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

使用平铺微阵列分析晶状体中的转录（芯片上的 ChIP）

• 批准号: 7230070
• 负责人: Ales Cvekl
• 金额: $ 18.55万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7230070
• 关键词: Analysis; transcription; lens; using; tiled

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.

描述（由申请人提供）：该 R21 申请的长期目标是使用染色质免疫沉淀 (ChlP) 对晶状体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）进行分析（芯片上的 ChLP）。该方法将用于在两种模型生物（小鼠和鸡）的脊椎动物晶状体发育过程中大规模加速数据收集、跨物种比较和调控网络的剖析。该提案将 (1) 确定调节晶状体谱系形成、Pax6 和 Sox2 以及分化（c-Maf、Proxl 和 pRb）的 5 个关键转录因子的结合位点概况，其中包含近 100 个编码晶状体发育和必需蛋白的基因/基因座。体内平衡； (2) 鉴定这些基因/位点的染色质结构，重点关注组蛋白 H3 K9 乙酰化、H3 K3/27 甲基化、DMA 甲基化、绝缘子蛋白 CTCF 和染色质激活剂 HMGN3 的结合，所有这些都参与表观遗传调控。这些相互作用将在小鼠和鸡晶状体中确定，每个基因座在 10 至 500 kb 之间，平铺在定制的微阵列上。这些基因座将包括所有晶状体结构基因（晶状体蛋白、膜和中间丝蛋白）、调节晶状体谱系形成的关键基因（例如 Pax6、Six3 和 Sox2）、其终末分化（Proxl、c-Maf 和 Sox1）以及信号成分传导网络（例如 FGFR1 至 4）在晶状体发育过程中活跃。这些数据将与 RNA 表达研究相关，并且选定的数据将使用分子生物学实验进行验证。这些数据将为体内各个基因座远端调控区域的功能识别、各种转录因子之间的交叉调控的识别以及功能丧失和功能获得模型中晶状体发育的系统分析奠定基础。这些数据的生成和分析将用于新的重点 R01 项目，以阐明先天性晶状体缺陷和白内障期间的正常晶状体发育和异常基因调控。