Functional analysis of DNA methylation and hydroxymethylation during eye development




Seritrakul, Pawat

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DNA methylation is an epigenetic mechanism known to play roles in regulating gene expression in various developmental and disease contexts. However, little is known about its function during eye development. 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. De novo methyltransferases (dnmt3 family) are responsible for the establishment of 5mC, while cytosine dioxygenases (tet family) convert 5mC into 5hmC, a stable epigenetic mark that can either remain on the genome or undergo subsequent demethylation. Here I performed gene expression and functional tests to elucidate the roles for both of these cytosine-modifying enzyme families during development, with an emphasis on the eye. All dnmt3-family and tet-family genes are expressed tissue-specifically in relevant domains during eye development. Single and double mutants for genes within dnmt3 family develop normally without any overt eye phenotype, indicating that these genes possess redundant functions during eye development. In contrast, in tet2-/-;tet3-/- mutants, retinal neurons are specified but most fail to terminally differentiate. Retinal ganglion cells lack a proper retino-tectal projection, and photoreceptors fail to generate outer segments. Mechanistically, mosaic analyses revealed a surprising cell non-autonomous requirement for tet activity during retinal neurogenesis. Through a combination of candidate gene analysis, transcriptomics and pharmacological manipulations, I identified candidate cell-extrinsic pathways regulated by tet2 and tet3. Additionally, genome-wide 5mC and 5hmC distribution profiles for retinal progenitor cells (RPCs) and differentiated retinal neurons are still currently unknown. To this end, I performed parallel bisulfite and oxidative bisulfite reactions followed by next-generation sequencing (BS/OXBS-seq) to generate the first nucleotide-resolution combined methylome/hydroxymethylome map of retinal cells during development and correlated these with gene expression. This genome- wide approach revealed expected 5mC/5hmC profiles of candidate retinal developmental genes, and identified several novel, uncharacterized genes with potential roles during RPC differentiation. These genes are candidates for further investigation to determine their functions during retinal neurogenesis. Data presented in this Dissertation uncover the role of DNA methylation and hydroxymethylation during eye development and provide the first epigenomic maps of 5mC/5hmC dynamics during retina formation.


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