a human body contains the same genetic information, but the diverse function of differentiated cells is made possible through precise control of gene activity. Proper gene regulation is crucial for the normal function of a cell, and human diseases can and do result from loss or circumvention of normal gene regulation. Our long term objective is therefore to understand how gene expression is regulated in eukaryotic cells. This grant proposal focuses on transcriptional coactivators which enable the TATA-binding protein (TBP) to bind to its TATA-box DMA binding site, a critical step in initiating gene transcription. Significant data indicates that the SAGA histone acetyltransferase (HAT) complex functions as a coactivator by delivering TBP to the promoter and by acetylating nucleosomes in the promoter region. Despite major advances in the last decade, the molecular mechanism for either activity is not well-understood. For example, we do not know precisely how SAGA recruits TBP to the core promoter. Similarly, while we do understand how HAT subunits recognize histone tail substrates, we do not have an equivalent understanding of how HAT or other chromatin enzymes recognize their physiological nucleosome substrate. Our specific aims are directed to address these deficiencies. In particular, we propose to: 1. Determine how SAGA's Spt8 subunit binds to TBP. We will perform mutagenesis and label transfer experiments to define the TBP and Spt8 surfaces that interact. 2. Determine how SAGA recruits TBP to a promoter TATA-box. We will develop and utilize in vitro assays to examine the molecular mechanism for how SAGA delivers TBP to a promoter. 3. Determine how the Ada2/Ada3/Gcn5 and Piccolo/NuA4 HAT enzymes interact with nucleosomes. We will use photoaffinity labeling to identify HAT complex subunits in close proximity to nucleosomes, and deletion and mutagenesis studies to define novel functions for the bromo and chromodomains in the binding and acetylation of nucleosomes. Our biochemical studies using defined components will complement the considerable available in vivo data by addressing mechanistic questions not easily tackled by other approaches.