Control of gene expression involves interactions between genomic cis-regulatory elements (CREs) and the transcription factors that bind them, while chromatin modifiers also modulate genome access to control cell type specification during development. Defining these regulatory controls is important, as most human genetic variation linked to disease is in non-protein coding sequences, but the locations and functionality of CREs that specify many developing human cell types has not yet been defined. In the cerebral cortex, balanced development of inhibitory cortical interneurons (cINs) and excitatory neurons (cEXs) is required for proper function. cIN development is susceptible to perturbation to cause multiple neurodevelopmental disorders (NDDs), while NDD contributory mutations are found in many genes encoding chromatin modifiers, linking disrupted epigenetic regulation of cIN development to NDD etiology. However, most aspects of molecular regulation of human cIN development remain undefined, including which regulators are required, the CREs through which these regulators act and networks of gene expression under their control, effects of their disruption on cIN development, and contributions of human mutations in these genes and CREs to NDDs. To elucidate these, we use a directed differentiation model that mimics many aspects of human cIN specification and differentiation, is robust and experimentally manipulable, and has high utility for studying these processes. Here, we begin to define the central regulatory logic underlying the cIN developmental program and build a resource to study how its disruption contributes to NDD etiology. We first integrate several types of genome- wide data to identify putative CREs controlling cIN specification. These data will be used to define how pathogenic mutations in both CREs and in genes encoding chromatin modifiers disrupt cIN development to cause NDDs. We next explore roles for CHD2, a gene encoding a chromatin remodeler mutated to cause several NDDs: we define direct targets of CHD2 regulation, their dysregulation in the context of different pathogenic CHD2 gene variants, the epigenetic mechanisms involved, and effects of these CHD2 pathogenic mutations on development and function of cINs and cEXs. This work will elucidate both CHD2?s required roles and mechanisms in cIN development and the basis of their disruption in NDDs. Finally, we conduct massively parallel reporter analysis: high throughput, quantitative, CRE activity testing is used to identify bona fide functional CREs, define cis-sequence requirements for CRE regulation during cIN specification, and compare CRE activity in cIN versus cEX progenitors, and with single or combinatorial transcription factor binding site mutation. A subset of these CREs is then validated by epigenome editing. Together, this work will elucidate the cis-regulatory logic of a human cell type-specific gene regulatory program central to neurodevelopment and disease, while building a data resource and experimental foundation for further dissection of how pathogenic variants both in human coding and non-coding genome sequences disrupt this program, contributing to NDDs.!
This work will define mechanisms that control gene expression to drive the development of human cortical interneurons. Mutations in the human genome sequence frequently disrupt these gene regulatory controls, perturbing interneuron development and contributing to disorders of the developing nervous system, such as pediatric epilepsies and autism spectrum and intellectual disability disorders. Many aspects of regulation of this developmental program appear to be unique to humans, and human genome sequence variants that cause disease are also often not present in other species. Therefore, modeling how this human neurodevelopmental program is regulated is necessary to build a foundation and resources for determining how such human- specific mutations disrupt this regulation to contribute to neurodevelopmental disorders.!
