CHD7 binds to insulators during neuronal differentiation.

Spiral ganglion neurons (SGNs) are crucial for hearing, and the loss of SGNs causes hearing loss. Stem cell-based therapies offer a promising approach for SGN regeneration and require understanding the mechanisms governing SGN differentiation. We investigated the chromatin remodeler CHD7 in neuronal differentiation using immortalized multipotent otic progenitor (iMOP) cells. We demonstrated that CHD7 knockdown impaired neuronal differentiation. Genome-wide analysis revealed CHD7 binding at diverse -regulatory elements, with notable enrichment at sites marked by the insulator-binding protein CTCF between topologically associating domains (TADs). Insulators marked by the enrichment of CHD7 and CTCF resided near genes critical for neuronal differentiation, including . Targeting these regulatory regions in iMOPs with CRISPR interference (CRISPRi) and activation (CRISPRa) increased miR-9 transcription, irrespective of the method. Blocking the CHD7 and CTCF marked sites suggested that the elements function as insulators to regulate gene expression. The study highlights CHD7 activity at insulators and underscores an unreported mechanism for promoting neuronal differentiation.