Transcriptional control of the mouse aA-crystallin locus

描述(申请人提供)：该计划的长期目标是阐明1Acrystallin(CryaA)基因座控制1Acrystallin在眼晶状体中表达的遗传和表观遗传调控成分。1Acrystallin或其突变体在晶状体中的表达缺失会影响晶状体透明度，导致晶状体混浊。1 Acrystallin在晶状体上皮细胞(晶状体前体细胞)和晶状体纤维细胞(终末分化晶状体细胞)中均有表达。由于其在晶状体初级纤维分化中的上调作用，它是研究纤维细胞分化和调控这一过程的上游信号通路的一个很好的标记物。这项工作的目的是识别1Acrystallin基因座的这些调节因子，并了解控制它们功能的分子机制。在1Acrystallin基因表达的众多调控因子中，我们现在已经发现了一个16kb的乙酰化组蛋白H3K9ac结构域，它含有晶状体染色质中的CryaA基因。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作为一种成纤维细胞生长因子调节的增强子发挥作用。DCR1/1.9kb的1Acrystallin启动子与EGFP报告基因偶联，实际上重现了1Acrystallin在晶状体上皮细胞和晶状体纤维中的表达模式。染色质免疫沉淀(ChIP)分析表明，1Acrystallin的高水平表达与c Maf与启动子结合的增加、组蛋白乙酰转移酶CBP对启动子的募集以及Pax6的稳定存在有关。为了实现这一长期目标，提出了以下具体目标：(1)利用原代晶状体外植体中的基因报告基因和体内的芯片技术来鉴定和鉴定DCR1中的FGF型顺式元件。我们还将通过以下方法在转基因小鼠中评估DCR1的功能：(1)利用绿色荧光蛋白(EGFP)整合报告从一个含有细菌人工染色体(BAC)的CryaA克隆中删除DCR1；(2)鉴定那些对晶状体中cMaf表达负责的依赖于FGF和不依赖于FGF的调控元件；以及(3)通过在小鼠中对CBP及其p300同源基因进行条件灭活来分析这些基因在晶状体发育过程中的功能，然后对与CryaA位点相关的核心组蛋白乙酰化和ATP依赖的染色质重塑酶BRG1和Snf2h进行分子分析。 公共卫生相关性：这项应用与人类健康相关，因为晶状体白内障是全球失明的主要原因。1Acrystallin是人类晶状体中含量最丰富的结构成分，其异常的功能和/或表达会导致晶状体混浊。这项研究中研究的晶状体调节蛋白基因突变，如PAX6、c MAF和CBP，已知会导致人类先天性白内障。

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.