DISSECTING THE CIS-REGULATORY ARCHITECTURE OF THE RETINA BY EPIGENOMIC PROFILING

 描述(申请人提供)：顺式调节元件(CRE)通过调节转录因子(TF)与其靶基因之间的相互作用，在基因表达调控中发挥关键作用。Cres内的突变可以破坏关键的TF结合位点，从而改变基因表达，从而导致疾病。在复杂疾病的全基因组关联研究中发现的绝大多数SNP属于非编码区，被认为影响Cres的活性。因此，更好地了解Cres的位置和功能对于解释非编码区内遗传变异的功能意义是至关重要的。Encode和其他基于联盟的项目已经开始绘制Cres在多个细胞系和整个器官中的位置。然而，我们在单个原代细胞类型水平上对顺式调控结构的理解是有限的。在这个方案中，我们将利用新开发的表观基因组图谱技术ATAC-seq，全面鉴定小鼠视网膜中所有七个主要细胞类别的Cres：视杆细胞、视锥细胞、水平细胞、双极细胞、Müller胶质细胞、无长突细胞和神经节细胞。ATAC-SEQ利用一种基于转座酶的方法来标记开放染色质的区域，并可以应用于通过荧光激活细胞分选(FACS)纯化的非常少量的细胞。这种变革性的方法将使我们能够探索染色质可及性、核小体定位和Tf结合在全基因组范围内在所有小鼠视网膜细胞类别中的复杂相互关系。此外，我们将利用这些数据来探索特定视网膜细胞类型中数千个CRE的功能，使用CRE-SEQ，一种用于高通量顺式调控分析的新技术。综上所述，这些研究将产生一张全面的视网膜Cres功能图，这将成为了解视网膜转录网络和非编码变体对疾病的影响的蓝图。此外，这些研究将为未来的工作奠定基础，这些工作将利用表观基因组图谱来表征发生的变化 在视网膜发育过程中和在退化过程中。

 DESCRIPTION (provided by applicant): Cis-regulatory elements (CREs) play a critical role in the regulation of gene expression by mediating the interaction between transcription factors (TFs) and their target genes. Mutations within CREs can disrupt key TF binding sites, thereby altering gene expression, and thus contributing to disease. The vast majority of SNPs identified in genome-wide association studies (GWAS) of complex disease fall within non-coding regions, and is thought to affect the activity of CREs. Thus, a better understanding of the location and function of CREs is essential for interpreting the functional significance of genetic variation within non-coding regions. ENCODE and other consortium-based projects have begun to map the location of CREs in multiple cell lines and whole organs. However, our understanding of cis-regulatory architecture at the level of individual primary cell types is limited. In this proposal,we will utilize a newly developed epigenomic mapping technology, ATAC-seq, to comprehensively identify the CREs of all seven major cell classes in the mouse retina: rods, cones, horizontal cells, bipolar cells, Müller glia, amacrine cells, and ganglion cells. ATAC-seq utilizes a transposase-based approach to tag regions of open chromatin and can be applied to very small numbers of cells purified by fluorescence-activated cell sorting (FACS). This transformative approach will permit us to probe the complex interrelations between chromatin accessibility, nucleosome positioning, and TF binding on a genome-wide scale in all mouse retinal cell classes. Furthermore, we will leverage these data to probe the function of thousands of CREs within specific retinal cell types, using CRE-seq, a novel technique for high-throughput cis-regulatory analysis. Taken together, these studies will generate a comprehensive, functional map of retinal CREs that will serve as a blueprint for understanding the transcriptional networks of the retina and the effects of non-coding variants on disease. In addition, these studies will se the stage for future work that will utilize epigenomic profiling to characterize changes that occur during the process of retinal development and in the course of degeneration.