Potentiated State of the HSP70 Heat Shock Gene

HSP70 热休克基因的增强状态

基本信息

批准号：
7892103
负责人：
DAVID Scott GILMOUR
金额：
$ 33.39万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-05-01 至 2014-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7892103
关键词：
Affect Animal Model Arginine BRCA1 gene Binding Binding Sites Biochemical Biological Models Biological Process Cell-Free System Cells Complex DNA Binding DNA Polymerase II DNA Sequence DNA-Binding Proteins Defect Development Disease Drosophila genus Elongation Factor Eukaryota Gene Expression Gene Expression Regulation Genes Genetic Techniques Genetic Transcription Goals HIV Heat-Shock Proteins 70 Heat-Shock Response Human In Vitro Kinetics Life Link Maps Methylation Modeling Modification Molecular Genetics Mutation Nuclear Extract Pathway Analysis Phase II Clinical Trials Phosphorylation Phosphotransferases Play Positive Transcriptional Elongation Factor B Post-Translational Protein Processing Process Proteins Proviruses Qualifying RNA RNA Binding Recruitment Activity Regulation Research Role Sequence-Specific DNA Binding Protein System TATA Box Techniques Testing Transcript Transcription Initiation Transcriptional Activation Transcriptional Regulation cancer therapy factor EF-P fly gene repression genome-wide in vivo inhibitor/antagonist insight malignant breast neoplasm mutant negative elongation factor novel promoter public health relevance reconstitution stem cell division

项目摘要

DESCRIPTION (provided by applicant): Control of gene expression is fundamental to life and this control usually involves transcriptional regulation. Much of the research on transcriptional regulation in eukaryotes over the past 2 decades has focused on regulatory mechanisms that ultimately affect transcription initiation by Pol II at protein-encoding genes. However, recent analyses of Pol II interactions in vivo using genome-wide mapping techniques such as ChIP- chip reveal that thousands of genes in human and Drosophila have Pol II concentrated at the 5' end. Much of this Pol II appears to have initiated transcription but paused in the promoter proximal region. Hence, a prominent control point in the transcription occurs after transcription initiation. This promoter proximal pausing and its regulation are poorly understood largely because it has gone unrecognized at most genes. The hsp70 gene of Drosophila is one of the few genes where promoter proximal pausing has long been known to occur, and it serves a paradigm for this process. The sequence-specific, DNA-binding proteins, GAGA factor and HSF, function respectively in the establishment and reactivation of the paused Pol II. The negative elongation factors, NELF and DSIF, participate in pausing by associating with Pol II. The positive elongation factor, P- TEFb, is thought to reactivate the paused Pol II by phosphorylating NELF, DSIF and Pol II. Even with the identification of these proteins, the mechanisms by which they control the Pol II are not known. The overall goal of this project is to elucidate mechanisms of promoter proximal pausing and its control. Aim 1 determines how DSIF and NELF cause Pol II to pause and how their activities are regulated by post-translational modifications. Aim 2 determines how GAGA factor and HSF control promoter proximal pausing. It also investigates the role of a novel sequence-specific, DNA-binding protein whose DNA binding site is detected in at the promoter of 20% of genes found to be associated with Pol II. Aim 3 will determine if the regulation of promoter proximal pausing involves kinetic competition between the rate of elongation and the rate at which DSIF and NELF capture the elongating Pol II. A combination of biochemical and in vivo approaches applied to the model organism, Drosophila, uniquely qualifies this project for obtaining significant insight into the mechanism of promoter proximal pausing, thereby having significant impact our fundamental understanding of gene regulation. In addition, NELF, DSIF and P-TEFb have each been linked to human maladies. One subunit of NELF associates with the BRCA1 and has been linked to breast cancer. DSIF and P-TEFb both contribute to transcription of the HIV provirus, and an inhibitor of P-TEFb is in phase 2 clinical trials for cancer therapy. PUBLIC HEALTH RELEVANCE: Transcriptional regulation plays a prominent role in the appropriate expression of genes, and many diseases arise because of defects in gene expression. Recent research has revealed that a mechanism of transcriptional regulation called promoter proximal pausing, once thought to occur at only a handful of genes, actually happens at thousands of genes including ones involved in development and stem cell renewal. Promoter proximal pausing will be studied in the model organism, Drosophila, which offers a unique combination of experimental approaches for understanding this important biological process.

描述（由申请人提供）：基因表达的控制是生命的基础，这种控制通常涉及转录调节。在过去的20年中，真核生物中转录调控的大部分研究都集中在调节机制上，这些机制最终会影响Pol II在蛋白质编码基因上的转录启动。然而，使用全基因组映射技术（例如芯片）在体内对POL II相互作用进行的分析表明，人和果蝇中成千上万的基因已集中在5'端。该Pol II的大部分似乎已经引发了转录，但在启动子近端区域停了下来。因此，转录开始后发生了一个突出的控制点。该启动子近端暂停及其调节的理解很少，因为它在大多数基因上都无法识别。果蝇的HSP70基因是众所周知，促进剂近端暂停的少数基因之一，并且为此过程提供了范式。序列特异性，DNA结合蛋白，GAGA因子和HSF分别在暂停的Pol II的建立和重新激活中起作用。 NELF和DSIF的负伸长因素通过与Pol II相关联参与了暂停。据认为，阳性伸长因子p-tefb可以通过磷酸化NELF，DSIF和POL II来重新激活暂停的Pol II。即使鉴定了这些蛋白质，也不知道它们控制POL II的机制。该项目的总体目标是阐明启动子近端暂停及其控制的机制。 AIM 1确定DSIF和NELF如何导致Pol II停顿，以及如何通过翻译后修改来调节其活动。 AIM 2确定GAGA因子和HSF控制启动子如何近端暂停。它还研究了一种新型序列特异性的DNA结合蛋白的作用，该蛋白的DNA结合位点在20％的基因的启动子中被发现与Pol II相关。 AIM 3将确定启动子近端暂停的调节是否涉及伸长率和DSIF和NELF捕获延长的Pol II之间的动力学竞争。果蝇的生化方法和体内方法的结合，独特的果蝇有资格获得对启动子近端暂停机制的重大见解，从而对我们对基因调节的基本理解产生了重大影响。此外，NELF，DSIF和P-TEFB都与人类疾病有关。 NELF与BRCA1的一个亚基，与乳腺癌有关。 DSIF和P-TEFB都有助于HIV病毒的转录，并且P-TEFB的抑制剂正在接受癌症治疗的第二阶段临床试验。公共卫生相关性：转录调控在基因的适当表达中起着重要的作用，并且由于基因表达缺陷而出现许多疾病。最近的研究表明，一种称为启动子近端暂停的转录调节机制，曾经被认为仅发生在少数基因上，实际上发生在数千种基因上，包括参与发育和干细胞更新的基因。在模型有机体果蝇中将研究启动子近端暂停，该果蝇提供了一种独特的实验方法组合，以理解这一重要的生物学过程。