Cell-specific DNA methylation patterns of retina-specific genes

Abstract

Many studies have demonstrated that epigenetic mechanisms are important in the regulation of gene expression during embryogenesis, gametogenesis, and other forms of tissue-specific gene regulation. We sought to explore the possible role of epigenetics, specifically DNA methylation, in the establishment and maintenance of cell type-restricted gene expression in the retina. To assess the relationship between DNA methylation status and expression level of retinal genes, bisulfite sequence analysis of the 1000 bp region around the transcription start sites (TSS) of representative rod and cone photoreceptor-specific genes and gene expression analysis were performed in the WERI and Y79 human retinoblastoma cell lines. Next, the homologous genes in mouse were bisulfite sequenced in the retina and in non-expressing tissues. Finally, bisulfite sequencing was performed on isolated photoreceptor and non-photoreceptor retinal cells isolated by laser capture microdissection. Differential methylation of rhodopsin (RHO), retinal binding protein 3 (RBP3, IRBP) cone opsin, short-wave-sensitive (OPN1SW), cone opsin, middle-wave-sensitive (OPN1MW), and cone opsin, long-wave-sensitive (OPN1LW) was found in the retinoblastoma cell lines that inversely correlated with gene expression levels. Similarly, we found tissue-specific hypomethylation of the promoter region of Rho and Rbp3 in mouse retina as compared to non-expressing tissues, and also observed hypomethylation of retinal-expressed microRNAs. The Rho and Rbp3 promoter regions were unmethylated in expressing photoreceptor cells and methylated in non-expressing, non-photoreceptor cells from the inner nuclear layer. A third regional hypomethylation pattern of photoreceptor-specific genes was seen in a subpopulation of non-expressing photoreceptors (Rho in cones from the Nrl -/- mouse and Opn1sw in rods). These results demonstrate that a number of photoreceptor-specific genes have cell-specific differential DNA methylation that correlates inversely with their expression level. Furthermore, these cell-specific patterns suggest that DNA methylation may play an important role in modulating photoreceptor gene expression in the developing mammalian retina.

Figure 1. DNA methylation of photoreceptor genes in human cell lines is inversely correlated with expression levels.

A, QPCR of OPN1LW, OPN1MW, OPN1SW, RHO and RBP3 in Y79, WERI, and HEK293 cells, normalized to expression of human retinal cDNA. B–F, % DNA methylation of photoreceptor genes in 3 cell lines plotted versus the CpG site position (noted by hash marks) relative to the TSS (position “0”). Expression level and % methylation are the average of biological triplicates ± SEM.

Figure 2. Bisulfite sequencing of mouse photoreceptor-specific genes and miRNAs shows increased DNA methylation in non-expressing tissues.

A–F, Genomic DNA was isolated from 4 mouse tissues and bisulfite sequenced. % DNA methylation of photoreceptor genes was plotted versus the CpG site position (noted by hash marks) relative to the TSS (position “0”) for genes or the 5′ end of the miRNA stem-loop sequence (position “0”). % methylation is the average of 3 animals ± SEM. Reliable sequence was not obtained for Rho sites −270, −166, and −129 due to lack of sequence complexity that preceded these sites.

Figure 3. Photoreceptor-specific genes are hypomethylated in photoreceptors and methylated in non-photoreceptor cells from the INL.

Cells from the ONL (expressing cells) and INL (non-expressing cells) were isolated by LCM from adult mouse retina. Genomic DNA was isolated and bisulfite sequenced. % DNA methylation of photoreceptor genes was plotted versus the CpG site position (noted by hash marks) relative to the TSS (position “0”) for A, Rbp3 and B, Rho. % methylation is the average of 3 animals ± SEM.

Figure 4. Z-score calculations show a variable correlation between the methylation status of RBP3 gene CpG sites.

Z-score of correlation between methylated sites for RPB3 in human cell lines HEK293 and Y79. A, Z-score of CpG site pairs (RPB3 CpG sites 314–737, and 777–957 bp downstream of the TSS, which were amplified as 2 separate PCR products). There was no Z-score if one site was extremely hypermethylated or hypomethylated. Z-scores ranged from 6 (red) to −2 (dark blue). B, Z-score as function of distance between CpG site pairs in base pairs (bp). Magenta circles represent smoothed values within 9-point smoothing window.

Figure 5. DNA hypomethylation correlates with expression of human photoreceptor-specific genes in tissue culture cells.

Bisulfite sequencing of DNA samples from 3 different human tissue culture cell lines was performed. Thirty sequences (10 clones for each of 3 biological replicates) were batched together in a true-to-scale map, which represents the % methylation for each CpG site by a color gradient (white, unmethylated; dark blue, 100% methylated).

Figure 6. Hypomethylation of mouse photoreceptor-specific genes in expressing photoreceptors and regional hypomethylation in non-expressing photoreceptors.

Bisulfite sequencing of photoreceptor-specific genes from non-expressing tissues (testes, kidney, and brain) and non-expressing cells from the INL, as well as expressing cells from the ONL (primarily rods from wild type mice and cones from Nrl −/− mice) was performed. Thirty sequences (10 clones for each of 3 biological replicates) were batched together in a true-to-scale map, which represents the % methylation for each CpG site by a color gradient (white, unmethylated; dark blue, 100% methylated). Opn1mw has only 2 CpG sites within the 1000 bp upstream of the TSS and none in the first exon. Both of these CpG sites (−762 and −510), were methylated in all tissues and cell types and the true-to-scale maps are not shown.