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.
Highlights
The vertebrate retina develops from an apparently homogeneous pool of pluripotent retinal neuroblasts [1]
Quantitative PCR (QPCR) was performed for RBP3 (Ensembl: ENSG00000107618), RHO (ENSG00000163914), OPN1LW (ENSG00000102076), OPN1MW (ENSG00000147380), and OPN1SW (ENSG00000128617) on the two human retinoblastoma (Rb) cell lines, WERI and Y79, and the human embryonic kidney cell line HEK293, which is known to have some neuronal features [15] (Fig. 1A)
Little is known about the function of DNA methylation and the role played by CpG-39 dinucleotide (CpG) islands in the establishment and regulation of tissue-specific expression
Summary
The vertebrate retina develops from an apparently homogeneous pool of pluripotent retinal neuroblasts [1]. To date, understanding gene regulation in the retina and the effects of intrinsic and extrinsic signaling molecules has primarily concentrated upon the role of specific DNA regulatory elements and the transcription factors with which they interact [2]. Other regions of the genome show tissue-specific differential methylation, the significance of this variance, especially for CpG-poor promoters, which are more often found in tissue-specific genes, remains unresolved [8]. These tissue-specific differentially methylated regions (T-DMRs), which have been identified by both restriction landmark genomic scanning [9,10,11] and microarraybased approaches [12,13,14], generally show an inverse relationship with differential gene expression. To investigate whether DNA methylation might play a role in the tissue-specific expression of photoreceptor genes, we investigated their methylation status in different tissues and cell types
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