Abstract
Purpose: :
Rod and cone opsin genes are expressed by retinal photoreceptors in a mutually exclusive manner. This is achieved by interactions between photoreceptor transcription factors and their responsive DNA elements. In mice, each opsin locus has a specific chromosomal looping organization in the photoreceptor subtype expressing it. This research was designed to test the hypothesis that active human opsin loci also undergo looping. Special attention was given to the OPN1LW (L) and OPN1MW (M) array, as their enhancer LCR pairs with either promoter for transcriptional activation.
Methods: :
To reveal looping interactions within each opsin locus, chromosomal conformation capture (3C) assays were performed on either human donor retinas or the Y79 retinoblastoma cell line. The donor retinas were dissected into three separate regions: Macula Lutea (ML, cone-rich), far periphery (FP, rod-rich), and peripheral Macula Lutea (PML, both rods and cones). Y79 cells were transfected with expression vectors with or without CRX. Quantitative PCR measured frequencies of chromosomal loops.
Results: :
In the rod-rich FP retinal samples, 3C assays revealed that the RHO enhancer RER contacts the RHO promoter and coding regions. These RER loops were also seen in the PML samples containing both rods and cones, but absent in the cone-rich ML samples, suggesting that silent RHO exists in the linear organization. In the ML samples, the L/M enhancer LCR loops with both L promoter (PL) / coding regions and M promoter (PM) / coding regions. Quantitative PCR showed that the difference in the looping frequency of LCR-PL versus LCR-PM is comparable to the reported mean L/M cone ratio. The LCR loops were barely detected in the rod-rich FP samples, suggesting that silent L/M loci exist in a linear organization. RHO and L/M loci also showed a linear organization in Y79 cells, where they are silenced. However, transient expression of CRX induced both opsin transcription and chromosomal looping.
Conclusions: :
Actively transcribed human opsin genes acquire a looping configuration, and CRX plays an important role in this process.
Keywords: opsins • transcription • transcription factors