Point mutations and structural variants that directly disrupt the coding
sequence of MEF2C have been associated with a spectrum of
neurodevelopmental disorders (NDDs). However, the impact of MEF2C
haploinsufficiency on neurodevelopmental pathways and synaptic processes is
not well understood, nor are the complex mechanisms that govern its
regulation. To explore the functional changes associated with structural
variants that alter MEF2C expression and/or regulation, we generated an
allelic series of 204 isogenic human induced pluripotent stem cell
(hiPSC)-derived neural stem cells and glutamatergic induced neurons. These
neuronal models harbored CRISPR-engineered mutations that involved direct
deletion of MEF2C or deletion of the boundary points for topologically
associating domains (TADs) and chromatin loops encompassing MEF2C.
Systematic profiling of mutation-specific alterations, contrasted to
unedited controls that were exposed to the same guide RNAs for each edit,
revealed that deletion of MEF2C caused differential expression of genes
associated with neurodevelopmental pathways and synaptic function. We also
discovered significant reduction in synaptic activity measured by
multielectrode arrays (MEAs) in neuronal cells. By contrast, we observed
robust buffering against MEF2C regulatory disruption following deletion of
a distal 5q14.3 TAD and loop boundary, whereas homozygous loss of a
proximal loop boundary resulted in down-regulation of MEF2C expression and
reduced electrophysiological activity on MEA that was comparable to direct
gene disruption. Collectively, these studies highlight the considerable
functional impact of MEF2C deletion in neuronal cells and systematically
characterize the complex interactions that challenge a priori predictions
of regulatory consequences from structural variants that disrupt
three-dimensional genome organization.