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Agustin Luz-Madrigal, Erika Grajales-Esquivel, Alexander McCorkle, Leah Stetzel, Sarah Kosse, Panagiotis A. Tsonis, Katia Del Rio-Tsonis; Re-patterning of histone modifications and DNA methylation during RPE to retina reprogramming. Invest. Ophthalmol. Vis. Sci. 2017;58(8):631.
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© ARVO (1962-2015); The Authors (2016-present)
The embryonic chick has the capacity to regenerate a complete neural retina after retinectomy via Retinal Pigmented Epithelium (RPE) reprogramming as long as a source of Fibroblast Growth Factor 2 (FGF2) is present. The RPE is “reset” and reprogrammed to give rise to a neuroepithelium that eventually differentiates to retina. We hypothesize that dynamic changes of DNA methylation/demethylation and histone modifications, primarily those found in “bivalent chromatin” (H3K4me3/ H3K27me3) are associated with the process of RPE reprogramming.
Retinectomies were performed at embryonic day 4 in the absence (injury only) or presence of FGF2, and eyes were collected at different times post-retinectomy (PR) to perform immunostaining and high-resolution three-dimensional (HR-3D) confocal microscopy. RPE was collected by laser capture microdissection (LCM) to perform RT-qPCR.
RT-qPCR showed a transient downregulation of genes involved in DNA methylation (dnmt1, dnmt3a and dnmt3b), upregulation of the ten-eleven translocation enzyme-3 (tet-3) and prdm14 that are important for the process of demethylation and epigenetic reprogramming, respectively, and upregulation of enzymes involved in base excision repair (BER) and thymine DNA glycosylase (TDG) including gadd45α, gadd45β, gadd45γ and tdg. Interestingly, this differential gene expression was observed even in the absence of FGF2. Immunostaining pinpointed the presence of bivalent domains (H3K27me3/H3K4me2 or H3K4me3), 5-methylcytosine (5mC), 5-hydroxymethylcytocine (5-hmC) and 5-Carboxycytosine (5caC) in the developing chick RPE. Following RPE dedifferentiation, the nuclear distributions of these epigenetic signatures changed dynamically and continued through RPE reprogramming at 3 days PR in the presence of FGF2. At this time, repression marks such as H3K27me3 and 5-methylcytosine (5mC) decreased in the newly regenerated neuroepithelium, while activation marks persisted (H3K4me2 or H3K4me3) along with 5caC suggesting a process of demethylation during RPE reprogramming.
We demonstrate dynamic changes and re-distribution of epigenetic marks during chick RPE reprogramming. Further analysis by ChIP-seq and whole genome bisulfite sequencing (WGBS) will provide insights to understand the epigenetic reprogramming of RPE during the process of retina regeneration.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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