Genomic Regulation of Chromatin Accessibility during Drosophila Development

描述（由申请人提供）：基因组在染色质中的组织在发育基因表达程序的调控中起着核心作用。一个被广泛接受的解释这种调节的信念是染色质可及性的协调控制。然而，几乎没有测量体内染色质可及性的高分辨率数据，大规模染色质可及性的调节因子在很大程度上是未知的，并且染色质可及性影响基因活性的分子机制也不清楚。该建议描述了一种基因组学方法，以直接测量染色质可及性在高分辨率，以开发一个系统，在实验上听话的果蝇模型，将检查染色质可及性和基因活性之间的关系。为了实现这一目标，我们将利用由Lieb实验室开发的称为FAIRE（甲醛辅助分离调节元件）的技术。我们在这里提供的数据不仅证明FAIRE能够可重复地分离“开放”染色质的离散区域，而且还能够识别具有染色质可及性特性的广泛染色体区域，即使使用少量细胞也是如此。 具体来说，我们将首先进行基因组学筛选，以确定新的调节染色质的可及性，通过使用RNAi介导的敲除果蝇S2细胞，然后通过分析FAIRE富集的DNA微阵列。第二，我们将产生基因组染色质可及性地图的不同组织和阶段的果蝇发展进行FAIRE随后高通量测序，提供一个框架，以评估染色质可及性和基因活性之间的关系在发展过程中，并确定顺式调控模块控制发育表达程序。最后，我们将开始通过突变分析来表征体内染色质可及性的潜在调节剂。 与公共卫生的相关性：许多发展途径和机制，如果调控不当，会导致人类疾病。因此，通过这些努力获得的知识将应用于理解疾病状态（如癌症）的机制。

DESCRIPTION (provided by applicant): The organization of the genome into chromatin plays a central role in the regulation of developmental gene expression programs. One widely held belief to explain this regulation is the coordinated control of chromatin accessibility. However, there is little high resolution data measuring chromatin accessibility in vivo, the regulators of large scale chromatin accessibility are largely unknown, and the molecular mechanisms by which chromatin accessibility affects gene activity are unclear. This proposal describes a genomics approach to directly measure chromatin accessibility at high resolution in order to develop a system in the experimentally tractable Drosophila model that will examine the relationship between chromatin accessibility and gene activity. To accomplish this objective, we will utilize a technique developed by the Lieb lab termed FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements). We present data here not only demonstrating the ability of FAIRE to reproducibly isolate discrete regions of 'open' chromatin, but also to identify broad chromosomal regions that share chromatin accessibility properties, even using small numbers of cells. Specifically, we will first perform a genomics screen to identify novel regulators of chromatin accessibility by using RNAi-mediated knockdown in Drosophila S2 cells, followed by analysis of FAIRE-enriched DNA on microarrays. Second, we will generate genomic chromatin accessibility maps for different tissues and stages of Drosophila development by performing FAIRE followed by high-throughput sequencing, providing a framework to assess the relationship between chromatin accessibility and gene activity during development and identifying cis-regulatory modules that control developmental expression programs. Finally, we will begin to characterize potential regulators of chromatin accessibility in vivo through mutational analyses. Relevance to Public Health: Many developmental pathways and mechanisms, when misregulated, contribute to human disease. Therefore, the knowledge gained by these efforts will have applications in understanding mechanisms that underlie disease states such as cancer.