ChIP-ChIP-Chip: A New Tool To Study RPE Gene Regulation

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): The objective of this proposal is to develop a new high-throughput approach for identifying in vivo direct targets of transcription factors in the retinal pigment epithelium (RPE). The RPE is important for the function and survival of retinal photoreceptors. Abnormalities in the RPE have been implicated in the pathogenesis of photoreceptor degeneration including age-related macular degeneration (AMD), the leading cause of irreversible blindness in elderly Americans. Despite the importance of the RPE in vision, our knowledge of the molecular mechanisms that regulate gene expression in the RPE is very limited. To better understand such mechanisms, I propose to generate a human genomic microarray (Chip), a high-throughput method involving hybridization of thousands of clones simultaneously, by using chromatin immunoprecipitation (CHIP), a technique for identifying genomic regions bound by a specific factor. In this proposal, I will first enrich "transcriptionally active" regions of chromatin from human RPE cells using ChIP with antibodies against acetylated histones H3 and H4 that have been found to be associated with active transcription and clone into a vector to construct human RPE "active promoter" libraries. I will then generate a genomic microarray using inserts of the libraries amplified by polymerase chain reaction (PCR). Finally, I will begin the process of identifying in vivo direct targets of microphthalmia associated transcription factor (MITF) using the genomic microarray and probes selected by the second ChIP with anti-MITF antibodies (ChiP-ChiP-Chip). This new strategy provides a very powerful tool enabling us to isolate multiple in vivo direct binding targets of any transcription activators in the RPE in a genomewide scale. In addition, since Mitf is well known to play an important role in the development and function of the RPE, isolation of multiple MI'I'F targets should give new insights into the functional networks of this important transcription factor in the RPE, leading to new areas of RPE biology and disease.