Abstract
Purpose: :
The rod and cone opsin genes are expressed in a mutually exclusive manner in their respective photoreceptor subtypes. Subtype specific transcription factors play important roles in this regulation. However, it is not clear how these factors differentially regulate rod vs. cone genes in a given subtype, and if chromatin configuration of the target genes contributes to this regulation. Our research goal was to determine if rod and cone opsin genes adopt different chromatin conformations in rods vs. cones, and if these conformations are altered in mutants defective in opsin transcription.
Methods: :
Physical interactions between chromatin segments of the rhodopsin or M-opsin gene locus were examined by chromosomal conformation capture (3C) assays (Dekker et al, Science 295:1306, 2002): Interacting chromatin segments in mouse retina were covalently linked using formaldehyde, digested with a restriction enzyme followed by intra-molecular ligation. The DNA was purified after de-crosslink and analyzed by PCR using primers specific for interacting candidate gene fragments. Three mouse lines at P14 provide the source of rods (wild-type), cones (Nrl-/-) and transcription defective rods (Crx-/-) for 3C.
Results: :
Rods and cones show different chromosomal organization of rhodopsin and M-opsin genes. In rods (wild-type retina), the rhodopsin enhancer (RER) loops out to interact with the rhodopsin promoter (RPPR) and coding exons, while the M-opsin chromatin maintains a linear configuration. In contrast, intrachromosomal loops were undetectable for rhodopsin in cones (Nrl-/- retina), where the M-opsin enhancer (locus control region, LCR) interacts with its promoter and coding exons. No intra-chromosomal loops were detected between the enhancer and regions 3' to the last coding exon of either opsin locus and between coding exons, adjacent or distant, suggesting that the long-range enhancer-promoter/exon interaction of the opsin genes represents a specific chromosomal conformation for transcriptional activation. This finding was further supported by 3C assays on Crx-/- retina, where the rhodopsin RER weakly interacts with RPPR but not the coding exons, correlating with defective rhodopsin transcription (10% of wild-type mRNA).
Conclusions: :
Rod and cone opsin genes undergo higher-order chromatin organization with intra-chromosomal loops in the respective photoreceptor subtypes expressing them. This epigenetic organization correlates with transcription and requires the action of photoreceptor transcription factors.
Keywords: transcription • photoreceptors • transcription factors