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，染色质重塑被调控并与基因表达变化相关的分子机制还知之甚少。酵母酸性磷酸酶基因PHO5的启动子是研究染色质结构和转录之间关系的经典模型，为基因调控和启动子活性提供了重要的见解。在磷酸盐饥饿时，PHO5被Pho4p激活，Pho4p是一种转录因子，介导启动子核小体的分解。虽然PHO5的调控已被广泛研究，但对激活的限速步骤、核小体分解的精确调控作用以及参与核小体去除的染色质重构体知之甚少。这项提案概述了一项研究计划，利用生化、遗传和成像技术的组合来研究PHO5启动子染色质的转变。我们将使用电子显微镜来确定在整个激活过程中PHO5处启动子核小体配置的可能性。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。我们还将产生并鉴定信息丰富的Pho4p激活结构域突变体。这些突变将被用于各种检测，以探索Pho4p在染色质重塑和转录中的作用。最后，我们将通过使用Pho4p激活域突变体进行抑制子筛选，系统地识别参与PHO5激活的基因。将对抑制物进行广泛的表征，并计划在未来的实验中探索它们对PHO5染色质转变的影响。这项拟议的研究有望拓宽我们对染色质重塑的理解，因为它与转录相关，并将提供关于转录激活物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变的新因素。基因调控异常在数十种人类疾病中普遍存在，包括几种类型的癌症。这项建议旨在阐明核小体介导的转录调控的影响，该调控可能适用于几个具有临床重要性的基因。基因表达与基因组DNA的包装和随后的可获得性有着千丝万缕的联系。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.