Investigation of the PHO5 promoter chromatic transition

描述（由申请人提供）： 转录激活通常伴随着启动子染色质状态的“重塑”。然而，染色质重塑的分子调控机制，并与基因表达的变化知之甚少。酵母酸性磷酸酶基因PHO5的启动子是研究染色质结构与转录之间关系的经典模型，为研究基因调控和启动子活性提供了重要的思路。在磷酸盐饥饿时，PHO5被Pho4p激活，Pho4p是一种介导启动子核小体解体的转录因子。虽然PHO5调控已被广泛研究，但对活化的限速步骤、核小体解体的精确调控作用以及核小体去除中涉及的染色质重塑知之甚少。该提案概述了一项研究计划，采用生物化学，遗传学和成像技术相结合，研究PHO5启动子染色质转换。我们将使用电子显微镜来确定在整个激活过程中的启动子核小体配置在PHO5的概率。这些研究将定量测试最近发表的描述PHO5启动子染色质随机性的数学模型。我们还将产生和表征信息Pho4p激活结构域突变体。这些突变将被用于各种检测，以探测Pho4p在染色质重塑和转录中的作用。最后，我们将系统地确定参与PHO5激活的基因进行抑制筛选，使用一个PHO4p激活结构域突变。抑制因子将被广泛表征，未来的实验计划探测它们对PHO5染色质转换的影响。拟议的研究有望拓宽我们对染色质重塑的理解，因为它与转录有关，并将提供有关转录激活因子的基本作用的有价值的信息。此外，这项工作有望揭示调节启动子染色质转换的新因子。异常基因调控在包括几种癌症在内的几十种人类疾病中普遍存在。该建议旨在阐明核小体介导的转录调控的影响，可能适用于几个基因，具有临床意义。基因表达与基因组DNA的包装和随后的可及性密不可分。通过研究调节DNA可及性的因素，我们增加了对发育，分化和疾病的潜在机制的理解。

DESCRIPTION (provided by applicant): Transcriptional activation is generally accompanied by the "remodeling" of promoter chromatin state. However, the molecular mechanisms by which chromatin remodeling is regulated and linked to changes in gene expression are poorly understood. The promoter of the yeast acid phosphatase gene PHO5 has served as a classical model to address the relationship between chromatin structure and transcription, providing major insights into gene regulation and promoter activity. Upon phosphate starvation, PHO5 is activated by Pho4p, a transcription factor that mediates promoter nucleosome disassembly. While PHO5 regulation has been widely investigated, little is known about the rate-limiting step of activation, the precise regulatory role of nucleosome disassembly, and the chromatin remodelers involved in nucleosome removal. This proposal outlines a research plan to employ a combination of biochemical, genetic, and imaging techniques to study the PHO5 promoter chromatin transition. We will use electron microscopy to determine the probabilities of promoter nucleosome configurations at PHO5 throughout the activation process. These studies will quantitatively test a recently published mathematical model describing chromatin stochasticity at the PHO5 promoter. We will also generate and characterize informative Pho4p activation-domain mutants. These mutations will be be used in a variety of assays to probe the role of Pho4p in chromatin remodeling and transcription. Finally, we will systematically identify genes involved in PHO5 activation by performing a suppressor screen, using a Pho4p activation-domain mutant. Suppressors will be extensively characterized, with future experiments planned to probe their effects on the PHO5 chromatin transition. The proposed research is expected to broaden our understanding of chromatin remodeling as it relates to transcription and will offer valuable information regarding the fundamental role of transcriptional activators. Furthermore, this work is expected to reveal novel factors that regulate promoter chromatin transitions. Aberrant gene regulation is ubiquitous in dozens of human diseases including several types of cancer. This proposal aims to shed light on the impact of nucleosome-mediated transcriptional regulation that may be applicable to several genes, of clinical importance. Gene expression is inextricably linked to the packaging and subsequent accessibility of genomic DNA. By studying factors that regulate DNA accessibility, we increase our understanding of the mechanisms underlying development, differentiation, and disease.