CCR5 Regulation and Promoter Variants in HIV-1

HIV-1 中的 CCR5 调控和启动子变体

• 批准号: 6779192
• 负责人: Sunil K Ahuja
• 金额: $ 29.19万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6779192
• 关键词: DNA methylation; HIV infections; clinical research; cytokine receptors; dendritic cells; female; gene expression; gene induction /repression; gene mutation; genetic polymorphism; genetic promoter element; helper T lymphocyte; high throughput technology; human immunodeficiency virus 1; human subject; macrophage; receptor expression; tissue /cell culture; transcription factor; transfection

DESCRIPTION (provided by applicant): An absolute requirement for HIV-1 infection is expression of CCR5 on the cell surface of specific leukocyte subsets. Delineating the molecular mechanisms that control the expression of CCR5 is of importance to our understandingHIV-1 pathogenesis, and this fact along with our quest to elucidate how genetic variation in the CCR5 gene influence its transcriptional/epigenetic machinery, and eventually its expression and coreceptor efficiency is the major focus of our research program and of this competing renewal. Our research will be guided by the hypotheses indicated in the following 2 specific aims: Specific Aim 1 will test the overall hypothesis that in primaryHIV-1 target cells the constitutive, inducible and cell-type specific expression of CCR5 is regulated by the intricate interplay of two promoters. There is currently only limited knowledge of the regulation of CCR5in primary cells, and this information is critical to understanding the in vivo determinants influencing CCR5 density on the cell surface. Thus, we propose to use novel cellular systems in conjunction with innovative primary cell transfection strategies, "phylogenetic footprinting" ,DNA-protein pull-down assays coupled to high-through put array based profiling of transcription factors (TFs), RNAi together with HIV-derived vector mediated transactivation techniques to manipulate primary cell TF levels, and ChIP assays of in vivo occupancy of TFs in order to elucidate the genetic machinery that regulatesCCR5gene expression in primary cells. We are well positioned to extend these studies to address the in vivo biological relevance of specific CCR5 activating or repressing TFs by determining their role in disease course. Specific Aim 2 will test the overall hypothesis that CCR5 polymorphisms mediate their HIV-phenotypic effects by influencing combinatorial transcriptional control and epigenetic events that collectively nfluenceCCR5surface expression and coreceptor activity. Given the increasing importance of epigenetics in controlling gene expression, in this aim we will use ChIP assays, DNase I hypersensitivity and methylation assays to test the novel hypothesis that mutations in the CCR5 promotermediatetheir effects by influencing epigenetic events such as chromatin remodeling, DNA accessibility, and methylation. We will also extend significantly our ongoing studies aimed at understanding the mechanisms by which CCR5 polymorphisms disrupt combinatorial transcriptional control in a cell-type specific manner. These in vitro studies will be eventually extended to evaluate the association between the altered transcriptional/epigenetic events to established clinical genotype-HIV phenotype relationships as well as CCR5 surface levels/coreceptor efficiency. The proposed research is significant because it will provide critical insights into the molecular mechanisms that regulate the constitutive, inducible and cell-type specific expression of CCR5, and elucidate the complex interplay between HIV infection/disease-modifying polymorphisms in CCR5 and their impact on gene expression in primary HIV-target cells. By bridging this knowledge gap we will have a better understanding of the CCR5- (e.g. SNPs) as well as non-CCR5-dependent mechanisms underlying variable susceptibility to HIV (e.g. variable levels of critical CCR5-regulatingTFs that in turn impact on CCR5 levels).

描述（由申请人提供）：HIV-1感染的绝对要求是在特定白细胞亚群的细胞表面上表达CCR5。描述控制CCR5表达的分子机制对于我们的理解性发病机理至关重要，以及这一事实以及我们寻求阐明CCR5基因的遗传变异如何影响其转录/表观遗传机械的遗传变异如何，并且最终是我们的表达和corepotor效率的主要重点，是我们研究计划的主要重点，并且是该竞争性的RENEW。我们的研究将由以下两个特定目的所示的假设进行指导：特定目标1将检验总体假设，即在初级HIV-1靶细胞中，CCR5的组成型，诱导和细胞类型的特异性表达受两个启动子的复杂相互作用调节。目前，对CCR5IN原代细胞的调节知识有限，此信息对于理解影响细胞表面CCR5密度的体内决定因素至关重要。 Thus, we propose to use novel cellular systems in conjunction with innovative primary cell tr​​ansfection strategies, "phylogenetic footprinting" ,DNA-protein pull-down assays coupled to high-through put array based profiling of transcription factors (TFs), RNAi together with HIV-derived vector mediated transactivation techniques to manipulate primary cell TF levels, and ChIP assays of in vivo occupancy of TFS为了阐明调节原代细胞中CCR5GENE的遗传机制。我们可以很好地扩展这些研究，以解决特定CCR5通过确定疾病过程中激活或抑制TF的体内生物学相关性。具体的目标2将检验总体假设，即CCR5多态性通过影响组合转录控制和表观遗传事件来介导其HIV - 原型型效应，这些事件统称Nfluenceccr5surface5surface5serface和corecector的活性。鉴于表观遗传学在控制基因表达方面的重要性越来越重要，在此目的中，我们将使用芯片测定，DNase I超敏反应和甲基化测定法测试CCR5中CCR5中突变促进促进摄影的新假设，通过影响表观遗传事件，例如染色质重塑，DNA访问性和甲基化和甲基化。我们还将显着扩展我们正在进行的研究，旨在了解CCR5多态性以细胞类型的特定方式破坏组合转录控制的机制。这些体外研究最终将扩展，以评估转录/表观遗传事件改变与已建立的临床基因型HIV表型关系以及CCR5表面水平/共核能效率之间的关联。拟议的研究之所以重要，是因为它将提供对CCR5的组成型，诱导和细胞类型的特异性表达的分子机制的关键见解，并阐明HIV感染/疾病感染的复杂相互作用，从而使CCR5中CCR5中的多态性及其对基因在原发性HIV靶向细胞中的影响之间的影响。通过弥合这个知识差距，我们将更好地了解CCR5-（例如SNP）以及对HIV的可变易感性基于的非CCR5依赖机制（例如，关键的CCR5-调节型TR的可变水平又对CCR5水平产生影响）。