5-Methylcytosine embedded in mammalian DNA represses local transcription by recruiting modification-specific binding partners. Its active removal is initiated by sequential oxidation of the 5-methyl group by TET enzymes to produce three oxidized species, collectively referred to as [ox]mC. Although rare, the distribution of [ox]mC modifications is tissue-, gene-, and coding strand-specific and distinct from 5-methylcytosine, suggesting unique functions. To examine this possibility, I fractionated mammalian brain extracts to discover, isolate and characterize binding partners specific for [ox]mC. This purification reveals remarkably specific factors that are selective for each of the three oxidation states and sensitive to the 5-modification state on each strand. I demonstrate that one such factor, WDR76, is a highly 5-hydroxymethylcytosine-specific binding protein. I have begun to lay the foundation for further mechanistic studies of these specific binding proteins in mouse embryonic stem cells and leukemia. My results provide an essential bridge from studies of the distribution of [ox]mC and the effects of TET knockouts, to the possible functions of [ox]mC recognition in gene regulation or chromatin signaling.




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