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

描述（由申请人提供）：本提案的目的是开发一种新的高通量方法，用于鉴定视网膜色素上皮（RPE）中转录因子的体内直接靶点。RPE对于视网膜光感受器的功能和存活是重要的。视网膜色素上皮细胞中的凋亡与光感受器变性的发病机制有关，包括年龄相关性黄斑变性（AMD），这是美国老年人不可逆失明的主要原因。 尽管RPE在视觉中的重要性，但我们对调节RPE中基因表达的分子机制的了解非常有限。为了更好地理解这种机制，我建议通过使用染色质免疫沉淀（CHIP）（一种用于识别特定因子结合的基因组区域的技术）来生成人类基因组微阵列（Chip），这是一种涉及同时杂交数千个克隆的高通量方法。在这个提议中，我将首先使用ChIP富集来自人RPE细胞的染色质的“转录活性”区域，该区域具有针对乙酰化组蛋白H3和H4的抗体，这些抗体已被发现与活性转录相关，并克隆到载体中以构建人RPE“活性启动子”文库。然后，我将使用聚合酶链反应（PCR）扩增的文库插入片段生成基因组微阵列。 最后，我将开始的过程中，确定在体内的直接目标，小眼相关转录因子（MITF）使用基因组芯片和探针选择的第二个ChIP与抗MITF抗体（芯片）。 这种新的策略提供了一个非常强大的工具，使我们能够在全基因组范围内分离RPE中任何转录激活因子的多个体内直接结合靶点。 此外，由于众所周知Mitf在RPE的发育和功能中起重要作用，因此分离多个MI“I”F靶点应该对RPE中该重要转录因子的功能网络提供新的见解，从而导致RPE生物学和疾病的新领域。

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.