Photoreceptor Transcription Factors Are Required for Configuring Intra-chromosomal Loops Associated With Transcriptional Activation of Photoreceptor Genes

Abstract

Purpose: : Rod and cone genes are expressed in a mutually exclusive manner in their respective photoreceptor subtypes, regulated by photoreceptor transcription factors. We previously showed that rhodopsin and M-opsin loci form intra-chromosomal loops in the appropriate photoreceptor subtype, which requires Nrl and Crx transcription factors. The goal of this research was to determine if other rod and cone genes adopt differential chromosomal conformations in rods vs. cones, and if the nuclear receptor Nr2e3 is also involved in regulating these chromatin conformations.

Methods: : Chromosomal conformation capture (3C) assays were used to reveal looping interactions between chromatin segments of two rod genes (rhodopsin and Gnat1) and three cone genes (M-opsin, S-opsin, and Gnat2). P14 retinae from four mouse lines provided sources of rods (C57BL/6), cones (Nrl^-/-), defective rods (Crx^-/-), and S-cone/rod hybrid cells (Nr2e3^rd7/rd7).

Results: : In normal rods (C57BL/6), the distal regulatory region of rod gene loci, rhodopsin and Gnat1, loops out to interact with the proximal promoter and coding exons. However, three cone gene loci maintain a linear configuration in rods. In contrast, rod gene chromosomal loops were undetectable in cones (Nrl^-/-), where the cone gene loci showed intra-chromosomal loops. These differential conformations of rod vs. cone genes in each photoreceptor subtype correlate with their transcription status, and are altered by photoreceptor transcription factor mutations. In Crx^-/- retina, the frequency of intra-chromosomal looping of the rod genes is reduced, which correlates with decreased expression levels. In Nr2e3 deficient (rd7) retina, both rhodopsin (Gnat1) and S-opsin loci adopt the loop conformation, correlating with dual expression of these genes in "hybrid S-cone/rod cells" (Chen et al, 2005; Corbo and Cepko, 2005) and suggesting that Nr2e3 is involved in regulating appropriate chromatin conformation of rod and cone genes.

Conclusions: : Transcriptionally active rod and cone genes undergo intra-chromosomal looping in the respective photoreceptor subtypes expressing them. Loop formation represents a specific chromosomal conformation for transcriptional activation, and requires the action of at least three photoreceptor transcription factors: Crx, Nrl, and Nr2e3.

Keywords: transcription factors • photoreceptors