A role for DNA methylation in regulation of EphA5 receptor expression in the mouse retina

Abstract

Understanding the mechanisms regulating expression of retinal ganglion cell (RGC) specific and axon-guidance genes during development and in retinal stem cells will be critical for successful optic nerve regeneration. Müller glia have some characteristics of retinal stem cell properties, but in mammals have demonstrated limited potential to differentiate into RGCs. Chromatin remodeling through histone deacetylation and DNA methylation are a potential mechanism for silencing genes necessary for neuronal differentiation of glial cells. We investigated DNA methylation as a mechanism for regulating expression of mouse EphA5, one member of a large family of ephrin receptor genes that regulate patterning of the topographic connections of RGCs during visual system development. We analyzed spatial and age-related patterns of EphA5 promoter methylation by bisulfite sequencing and mRNA expression by quantitative RT-PCR in the mouse retina. The CpG island in the EphA5 promoter was hypomethylated in the retina and showed no change in overall methylation with age, despite a decline in EphA5 mRNA expression levels in the adult retina. In the nasal retina of post-natal day 0 mice, there was a modest, but statistically significant increase in methylation. Increased methylation corresponded with lower levels of receptor mRNA expression in the nasal retina. We cloned the EphA5 promoter and found that site-specific differences in methylation could preferentially activate or repress promoter activity in transient transfections of rat retinal progenitor cells (R28) using luciferase assays. In sphere cultures generated by EGF/FGF2 stimulation of conditionally immortalized mouse Müller glia (ImM10), EphA5 promoter was hypermethylated and EphA5 mRNA was not detected. Demethylation using 5-azadeoxycytidine (AzadC) resulted in a significant decrease of methylation of the EphA5 promoter and re-expression of the EphA5 mRNA. The inverse relationship between EphA5 promoter methylation and mRNA expression is consistent with a role for DNA methylation in modulating the spatial patterns of EphA5 gene expression in the retina and in silencing EphA5 expression in ImM10 cells. The robust up regulation of EphA5 in ImM10 cells following demethylation suggests that modulation of chromatin structure may be a useful approach for promoting expression of silenced developmental genes and increasing the neurogenic potential of Müller glia.

Novel Roles and Mechanism for Krüppel-like Factor 16 (KLF16) Regulation of Neurite Outgrowth and Ephrin Receptor A5 (EphA5) Expression in Retinal Ganglion Cells

“…3A). Interestingly, the GT box is positioned immediately 3Ј to a cluster of CpG dinucleotides that we previously found to have increased methylation in genomic DNA from the nasal but not temporal retina (25). Therefore, subsequent analysis focused on this site.…”

“…There is evidence for silencing of EphA5 expression by hypermethylation in cancer (22)(23)(24). We previously showed that total CpG methylation abolishes activity of mEphA5 promoter-reporter constructs in R28 retinal progenitor cells and that transcriptional silencing of the endogenous EphA5 gene in a mouse retinal cell line (ImM10) is reversed by genome-wide demethylation (25). Intriguingly, our prior analysis also showed that in the nasal retina, where EphA5 expression is lowest, CpG methylation is increased specifically in a cluster of CpG dinucleotides immediately downstream of the EphA5 transcription start site.…”

Regulation and misregulation of Eph/ephrin expression

“…67 New findings have shown that CpG islands in the ephA5 promoter display site-specific differences in methylation that could preferentially activate or repress promoter activity and may contribute to graded ephA5 gene expression in the tectum. 68 More studies, however, have focused on aberrant methylation patterns of Eph receptor and ephrin genes in cancer. Methylation of CpG promoter sequences of ephA1, ephA2, ephA7, ephB2, ephB4 and ephB6 has been reported in several human tumors, including colorectal, prostate and breast cancer.…”

Epigenetic regulation of retinal development and disease

“…How might DNA methylation regulate particular RGC genes? To date, only one study described DNA methylation changes-via bisulfite specific sequencing (a modified PCR method by which methylated CpG regions in primer-specific sequences can be amplified and identified)-in an RGC-expressed gene, EphA5 [80]. EphA5 is a member of the ephrin receptor family of tyrosine kinase receptors, which induce intracellular signaling cascades via binding of ephrin ligands.…”

“…In a Müller cell line, the EphA5 promoter is highly methylated; 5-azacytidineinduced demethylation allowed upregulation of EphA5 mRNA. The authors propose that DNA demethylation may "unlock" the neurogenic potential of Müller cells-considered by some researchers to be multipotent progenitors-and coax these cells to differentiate to RGC-like cells in injury or neurodegeneration [80].…”