Date of Award
Doctor of Philosophy (PhD)
Compensation among paralogous transcription factors (TFs) confers genetic robustness of cellular processes. Despite the prevalence of this phenotypic phenomenon, an in vivo genome-scale understanding of how TFs dynamically respond within the chromatin context to paralog depletion is still lacking. We explore this question in the mammalian brain by studying the highly conserved MEF2 family of TFs, which confer phenotypic robustness for neuronal processes across multiple brain regions. The paralogous TFs MEF2A and MEF2D are strongly co-expressed in granule neurons of the cerebellum, the most abundant neurons in the brain. Employing single and double conditional knockout of MEF2A and MEF2D in granule neurons of the mouse cerebellum, we find MEF2A and MEF2D play functionally redundant roles in cerebellar-dependent motor learning. To explore the molecular basis for MEF2 paralogous phenotypic redundancy, we systematically characterize in vivo genome-wide occupancy of MEF2A and MEF2D in the presence or absence of one another. Although highly co-expressed in granule neurons, MEF2D is the predominant genomic regulator of gene expression. Strikingly, upon depletion of MEF2D, the occupancy of MEF2A robustly increases at a subset of sites normally bound to MEF2D. Epigenome and transcriptome analyses reveal that sites experiencing compensatory MEF2A occupancy undergo functional compensation for genomic activation and gene expression. In contrast, a distinct population of sites without compensatory MEF2A activity undergo significant dysregulation upon loss of MEF2D. The two populations of MEF2 target sites are further stratified by relative chromatin accessibility, with compensatory MEF2A activity concentrated within more open chromatin. Finally, we reveal that motor activity-induced changes in neuronal state induce a dynamic switch from non-compensatory to compensatory MEF2-dependent gene regulation, demonstrating the context-dependent nature of paralogous TF interdependency. Collectively, these studies provide in vivo genome-wide characterization of functional interdependency between paralogous TFs. We have uncovered highly divergent compensatory activities at MEF2-target sites that stratify with chromatin accessibility. In addition, we propose that compensatory activity dynamically responds to changes in cellular context, thus providing a molecular basis for the functionally redundant regulation of cerebellar-dependent learning by MEF2 TFs. The permissibility of TF binding depends on the chromatin environment. In the nucleus, arrays of nucleosomes tightly package DNA into condensed chromatin, which can occlude many DNA regulatory elements from nuclear factors, such as TFs. To overcome this issue and allow dynamic access to condensed DNA, a process known as chromatin remodeling facilitates access of nucleosomal DNA by remodeling the structure, composition and positioning of nucleosomes. The latter half of this dissertation focuses on an enzymatic mediator of this process, the ATP-dependent chromatin remodeler CHD7, in the context of mammalian brain development. Recent studies employing genetic manipulation and biochemical characterization of the chromodomain helicase DNA-binding (CHD) family of ATP-dependent chromatin remodelers have revealed their significant roles in neural development. Mutations in CHD7 are found in nearly 90% of patients with CHARGE syndrome, diagnosed in 1: 10,000 newborns. CHARGE syndrome is a clinically heterogeneous disorder named after several prevalent defects, including coloboma of the eye, heart defects, atresia of the choanae, retardation of growth or development, genital or urinary defects, and ear anomalies or deafness. In addition, neurodevelopmental defects and neurological signs suggest involvement of the central nervous system. Recent MRI studies have identified cerebellar hypoplasia and foliation defects among the more prominent abnormalities of the central nervous system in CHARGE patients. To elucidate the role of CHD7 in the pathogenesis of cerebellar defects in CHARGE syndrome, we perform conditional knockout of CHD7 (CHD7 cKO) in cerebellar granule cell precursors, which are enriched for CHD7 expression. We define the role of CHD7 in the regulation of epigenomic determinants of enhancer activation in granule cell precursors and consequent morphogenesis of the cerebellar cortex. Using ATAC-seq and ChIP-seq analyses of the anterior cerebellum in conditional CHD7 knockout mice, we show that CHD7 robustly promotes the accessibility and stimulates active histone modifications of gene enhancers in granule cell precursors. Remarkably, genome architecture analyses of the anterior cerebellum reveal that CHD7-dependent enhancer activation correlates with local alterations of chromatin. Genome and gene ontology studies demonstrate that CHD7-regulated enhancers and genes in granule cell precursors prominently include cerebellar morphogenesis genes. Accordingly, we discover that conditional CHD7 knockout triggers a striking cerebellar polymicrogyria phenotype, which we have also found in a case of CHARGE syndrome. These findings elucidate CHD7 functions in gene enhancer regulation and tissue morphogenesis in the cerebellum, with potential implications for our understanding of CHARGE syndrome. Fortuitously, this provides the first model in which a genomic perturbation leads to fully penetrant and spatially consistent folding patterns in the brain. Thus, the CHD7 cKO model serves as a uniquely tractable model to study the cellular and molecular underpinnings of neural folding, a process that has largely remained a mystery to scientists. Collectively, our studies on MEF2 and CHD7 provide novel insights into the role of two critical classes of gene regulators in cerebellar development and function: TFs and chromatin remodelers, respectively. We anticipate that future studies will clarify how the coordinated deployment of these two types of regulators governs precise patterns of gene expression in the brain.
Chair and Committee
Aaron DiAntonio, Robi Mitra, Joseph Corbo, Harrison Gabel,
Majidi, Shahriyar, "Transcriptional and Epigenetic Regulation of Cerebellar Development and Function" (2021). Arts & Sciences Electronic Theses and Dissertations. 2440.