Genome-wide analysis of Mecp2 dependent DNA methylation and hydroxymethylation at base-resolution in neurons. KE. Szulwach1, M. Yu2, X. Li1, CR. Street1, C. He2, P. Jin1 1) Dept Human Gen, Emory Univ, Atlanta, GA; 2) Dept of Chem and Inst for Biophysical Dynamics, The University of Chicago, Chicago, IL.

   5-methylcytosine (5mC) imparts epigenetic function to genomic elements and has been implicated in an array of human neurological disorders. In neurons, 5mC exhibits stimulus dependent dynamics and is enzymatically oxidized to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by TET-family proteins. When coupled with base-excision repair, TET-mediated oxidation of 5mC can result in active DNA demethylation. We have established whole-genome DNA methylation states at base-resolution using complementary methods to distinguish 5mC and 5hmC (WGBS and TAB-Seq, respectively) in mature neurons isolated from adult mouse hippocampal dentate gyrus as well as from a mouse model of the human neurological disease, Rett Syndrome (RTT). RTT is primarily caused by mutations in MECP2, which encodes a 5mCpG specific binding protein. In brain, Mecp2 expression is inversely correlated with 5hmC levels, and Mecp2 has affinity for 5hmC, implicating it in the dynamic regulation of 5mC. Our base-resolution analysis of 5mC and 5hmC indicate that, although Mecp2 is extremely abundant in mature neurons, DNA methylation states are largely preserved in Mecp2-/y mice (Pearson 0.99). Yet, stringent identification of differentially methylated regions (DMRs) consistent among biological replicates identified 5,126 DMRs within a small fraction of the genome (~2.5MB in total). These DMRs exhibited three key features; 1.) DMRs were distal to promoters 2.) DMRs were initially depleted of 5mC relative to the genome-wide average in wildtype neurons 3.) DMRs were preferentially hypomethylated in Mecp2-/y neurons. These features indicate Mecp2 dependent regulation of DNA methylation at sites likely undergoing dynamic regulation under normal conditions, which is supported by the enrichment of 5hmC at DMRs. Lastly, DMRs that become hypomethylated in Mecp2-/y neurons are enriched for a distinct set of transcription factor binding motifs (P 1e-6), including Creb1 and CTCF, among others. These DMRs, therefore, represent sites of Mecp2 dependent dynamic DNA methylation, providing links to the underlying gene regulatory networks dysregulated in Mecp2-/y mice. Overall, our data demonstrate a novel approach whereby genome-wide analyses of 5mC and 5hmC can be utilized to define DMRs reflective of the underlying alterations in the transcriptional regulatory circuitry, supporting a role for Mecp2 in localized DNA methylation dynamics at key gene regulatory regions.

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