Harrison W. Gabel, Ph.D. - US grants

Abstract Advances in human genetics have led to the identification of a large number of genes associated with autism spectrum disorder (ASD), intellectual disability (ID), and related neurodevelopmental diseases. A critical new challenge is to understand the molecular pathways these genes contribute to in neurons and to dissect how their disruption alters nervous system function. Methylation of cytosines in DNA classically occurs only at CG dinucleotides, but recent studies have also uncovered functions for unique non-CpG DNA methylation in neurons (which occur largely at CA dinucleotides, mCA). We have discovered that the methyl-DNA-binding protein MeCP2, which is critical for proper nervous system function, binds to mCA within the transcribed regions of genes in neurons to downregulate their expression. New studies have identified mutations in DNMT3A, the DNA methyltransferase required for the deposition of mCA in the brain, in individuals with ID and ASD. These findings support the hypothesis that disruption of gene regulation mediated by mCA contributes to disorders caused by mutation of DNMT3A. Our proposed studies will test this hypothesis by pursuing three specific aims: Aim 1 will use in vitro cell culture systems to determine how disease-associated mutations in DNMT3A affect enzyme function and alter deposition of mCA in neurons. Aim 2 will employ newly-generated transgenic mice carrying heterozygous mutations in DNMT3A to determine the effects of this disruption on neuronal DNA methylation, chromatin structure, transcription and cellular functions. Aim 3 will interrogate the mechanisms by which mCA and MeCP2 directly regulate transcription in neurons, testing the hypothesis that these components interact with novel gene-regulatory sites to control transcription. Together these studies will determine the molecular mechanisms by which mCA regulates gene expression in neurons, and define a site of molecular pathology in ASD and ID.

The long-term goals of the proposed research are to elucidate the epigenetic mechanisms that control neuronal connectivity in the brain. We have recently discovered essential roles for the chromatin remodeling enzyme Chd4 in granule neuron connectivity in the mouse cerebellum. Strikingly, genome-wide analyses of the cerebellum in conditional Chd4 knockout mice reveal that Chd4 triggers deposition of the histone variant H2A.z at promoters of neuronal activity-dependent genes in vivo, thereby triggering their shutoff. Purification of ribosome-associated mRNAs from synchronously developing granule neurons shows that conditional knockout of Chd4 impairs shutoff of activity-dependent genes when neurons undergo dendrite pruning in vivo. Accordingly, Chd4-dependent shutoff of activity genes drives granule neuron dendrite pruning in vivo. Our findings define an epigenetic mechanism that shuts off activity-dependent transcription and thereby regulates dendrite patterning in the brain. These findings also raise fundamental questions on the regulation and mechanisms of Chd4-control of gene expression and neuronal connectivity in the brain. The ATPase Chd4 represents the core subunit of the nucleosome remodeling and deacetylase (NuRD) complex. We will elucidate the role of the protein Mbd3, which is required for the assembly of the NuRD complex, in Chd4-induced H2A.z- dependent shutoff of activity genes and granule neuron dendrite patterning in the mouse brain in vivo. We will also determine whether Chd4, like the chromatin remodeling enzyme p400, directly incorporates H2A.z into nucleosomes, and assess the role of p400 in the Chd4/H2A.z epigenetic pathway. Finally, we will characterize the biological role of the Chd4/H2A.z epigenetic pathway in granule neuron responses in the context of cerebellar circuitry. The proposed research will advance our understanding of the epigenetic mechanisms that control neuronal connectivity in the brain. Because mutations of epigenetic regulators including Chd4 cause neurodevelopmental disorders of cognition including autism and intellectual disability, our studies will also shed light on pathogenic mechanism underlying these major disorders of the brain.