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Stylianos Michalakis, Arshan Perera, Susanne Koch, Mirko Wagner, Lukas Windhager, Kerstin Nagel-Wolfrum, Tim Strom, Ralf Zimmer, Thomas Carell, Martin Biel; Role of 5-hydroxymethylcytosine during postnatal retinal development. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2618.
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5-hydroxymethylcytosine (5hmC), also known as the sixth base of the genome, is a recently discovered oxidative product of 5-methylcytosine (5mC) generated by the enzymatic action of Ten Eleven Translocation (TET) family members. The functional role of 5hmC is largly unknown. However, studies in embryonic stem cells and cancer tissues suggest that TET enzymes and 5hmC may be involved in gene regulation. Eye opening at postnatal week (pw) 2 is a key time point during mouse retinal development. Maturation of retinal cells as well as formation of the retinal network is ongoing after eye opening and is completed only one to two weeks later. Here, we analyzed the role of 5hmC during this phase of postnatal retinal development in the mouse.
5hmC was localized to specific retinal cell types using immunohistochemistry. Global 5hmC levels in retinal samples were quantified using ultra high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). 5hmC marks were mapped to the retinal genome by hydroxymethylated DNA immunoprecipitation (hMeDIP) followed by next generation sequencing and were subsequently correlated with retinal gene expression data obtained from microarray experiments.
At pw 2 5hmC weakly localized to nuclei of cells within the ganglion cell layer and the inner nuclear layer of the retina. The levels of 5hmC in these cells were increased at pw 3. In addition, 5hmC was now detectable in retinal photoreceptors were it co-localized with histone marks of the euchromatin. Global levels of 5hmC also increased from pw 2 to pw 3, whereas 5-methylcytosine was unchanged. hMeDIP experiments revealed a developmentally programmed acquisition of 5hmC during retinal maturation at gene-rich regions and in genes containing activating histone marks.
Our data suggest that 5hmC is dynamically regulated during postnatal retinal development and is capable to elevate gene expression of retina-specific genes.
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