Title: Chromatin-modifying enzymes of the 2-oxoglutarate and iron(II)-dependent oxygenase superfamily
Description: Chromatin dynamics are critically influenced by the covalent modifications of both histone tails and DNA. Methylation of lysine residues in histone tails is dynamically regulated by the opposing activities of histone methyltransferases and histone demethylases. In the first part of this thesis, I show that JARID1C/SMCX, a JmjC domain-containing protein implicated in X-linked mental retardation (XLMR), possesses 2-oxoglutarate (20G)- and Fe(II)-dependent H3K4 tri-demethylase activity and functions as a transcriptional repressor. An SMCX complex isolated from HeLa cells contains additional chromatin modifiers (the histone deacetylases, HDAC1/2 and the histone H3 lysine 9 methyltransferase, G9a) and the transcriptional repressor REST, suggesting a direct role for SMCX in chromatin dynamics and REST-mediated repression. Chromatin immunoprecipitation (ChIP) reveals that SMCX and REST co-occupy the neuron-restrictive silencing elements (NRSE) in the promoters of a subset of REST target genes. RNAi-mediated depletion of SMCX derepresses several of these targets and concomitantly elevates H3K4 trimethylation at sodium channel type 2A ( SCN2A ) and synapsin I ( SYN1 ) promoters. We propose that loss of SMCX activity impairs REST-mediated neuronal gene regulation, thereby contributing to SMCX-associated XLMR. DNA cytosine methylation is crucial for retrotransposon silencing and mammalian development.

In a computational search for enzymes that could modify 5-methylcytosine (5mC), we identified TET proteins as mammalian homologs of the trypanosome proteins JBP1 and JBP2, that have been proposed to oxidize the 5-methyl group of thymine. In the second part of this thesis, I show that TET1 is a 2OG- and Fe(II)-dependent enzyme that catalyzes conversion of 5mC to 5-hydroxymethylcytosine (hmC) in cultured cells and in vitro . Expression of TET1 in HEK293 cells led to a decrease in 5mC as judged by immunocytochemistry, with concomitant appearance of a novel nucleotide identified by mass spectrometry as hmC. The catalytic domain of TET1 converted 5mC to hmC in vitro , whereas a variant with substitutions in residues predicted to bind Fe(II) did not generate hmC. hmC is present in the genome of mouse ES cells, and hmC levels decrease upon RNAi-mediated depletion of TET1. Thus TET proteins have potential roles in epigenetic regulation through modification of 5mC to hmC.