Structure and Function of Gene Regulatory Complexes

描述（由申请人提供）：人体中的每个体细胞都含有相同的遗传信息，但通过精确控制基因活性，分化细胞的不同功能成为可能。适当的基因调控对细胞的正常功能至关重要，人类疾病可以而且确实是由于正常基因调控的丧失或规避而导致的。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。这项拨款建议的重点是转录辅激活因子，使TATA结合蛋白（TBP）结合到其TATA盒DMA结合位点，启动基因转录的关键步骤。重要的数据表明，佐贺组蛋白乙酰转移酶（HAT）复合物的功能作为一个辅激活剂，提供TBP的启动子和乙酰化的启动子区域的核小体。尽管在过去的十年中取得了重大进展，但这两种活性的分子机制都没有得到很好的理解。例如，我们不知道佐贺是如何将TBP募集到核心启动子的。类似地，虽然我们确实了解HAT亚基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理解。我们的具体目标是解决这些缺陷。具体而言，我们建议：1.确定佐贺的Spt 8亚基如何与TBP结合。我们将进行诱变和标签转移实验，以确定TBP和Spt 8表面的相互作用。2.确定佐贺如何将TBP招募到启动子TATA盒。我们将开发和利用体外试验来研究佐贺如何将TBP传递到启动子的分子机制。3.确定Ada 2/Ada 3/Gcn 5和Piccolo/NuA 4 HAT酶如何与核小体相互作用。我们将使用光亲和标记，以确定HAT复合物亚基在接近核小体，和删除和诱变研究，以确定新的功能的溴和chromodomains的结合和乙酰化的核小体。我们的生物化学研究，使用定义的组件将补充大量可用的体内数据，解决机制问题，不容易解决的其他方法。

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.