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Stylianos Michalakis, Arshan Perera, David Eisen, Silvia K Laube, Mirko Wagner, Markus Müller, Martin Biel, Thomas Carell; The epigenetic mark 5hmC recruits a dynamic set of readers in the retina. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4997.
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Hydroxylation of genomic 5-methylcytosine (5mC) by Tet enzymes has been proposed to play a key role in the formation, plasticity and function of neuronal circuits. The exact mechanisms controlling Tet enzyme activity and 5-hydroxymetylcytosine (5hmC) levels in neurons are not known. Here, we used the mouse retina to study the role of 5hmC during maturation of neuronal circuits.
To identify specific readers of 5hmC and 5mC in retinal cells we applied a protein pull-down experiment combined with mass spectrometry (MS) at two developmental time points: before eye opening (postnatal day (p) 11) and at a time points when retinal maturation is completed (p21). We incubated biotin-coupled double-stranded DNA oligos containing 5hmC or 5mC or unmodified cytosines with nuclear protein extracts from p11 or p21 mouse retina to precipitate and purify DNA binding proteins. After tryptic digestions peptides were chemically labeled with tandem mass tags in forward and reverse experiments and subsequently analyzed using liquid chromatography coupled to MS.
We identified 212 specific readers of 5hmC and 43 specific readers of 5mC in the mouse retina. Notably, the p11 retina contained more 5mC readers (35) than 5hmC readers (26). Conversely, at p21 the number of 5hmC readers increased dramatically to 195 whereas the number of 5mC readers dropped to 8. The majority of readers appeared to be specific for a particular cytosine modification whereas 10 of the identified proteins were readers of both 5mC and 5hmC. There was no overlap between 5mC readers at the two developmental stages. However, eight 5hmC readers were conserved between the two ages. Gene ontology analysis revealed that biological functions related to cellular assembly and organization, DNA replication, recombination and repair or cellular morphology were significantly enriched within the set of 5hmC-specific readers whereas the set of 5mC readers was enriched in proteins involved in chromatin or histone modification or negative regulation of gene expression. In total, 23 (4 at p11 and 19 at p21) of the 5hmC readers identified in the retina are regulators of transcription. In contrast, only five 5mC readers at p11 and one at p21 could be classified as regulators of transcription.
In summary, our data provide strong evidence for a role of 5hmC as epigenetic regulator in the retina.
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