Analysis of transcription in lens using tiled microarrays (ChIP on chip)

描述（由申请方提供）：该R21申请的长期目标是使用染色质免疫沉淀（ChlPs）对透镜中的基因表达进行综合探索性研究，然后使用Nimblegen开发的高密度平铺寡核苷酸阵列（芯片上ChlPs）进行分析。这种方法将用于大规模加速数据收集，跨物种的比较和解剖过程中的脊椎动物透镜发展的监管网络在2个模式生物，小鼠和鸡。本研究的目的是：（1）确定调控透镜谱系形成和分化的5个关键转录因子Pax 6和Sox 2的结合位点（c-Maf、Proxl和pRb）具有近100个编码透镜发育和稳态所必需的蛋白质的基因/位点;和（2）鉴定这些基因/基因座的染色质结构，集中于组蛋白H3 K9乙酰化、H3 K3/27甲基化、DMA的甲基化，绝缘子蛋白CTCF和染色质激活剂HMGN 3的结合，所有这些都参与了表观遗传调控。这些相互作用将在小鼠和鸡透镜中测定，每个位点在10至500 kb之间，平铺在基于定制的微阵列上。这些基因座将包括所有透镜结构基因（晶状体蛋白、膜和中间丝蛋白）、调节透镜谱系形成的关键基因（例如，Pax 6、Six 3和Sox 2）、其终末分化（Prox 1、c-Maf和Sox 1）和信号转导网络的组分（例如，FGFR 1至4）在透镜发育期间是活性的。这些数据将与RNA表达研究相关，并将使用分子生物学实验验证选定的数据。这些数据将为在体内鉴定单个基因座的远端调控区的功能、鉴定各种转录因子之间的交叉调节以及系统分析功能丧失和获得模型中的透镜发育奠定基础。这些数据的生成和分析将用于新的重点R 01项目，以阐明先天性透镜缺陷和白内障期间正常的透镜发育和异常基因调节。

DESCRIPTION (provided by applicant): The long-term goal of this R21 application is to perform integrative exploratory studies of gene expression in lens using chromatin immunoprecipitations (ChlPs) followed by analysis using high-density tiled oligonucleotide arrays (chips) developed by Nimblegen (ChlPs on chip). This approach will be used for massive acceleration of data collection, cross-species comparisons and dissection of regulatory networks during vertebrate lens development in 2 model organisms, mouse and chicken. This proposal will (1) Determine the profiles of binding sites of 5 key transcription factors regulating lens lineage formation, Pax6 and Sox2, and differentiation (c-Maf, Proxl and pRb) with nearly 100 genes/loci encoding proteins essential for lens development and homeostasis; and (2) Identify chromatin structure of these genes/loci focusing on histone H3 K9 acetylation, H3 K3/27 methylation, methylation of DMA, binding of insulator protein CTCF and chromatin activator HMGN3, all involved in epigenetic regulation. These interactions will be determined in mouse and chicken lens with each locus between 10 to 500 kb, tiled on custom-based microarrays. These loci will include all lens structural genes (crystallins, membrane and intermediate filament proteins), key genes regulating lens lineage formation (e.g., Pax6, Six3 and Sox2), its terminal differentiation (Proxl, c-Maf and Sox1) and components of signal transduction networks (e.g., FGFR1 to 4) active during lens development. The data will be correlated with RNA expression studies and selected data will be validated using molecular biology experiments. This data will lay a foundation for the functional identification of distal regulatory regions of individual loci in vivo, identification of cross-regulation between various transcription factors, and systemic analysis of lens development in loss-of-and gain-of-function models. Generation and analysis of this data will be used for new focused R01 projects to elucidate normal lens development and abnormal gene regulation during congenital lens defects and in cataracts.