July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Epigenomic profiling of zebrafish retinal progenitor cells during neurogenesis
Author Affiliations & Notes
  • Pawat Seritrakul
    Ophthalmology , University of Pittsburgh , Pittsburgh, Pennsylvania, United States
    Molecular Biosciences , University of Texas at Austin, Austin, Texas, United States
  • Dennis Kostka
    Developmental Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Jeffrey M Gross
    Ophthalmology , University of Pittsburgh , Pittsburgh, Pennsylvania, United States
  • Footnotes
    Commercial Relationships   Pawat Seritrakul, None; Dennis Kostka, None; Jeffrey Gross, None
  • Footnotes
    Support  RO1-EY18005
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 5386. doi:
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      Pawat Seritrakul, Dennis Kostka, Jeffrey M Gross; Epigenomic profiling of zebrafish retinal progenitor cells during neurogenesis. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5386.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Retinal neurons undergo cellular differentiation from a pool of retinal progenitor cells (RPCs), and this serves as an ideal process in which to study the epigenetic basis of neurogenesis. DNA methylation is an epigenetic mechanism that has been shown to regulate gene expression in various contexts. Two types of methylation marks: 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are thought to serve as silencing and activating signals for gene regulation, respectively. However, genome-wide 5mC and 5hmC profiles of RPCs and differentiated retinal neurons are still currently unknown. The purpose of this study is to profile and correlate DNA methylation (5mC and 5hmC) and gene expression patterns during retinal neurogenesis.

Methods : We generated nucleotide-resolution maps of whole-genome 5mC (methylome) and 5hmC (hydroxymethylome) from FACS-purified zebrafish retinal cells. RPCs were isolated using transgenic vsx2:GFP which is expressed early at 22 hours post-fertilization (hpf) before the onset of differentiation. Genomic DNA was extracted and processed, in parallel, for whole-genome bisulfite and oxidative bisulfite conversion followed by next-generation sequencing. Sequences were mapped using bisulfite-specific aligners, and percentage of C/5mC/5hmC calculated at single nucleotide resolution. Gene expression was quantified via RNA-seq.

Results : Similar to human and mouse, the zebrafish genome is overall hypermethylated. In 22hpf RPCs, genes highly expressed in RPCs (vsx2 and pax6a) are devoid of 5mC (demethylated) around the TSS and CpG islands, and they accumulate 5hmC. Genes involved in differentiation, such as atoh7, exhibit signs of pre-activation, which includes accumulation of 5hmC and partial demethylation. Conversely, genes silenced in RPCs such as crystallins remain fully methylated.

Conclusions : This work is the first to profile retinal methylome and hydroxymethylome at single nucleotide-resolution in RPCs, and show correlation between these epigenetic marks and gene expression. Ongoing work is aimed at generating epigenomic profiles at higher sequencing depth to increase statistical power, identify differentially methylated and hydroxymethylated regions, and expand the study to late RPCs (27hpf) and differentiated retinal neuron types.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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