Crx ChIP-Seq Reveals Combinatorial Architecture of the Photoreceptor Cis-Regulatory Network

Abstract

Purpose: : Nearly 200 different human genes have been implicated in retinal disease and the majority of those are expressed in photoreceptors. The transcription factor Crx is a master regulator of photoreceptor gene expression, but its in vivo binding sites have not previously been mapped on a genome-wide scale.

Methods: : In order to define the genomic targets of Crx, we carried out Crx chromatin-immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) of eight week old mouse retinas using an Illumina GAII sequencer. Sequence reads were mapped to the genome and 'peaks' were identified. These data were subjected to extensive bioinformatic analysis. In addition, selected peaks were experimentally tested for cis-regulatory activity by electroporation as promoter-reporter fusions into living mouse retinas.

Results: : Over 5,000 Crx-bound regions (CBRs) were identified throughout the mouse genome. Many clusters of CBRs occur specifically around photoreceptor genes. In fact, Crx directly regulates the majority of known photoreceptor transcription factors as well as most known photoreceptor disease genes. Cis-regulatory analysis revealed that individual CBRs contribute in a combinatorial fashion to the overall activity of the gene they control. In addition, multiple Crx binding sites within CBRs cooperatively interact via precise spacing rules to generate optimal levels of transcription.

Conclusions: : Using Crx ChIP-Seq analysis we have mapped Crx binding sites on a genome-wide scale and have shown that photoreceptor cis-regulatory regions have a complex combinatorial architecture. Not only has this study identified numerous novel retinal disease gene candidates, but it has pinpointed those non-coding regions of the genome that may harbor mutations contributing to both Mendelian as well as complex retinal disease.

Keywords: photoreceptors • transcription factors • gene/expression