Abstract
BackgroundVertebrate genomes undergo epigenetic reprogramming during development and disease. Emerging evidence suggests that DNA methylation plays a key role in cell fate determination in the retina. Despite extensive studies of the programmed cell death that occurs during retinal development and degeneration, little is known about how DNA methylation might regulate neuronal cell death in the retina.MethodsThe developing chicken retina and the rd1 and rhodopsin-GFP mouse models of retinal degeneration were used to investigate programmed cell death during retinal development and degeneration. Changes in DNA methylation were determined by immunohistochemistry using antibodies against 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC).ResultsPunctate patterns of hypermethylation paralleled patterns of caspase3-dependent apoptotic cell death previously reported to occur during development in the chicken retina. Degenerating rd1 mouse retinas, at time points corresponding to the peak of rod cell death, showed elevated signals for 5mC and 5hmC in photoreceptors throughout the retina, with the most intense staining observed in the peripheral retina. Hypermethylation of photoreceptors in rd1 mice was associated with TUNEL and PAR staining and appeared to be cCaspase3-independent. After peak rod degeneration, during the period of cone death, occasional hypermethylation was observed in the outer nuclear layer.ConclusionThe finding that cell-specific increases of 5mC and 5hmC immunostaining are associated with the death of retinal neurons during both development and degeneration suggests that changes in DNA methylation may play a role in modulating gene expression during the process of retinal degeneration. During retinal development, hypermethylation of retinal neurons associates with classical caspase-dependent apoptosis as well as caspase-3 independent cell death, while hypermethylation in the rd1 mouse photoreceptors is primarily associated with caspase-3 independent programmed cell death. These findings suggest a previously unrecognized role for epigenetic mechanisms in the onset and/or progression of programed cell death in the retina.
Highlights
Epigenetic modifications to genomic DNA and associated histone proteins dictate chromatin structure and regulate gene expression across a range of cellular processes [1]
A striking pattern of intensely labeled cells was conspicuously present in the ventral region of the embryonic days 3 (E3) neural retina where a large cluster of cells staining positive for 5methyl cytosine (5mC) was present along the morphogenic furrow (Figure 1E2; downward arrow), a region long-recognized to undergo a wave of cell death [14]. 5mC positive cells in the lens were evident at this time
Double labeling with antibodies against 5mC and 5hmC confirmed that these signals colocalized with one another in individual chicken retinal neurons
Summary
Epigenetic modifications to genomic DNA and associated histone proteins dictate chromatin structure and regulate gene expression across a range of cellular processes [1]. The recent discovery that 5mC can be further modified to 5-hydroxymethylcytosine (5hmC), 5formylcytosine (5fC) and carboxylcytosine (5caC) through the activity of the Tet (ten eleven translocation) proteins increases the complexity by which epigenetically modified cytosine bases can participate in gene regulation [3,4]. Genome-wide profiles in plants and vertebrates have demonstrated an inverse correlation between transcriptional activity and the accumulation of 5mC in upstream regulatory regions of genes [5,6]. Emerging evidence demonstrates a positive correlation between transcription and 5hmC in upstream regulatory regions of genes [7]. Despite extensive studies of the programmed cell death that occurs during retinal development and degeneration, little is known about how DNA methylation might regulate neuronal cell death in the retina
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