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

描述(申请人提供)：这项建议的目标是开发一种新的高通量方法，用于在体内识别视网膜色素上皮(RPE)中转录因子的直接靶标。视网膜色素上皮对视网膜光感受器的功能和存活具有重要意义。视网膜色素上皮的异常与包括老年性黄斑变性(AMD)在内的光感受器变性的发病机制有关，AMD是美国老年人不可逆性失明的主要原因。尽管RPE在视觉上很重要，但我们对RPE中调控基因表达的分子机制的了解非常有限。为了更好地理解这些机制，我提议通过使用染色质免疫沉淀(CHIP)来生成人类基因组微阵列(CHIP)，这是一种高通量方法，涉及同时杂交数千个克隆。染色质免疫沉淀(CHIP)是一种识别特定因子结合的基因组区域的技术。在这项提议中，我将首先使用含有已被发现与活性转录相关的乙酰化组蛋白H3和H4的抗体的芯片，从人类RPE细胞中丰富染色质的“转录活性”区域，并将其克隆到载体中，以构建人类RPE“活性启动子”文库。然后，我将使用通过聚合酶链式反应(PCR)扩增的文库插入片段来生成基因组微阵列。最后，我将开始使用基因组微阵列和抗MITF抗体的第二芯片(CHIP-CHIP-CHIP)选择的探针在体内鉴定MITF的直接靶点。这一新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中任何转录激活子的多个体内直接结合靶点。此外，由于MITF在RPE的发育和功能中发挥重要作用是众所周知的，分离多个MI I‘F靶标将使人们对这一重要转录因子在RPE中的功能网络有新的见解，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.