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. ? ? ? ?
