Analyzing cell-specific patterns of DNA methylation in human retinal neurons

Penelope-Marie Clanor

Abstract

DNA methylation is an epigenetic modification that contributes to regulating gene expression by the process of an addition of a methyl group to cytosine nucleotides in genomic DNA. In retinal neurons, the accumulation of DNA methylation on gene regulatory sequences has been identified having an inverse relationship with gene expression. The cone-rod homeobox (CRX) transcription factor is a critical regulator of photoreceptor (PR) neuron differentiation and maturation. CRX functions by binding to PR-specific cis-regulatory elements and recruiting histone acetyltransferases that ultimately remodel local chromatin and initiate transcription. Much less is known about how CRX binding to regulatory elements is regulated and how DNA methylation impacts this affinity. The goal of this project is to quantitatively measure DNA methylation at CRX binding regions (CBRs) that regulate PR-specific genes. DNA collected from cone and rod enriched postmortem human retina was collected and treated with sodium bisulfite (BS) to mutate unmethylated cytosines into uracil, leaving 5mCs in the genome intact. Following BS treatment, CBRs were amplified via PCR, and DNA methylation was measured using quantitative pyrosequencing. Electrophoretic mobility shift assays (EMSAs) used purified recombinant human CRX-DBD-MBD fusion protein bound to synthetic dsDNA oligonucleotide probes identified functional CRX binding motifs. The EMSAs validated the functionality of these CBRs in 7 human PR-specific genes, RHO, PDE6B, PDE6A, GUCA1B, NR2E3, RCVRN, and SAG. Results demonstrate a distinct pattern of reduced DNA methylation on the 7 rod specific CBRs predicted in rod-enriched peripheral retina tissue relative to cone-enriched macula and cornea tissue, suggesting CRX may be acting as a transcriptional repressor at certain loci.