Analysis of transcription by ChIP‐seq in the eye: Progress and challenges

Abstract

AbstractPurposeChIP‐seq experiments are designed to map in vivo localization of DNA‐binding transcription factors, their associated chromatin remodeling enzymes, RNA polymerase, and histone posttranslational modifications, at the genome scale. The results, coupled with analysis of gene loss‐of‐function studies by RNA‐seq, are used to reconstruct gene regulatory networks (GRNs) that govern cellular development and differentiation. In the eye, the majority of available data include lens and retina. Although some of the chromatin‐based assays can be conducted at single cell level (e.g. ATAC‐seq), mapping of transcription factors and modified histones require large numbers of cells.MethodsStandard ChIP‐seq experiments require millions of cells. In contrast, recent modifications allow the use of <100 000 cells. Complementary ATAC‐seq experiments employ 1–50 000 individual cells. RNA‐seq experiments are included in the analysis to distinguish directly and indirectly regulated specific targets of these transcription factors.ResultsIn lens, standard ChIP‐seq data exist for Pax6, H3K4me1, H3K4me3, H3K27ac, H3K27me3, and RNA polymerase II. Using ATAC‐seq, we found that Pax6 was bound both in “open” and “closed” lens chromatin domains in agreement with its dual roles as transcriptional activator and repressor.ConclusionsReconstructions of GRNs that govern lens and retinal morphogenesis are in progress and require joint efforts of multiple research teams. Ongoing studies to use smaller numbers of cells will be reported. Together, these data open the field for mechanistic studies of lens‐ and retinal‐specific promoters and enhancers.