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: 8065975
• 负责人: Ales Cvekl
• 金额: $ 51.39万
• 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/8065975
• 关键词: 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; 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; 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; 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; public health relevance; 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（由申请方提供）：本项目的长期目标是阐明控制眼透镜中1-乙酰半胱氨酸表达的1-乙酰半胱氨酸（Cryaa）基因座的遗传和表观遗传调控组分。透镜中1-聚乙烯醇蛋白表达的缺失或其突变体的表达损害了透镜的透明度并导致透镜混浊。在透镜上皮细胞（透镜前体细胞）和透镜纤维细胞（终末分化的透镜细胞）中均表达1-Accelallin。由于其在分化的透镜初级纤维中的上调，它是纤维细胞分化和调节该过程的上游信号传导途径的研究的极好标记物。这项工作的目标是确定这些调节器的1Accellalin基因座和了解的分子机制，管理他们的功能。在众多的1-acetylalin基因表达调节因子中，我们已经鉴定出一个16 kb的乙酰化组蛋白H3 K9 ac结构域，它在透镜染色质中含有Cryaa基因座。我们还发现Cryaa基因座的5 '/3'边界由两个发育控制的增强子DCR 1和DCR 3产生。我们已经表明，DCR 1作为FGF调节的增强子发挥作用。DCR 1/1.9 kb的1-acetylalin启动子与EGFP报告基因偶联，基本上再现了1-acetylalin在透镜上皮和透镜纤维中的表达模式。染色质免疫沉淀（ChIP）分析表明，高水平的1 Accumulallin表达与c Maf的启动子，组蛋白乙酰转移酶CBP的启动子，和稳定的Pax 6的存在下，招聘的结合增加。为了实现这一长期目标，提出了以下具体目标：（1）在原代透镜外植体中使用基因报告分析和在体内使用ChIP分析来鉴定和表征DCR 1中的FGF应答顺式元件。还将在转基因小鼠中通过从带有EGFP整合的报道基因的Cryaaharboring细菌人工染色体（BAC）克隆中缺失DCR 1来评估DCR 1的功能。（2）为了鉴定负责透镜中cMaf表达的那些FGF依赖性和FGF非依赖性调节元件，以及（3）通过对CBP及其p300同源基因的条件性失活及核心组蛋白的分子分析，探讨CBP及其p300同源基因在小鼠透镜发育中的功能乙酰化和ATP依赖性染色质重塑酶Brg 1和Snf 2 h与Cryaa基因座相关。 公共卫生相关性：由于透镜白内障是全球范围内致盲的主要原因，因此本申请与人类健康相关。1-acetylalin是人类透镜中最丰富的结构成分;其异常功能和/或表达导致透镜混浊。已知编码透镜调节蛋白如PAX 6、c MAF和CBP的基因突变会导致人类先天性白内障。