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Cell-specific DNA methylation patterns of retina-specific genes.

Merbs SL, Khan MA, Hackler L, Oliver VF, Wan J, Qian J, Zack DJ - PLoS ONE (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America. smerbs@jhmi.edu

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.

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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.
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pone-0032602-g003: 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.

Mentions: To explore the methylation profile of Rbp3 and Rho in the mouse retina in more detail, expressing photoreceptors from the ONL and non-expressing, non-photoreceptor cells from the INL were isolated by LCM from three different adult mouse retinas. Bisulfite sequencing of the genomic DNA from each cell population demonstrated that the Rbp3 and Rho were mostly unmethylated around the TSS in photoreceptors (primarily rods) and mostly methylated in non-photoreceptor cells isolated from the INL (Fig. 3A–B), again demonstrating an inverse correlation between DNA methylation and cell-specific gene expression; however, not all Rbp3 and Rho CpG sites sequenced were unmethylated in photoreceptors. CpG sites −475 and −294 in Rbp3 and site −468 in Rho were methylated in photoreceptors, in addition to cells from the INL. In Rho, CpG site −128 had similar levels of methylation (∼20%) in both cell populations.


Cell-specific DNA methylation patterns of retina-specific genes.

Merbs SL, Khan MA, Hackler L, Oliver VF, Wan J, Qian J, Zack DJ - PLoS ONE (2012)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293830&req=5

pone-0032602-g003: 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.
Mentions: To explore the methylation profile of Rbp3 and Rho in the mouse retina in more detail, expressing photoreceptors from the ONL and non-expressing, non-photoreceptor cells from the INL were isolated by LCM from three different adult mouse retinas. Bisulfite sequencing of the genomic DNA from each cell population demonstrated that the Rbp3 and Rho were mostly unmethylated around the TSS in photoreceptors (primarily rods) and mostly methylated in non-photoreceptor cells isolated from the INL (Fig. 3A–B), again demonstrating an inverse correlation between DNA methylation and cell-specific gene expression; however, not all Rbp3 and Rho CpG sites sequenced were unmethylated in photoreceptors. CpG sites −475 and −294 in Rbp3 and site −468 in Rho were methylated in photoreceptors, in addition to cells from the INL. In Rho, CpG site −128 had similar levels of methylation (∼20%) in both cell populations.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America. smerbs@jhmi.edu

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.

Show MeSH
Related in: MedlinePlus