Mechanistic dissection of chromatin topology disruption in the 5q14.3 MEF2C locus as an indirect driver of neurodevelopmental disorders

Structural variants have the potential to create long-range positional effects, uncouple genes from regulatory elements, and facilitate aberrant 3D chromatin folding. In an independent study, we revealed a significant enrichment of intergenic balanced chromosomal abnormality (BCA) breakpoints from congenital anomaly cases at chromosome 5q14.3. All 11 5q14.3 BCA carriers had breakpoints disrupting the topologically associated domain (TAD) housing MEF2C, a known driver of neurodevelopmental disorders. In a second study, we showed, using 4C-seq, that MEF2C forms proximal and distal interaction loops within this TAD, contacting multiple neuronal enhancers. To understand the functional impact of these BCAs, we performed a mechanistic dissection of the 3D regulatory network at the 5q14.3 locus and its constituent functional elements using Cas9-based genome editing. We generated >200 cell lines, representing deletions of MEF2C alongside five MEF2C TAD boundary and interaction loop targets in iPS-derived neural stem cells (NSCs) and cortical induced neurons (iNs). We profiled changes in expression and 3D chromatin interactions within these models using RNA-seq and 4C-seq. MEF2C was variably differentially expressed upon deletion of proximal and distal MEF2C loop regions, with effects depending on cell type and variant class. Underlying chromatin interaction patterns revealed evidence of loop maintenance in these models, possibly via CTCF buffering, highlighting compensatory mechanisms against 3D chromatin disruption. In contrast, deletion of the proximal TAD boundary facilitated increased contacts with predicted enhancers in the adjacent TAD. Our results suggest novel regulatory mechanisms driving phenotypic outcomes for the 5q14.3 region, with significant implications for interpretation of pathogenic structural variation.