DNA Methylation Dynamics During the Differentiation of Retinal Progenitor Cells Into Retinal Neurons Reveal a Role for the DNA Demethylation Pathway.

To evaluate the contribution of the DNA methylation and DNA demethylation pathways in retinal development, we studied DNA methylation in retinal progenitor cells (RPCs) and retinal neurons using a combination of whole genome bisulfite sequencing (WGBS) data obtained in our study and WGBS data collected from previous studies. The data was analyzed using Hidden Markov Model- and change point-based methods to identify methylome states in different segments of the studied genomes following genome annotation. We found that promoters of rod and cone phototransduction genes and rod photoreceptor genes, but not genes required for the development and function of other retinal phenotypes, were highly methylated in DNA isolated from human and murine fetal retinas (which mostly contain RPCs) and postnatal murine RPCs. While these highly methylated genomic regions were inherited by non-photoreceptor phenotypes during RPC differentiation, the methylation of these promoters was significantly reduced during RPC differentiation into photoreceptors and accompanied by increased expression of these genes. Our analysis of DNA methylation during embryogenesis revealed low methylation levels in genomic regions containing photoreceptor genes at the inner cell mass stage, but a sharp increase in methylation at the epiblast stage, which remained the same later on (except for DNA demethylation in photoreceptors). Thus, our data suggest that the DNA demethylation pathway is required for photoreceptor phenotypes in the developing retina. Meanwhile, the role of the DNA methylation and DNA demethylation pathways during RPC differentiation into non-photoreceptor retinal phenotypes might be less important.

Development and epigenetic plasticity of murine Müller glia.

The ability to regenerate the entire retina and restore lost sight after injury is found in some species and relies mostly on the epigenetic plasticity of Müller glia. To understand the role of mammalian Müller glia as a source of progenitors for retinal regeneration, we investigated changes in gene expression during differentiation of retinal progenitor cells (RPCs) into Müller glia and analyzed the global epigenetic profile of adult Müller glia. We observed significant changes in gene expression during differentiation of RPCs into Müller glia in only a small group of genes and found a high similarity between RPCs and Müller glia on the transcriptomic and epigenomic levels. Our findings also indicate that Müller glia are epigenetically very close to late-born retinal neurons, but not early-born retinal neurons. Importantly, we found that key genes required for phototransduction were highly methylated. Thus, our data suggest that Müller glia are epigenetically very similar to late RPCs; however, obstacles for regeneration of the entire mammalian retina from Müller glia may consist of repressive chromatin and highly methylated DNA in the promoter regions of many genes required for the development of early-born retinal neurons. In addition, DNA demethylation may be required for proper reprogramming and differentiation of Müller glia into rod photoreceptors.

The epigenetic basis for the impaired ability of adult murine retinal pigment epithelium cells to regenerate retinal tissue.

The epigenetic plasticity of amphibian retinal pigment epithelium (RPE) allows them to regenerate the entire retina, a trait known to be absent in mammals. In this study, we investigated the epigenetic plasticity of adult murine RPE to identify possible mechanisms that prevent mammalian RPE from regenerating retinal tissue. RPE were analyzed using microarray, ChIP-seq, and whole-genome bisulfite sequencing approaches. We found that the majority of key genes required for progenitor phenotypes were in a permissive chromatin state and unmethylated in RPE. We observed that the majority of non-photoreceptor genes had promoters in a repressive chromatin state, but these promoters were in unmethylated or low-methylated regions. Meanwhile, the majority of promoters for photoreceptor genes were found in a permissive chromatin state, but were highly-methylated. Methylome states of photoreceptor-related genes in adult RPE and embryonic retina (which mostly contain progenitors) were very similar. However, promoters of these genes were demethylated and activated during retinal development. Our data suggest that, epigenetically, adult murine RPE cells are a progenitor-like cell type. Most likely two mechanisms prevent adult RPE from reprogramming and differentiating into retinal neurons: 1) repressive chromatin in the promoter regions of non-photoreceptor retinal neuron genes; 2) highly-methylated promoters of photoreceptor-related genes.