Transcriptional control of the mouse aA-crystallin locus

小鼠aA-晶状体蛋白基因座的转录控制

• 批准号: 7458344
• 负责人: Ales Cvekl
• 金额: $ 49.48万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7458344
• 关键词: A Mouse; ATP phosphohydrolase; Bacterial Artificial Chromosomes; Binding; Binding Sites; Biological Assay; Blindness; Bromodomain; CREB-binding protein; Cataract; Cell Differentiation process; Cells; Chromatin; Chromatin Structure; Control Locus; Coupled; Crystalline Lens; Crystallins; DNA; Development; Distal; EP300 gene; Embryo; Enhancers; Enzymes; Epigenetic Process; Epithelium; Eye Development; FGF2 gene; Family; Fiber; Fibroblast Growth Factor; Fibroblast Growth Factor 2; Gene Expression; Gene Expression Regulation; Gene Silencing; Genes; Genetic; Genetic Enhancer Element; Genetic Transcription; Goals; Growth Factor; Health; Histone Acetylation; Histone Code; Histone H3; Histones; Homologous Gene; Human; Indium; Lens Fiber; MAP Kinase Gene; Mediating; Modeling; Molecular; Molecular Analysis; Mus; Mutation; Pathway interactions; Pattern; Play; Process; Proteins; Public Health; Rattus; Recruitment Activity; Regulation; Regulatory Element; Reporter; Reporter Genes; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; System; TNFRSF6B gene; Tail; Time; Tissues; Transcription Factor AP-1; Transcriptional Regulation; Transgenic Mice; Up-Regulation; Vesicle; Work; Yang; base; chromatin immunoprecipitation; chromatin remodeling; congenital cataract; enhanced green fluorescent protein; fiber cell; genetic regulatory protein; histone acetyltransferase; human CREBBP protein; in vivo; insight; lens; lens transparency; member; mutant; novel; precursor cell; programs; promoter; transcription factor

DESCRIPTION (provided by applicant): The longterm goal of this program is to elucidate those genetic and epigenetic regulatory components of the 1Acrystallin (Cryaa) locus that control expression of 1Acrystallin in the ocular lens. A loss of 1Acrystallin expression or expression of its mutants in lens compromises lens transparency and results in lens opacification. Expression of 1Acrystallin occurs both in the lens epithelium (lens precursor cells) and in the lens fiber cells (terminally differentiated lens cells). Due to its upregulation in differentiating lens primary fibers, it is an excellent marker for fiber cell differentiation and studies of upstream signaling pathways regulating this process. The goal of this work is to identify these regulators of the 1Acrystallin locus and to understand the molecular mechanisms that govern their functions. Among the many regulators of 1Acrystallin gene expression, we have now identified a 16 kb domain of acetylated histone H3 K9ac that harbor the Cryaa locus in lens chromatin. We have also found that the 5'/3' borders of the Cryaa locus are generated by two developmentally controlled enhancers, DCR1 and DCR3. We have shown that DCR1 functions as an FGF regulated enhancer. A DCR1/1.9 kb 1Acrystallin promoter coupled with a EGFP reporter gene virtually recapitulated the expression pattern of 1Acrystallin in lens epithelium and lens fibers. Chromatin immunoprecipitation (ChIP) assays showed that high levels of 1Acrystallin expression correlate with increased binding of c Maf to the promoter, recruitment of histone acetyltransferase CBP to the promoter, and stable presence of Pax6. In order to carry out this longterm goal, the following specific aims are proposed: (1) To identify and characterize FGFresponsive ciselements in DCR1 using gene reporter assays in primary lens explants and in vivo by ChIP assays. The function of DCR1 will be also assessed in transgenic mice through its deletion from a Cryaaharboring bacterial artificial chromosome (BAC) clone with an EGFP integrated reporter, (2) To identify those FGF dependent and FGF independent regulatory elements responsible for cMaf expression in the lens, and (3) To analyze the function of CBP and its p300 homologue gene during lens development by conditional inactivation of these genes in mouse followed by molecular analysis of core histone acetylations and ATP dependent chromatin remodeling enzymes Brg1 and Snf2h associated with the Cryaa locus. PUBLIC HEALTH RELEVANCE: This application is relevant to human health as lens cataract is a major cause of worldwide blindness. The 1Acrystallin is the most abundant structural component of the human lens; its abnormal function and/or expression causes lens opacification. Mutations in genes encoding lens regulatory proteins such as PAX6, c MAF and CBP studied here are known to cause human congenital cataracts.

描述（由申请人提供）：本项目的长期目标是阐明控制晶状体中1Acrystallin表达的基因座1Acrystallin的遗传和表观遗传调控成分。晶状体中1crystin表达缺失或其突变体表达降低晶状体透明度，导致晶状体混浊。晶状体上皮（晶状体前体细胞）和晶状体纤维细胞（终末分化的晶状体细胞）均表达1crystallin。由于其在晶状体原纤维分化过程中表达上调，因此它是纤维细胞分化和研究这一过程的上游信号通路的良好标记物。这项工作的目的是确定1crystallin位点的这些调节因子，并了解控制其功能的分子机制。在1crystallin基因表达的许多调节因子中，我们现在已经确定了一个16kb的乙酰化组蛋白h3k9ac结构域，该结构域在晶体染色质中包含Cryaa位点。我们还发现Cryaa基因座的5‘/3’边界是由两个发育控制的增强子DCR1和DCR3产生的。我们已经证明DCR1作为FGF调节增强子起作用。一个DCR1/1.9 kb的1Acrystallin启动子与一个EGFP报告基因结合，几乎再现了1Acrystallin在晶状体上皮和晶状体纤维中的表达模式。染色质免疫沉淀（ChIP）分析显示，高水平的1crystallin表达与c Maf与启动子结合增加、组蛋白乙酰转移酶CBP向启动子募集以及Pax6的稳定存在相关。为了实现这一长期目标，我们提出了以下具体目标：(1)在初级晶状体外植体中使用基因报告基因检测和在体内使用ChIP检测来鉴定和表征DCR1中FGFresponsive顺式元件。DCR1的功能也将通过从含有EGFP整合报告基因的cryaahartting细菌人工染色体（BAC）克隆中删除其在转基因小鼠中的作用进行评估。(2)为了鉴定晶状体中负责cMaf表达的FGF依赖性和FGF非依赖性调节元件。(3)通过条件失活小鼠CBP及其p300同源基因，分析CBP及其p300同源基因在晶状体发育过程中的功能，并对Cryaa位点相关核心组蛋白乙酰化和ATP依赖性染色质重塑酶Brg1和Snf2h进行分子分析。