Chromatin Regulation of Transcriptional Repression in the Developing Brain

Abstract

The complex organization of the brain is enabled by the precise coordination of developmental and activity-dependent gene programs. Mechanisms acting at the level of chromatin play a key role in promoting, repressing, or stabilizing levels of transcriptional output, and these mechanisms ensuring transcriptional fidelity have proved to play a key role in brain development. Disruptions to chromatin have emerged as a major cause of neurodevelopmental disorders, emphasizing a particular dependence of the nervous system on the precise coordination of gene expression. Here we investigate the mechanistic basis of transcriptional control by chromatin regulators that play essential roles in brain development. Modifications to DNA itself are a major form of chromatin regulation. Cytosine methylation is an epigenetic modification classically occurring at CG dinucleotides, but neuronal chromatin shows a dramatic expansion of methylation in the CA sequence context during postnatal development. To better understand the role of mCA in the brain, we characterized protein-protein interactions and post-translational modifications of the DNA methyltransferase Dnmt3a to identify modifications enriched at a key critical period of neuronal CA methylation. Performing a proteomic screen for readers of CG and CA-methylation, we confirmed that the Rett Syndrome-associated factor MeCP2 is the major reader of CA methylation in the brain. Finally, to better understand the role of MeCP2 in transcriptional regulation, we used base-resolution genomic methods to map RNA polymerase distribution and quantify rates of transcriptional initiation and elongation. We unexpectedly found that MeCP2 acts to repress the rate of transcriptional initiation, providing greater insight into a mechanism of transcriptional regulation directly implicated in the pathophysiology of a significant neurological disorder. Beyond DNA, histone tails provide an extensive platform for the recruitment of regulatory factors. Reader proteins bind histone modifications at specific sites to direct regulatory inputs that are sensitive to genomic context. One such chromatin reader, the zinc finger protein Zmynd11, has been identified as a selective reader of trimethylation at lysine 36 on the histone variant H3.3 (H3.3K36me3), and in recent years mutations within the gene encoding Zmynd11 have been identified as the cause of a syndromic intellectual disability. We developed the first mouse model to investigate Zmynd11 function in vivo and identified an extensive gene program misregulated upon its loss. Using quantitative proteomics we found that Zmynd11 interacts with a broad network of chromatin-modifying enzymatic machinery, and that a disease-associated point mutation disrupts only a subset of these interactions, including the histone lysine methyltransferase Kmt2a/MLL1. We confirmed in the brain the physical interaction and extensive genomic co-binding of Zmynd11 and Kmt2a, and showed that deletion of Zmynd11 leads to a loss of Kmt2a genomic occupancy and concomitant increases in H3K4 trimethylation and gene transcription. We propose a model in which Zmynd11 inhibits the ability of Kmt2a to activate target genes, implicating MLL1 inhibition as a mechanism of particular relevance to neuronal development and the pathophysiology of Zmynd11-linked developmental disorders.