The cis-regulatory grammar and epigenetic control of human interneuron progenitor specification

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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 foundation for studying how its disruption contributes to NDD etiology. We first integrate several types of genome-wide data to define putative CREs controlling cIN specification. These data will be used to assess 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 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.