Mechanistic dissection of direct MEF2C disruption and long-range regulatory alterations within the chromosome 5q14.3 locus as strong effect drivers of neurodevelopmental disorders

Abstract

Direct and indirect point mutations and structural variants that cause haploinsufficiency of MEF2C have been implicated as causes of neurodevelopmental disorders (NDDs). However, the impact of these mutations on neurodevelopmental pathways and synaptic processes is not well understood, nor are the complex mechanisms that govern indirect regulation of MEF2C. We generated an allelic series of 204 isogenic iPSC-derived neural cell lines harboring CRISPR-engineered mutations that directly delete predominant isoforms of MEF2C, as well as the boundaries of topologically-associating domains (TADs) and chromatin loops encompassing MEF2C. We then performed a systematic dissection of mutation-specific alterations to transcriptional signatures, regulatory interactions, chromatin contacts, and electrophysiological effects. Our analyses reveal that direct MEF2C disruption causes differential expression of genes enriched for neurodevelopmental and synaptic-associated terms, accompanied by a reduction in synaptic firing and synchrony in neuronal cells. By contrast, we observe robust buffering against indirect MEF2C disruption upon 5q14.3 TAD and loop boundary disruption; however, homozygous loss of a proximal loop boundary in an intron of MEF2C results in significant alterations to expression and function of the gene, comparable to its direct disruption. Furthermore, we analyze long-range chromatin contacts and MEF2C expression from patients with hypothesized MEF2C haploinsufficiency caused by positional effect. Considering six NDD cases without direct MEF2C disruption, we observe variable long-range contacts and MEF2C expression per case despite disruption of the MEF2C-containing TAD. Collectively, our findings demonstrate the functional impact of MEF2C haploinsufficiency in human-derived neural models and patient materials, and highlight the complex interactions of gene regulation and chromatin topology that challenge a priori regulatory predictions of structural variant disruption to three-dimensional genome organization.