Date of Award
Functions of the nervous system are accompanied at the cellular level by changes in gene expression, regulated by transcription factors and epigenetic mechanisms, such as histone modifications and DNA methylation, that are frequently altered in neurological disorders. 5-hydroxymethylcytosine (5hmC), a recently identified DNA base derived from 5-methylcytosine, accounts for ~40% of modified cytosines in the neuronal genomes, suggesting that 5hmC is a stable epigenetic mark and its interpretation in the nervous system may differ from the other tissues. This hypothesis was supported by the recent findings showing that 5hmC is enriched over the bodies of active genes within euchromatin in a cell-specific manner. In the first part of this study, we identify the methyl-CpG binding protein 2 (MeCP2) as the major reader of this activation mark and demonstrate that MeCP2 is the only methyl-CpG binding domain (MBD) protein that binds 5hmC and, 5mC, with high affinity. We reveal strong preferential inhibition of MeCP2 affinity to 5hmC by a Rett-syndrome-causing mutation, R133C. We then show that MeCP2 recognizes 5hmC and 5mC within CpA context. Modified CpAs were recently shown to exist in embryonic stem and neuronal cells, where it localizes to actively transcribed gene bodies. Together these data support a model where 5hmC and MeCP2 formulate a cell-specific epigenetic mechanism for establishing active chromatin states that facilitate gene expression. This is supported by our observation of reduced chromatin accessibility of 5hmC containing loci in the absence of MeCP2. In the second part of the study, we discover a complex in the brain nuclear extract that assembles specifically in the presence of 5hmC. We purify and identify components of this complex: the purine-rich element binding protein (Pur) α and β, which are required for the proper development of neuronal cell types. We verify the increased affinity of recombinant Pur α and Purβ to 5hmC and support a binding mechanism where they separate two strands of DNA and recognize specific sequences. These findings offer a previously unknown function for Pur proteins via binding to 5hmC. This study presents new decoders of a novel epigenetic mark, 5hmC, that is effectively and differentially employed in the brain. Unlike previous studies, we introduce readers of 5hmC as a stable activation mark. In addition, by mechanistic characterization of our model we link 5hmC to an autism spectrum disorder and offer a new avenue toward investigation of its pathophysiology.
Ayata, Pinar, "Decoding 5HMC as an Active Chromatin Mark in the Brain and its Link to Rett Syndrome" (2013). Student Theses and Dissertations. 238.