CHD7 binds to insulators during neuronal differentiation [CUT&Tag]

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 SGN differentiation using immortalized multipotent otic progenitor (iMOP) cells. We demonstrate that CHD7 knockdown significantly impairs neuronal differentiation. Genome-wide analysis reveals CHD7 enrichment at diverse cis-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 Mir9-2. Targeting these regulatory regions in iMOPs with CRISPR interference (CRISPRi) and activation (CRISPRa) increased miR9 transcription, irrespective of the method. The occlusion of the regulatory elements suggested that the insulators function to regulate gene expression. The study highlights CHD7 activity at insulators and underscores its importance in promoting neuronal differentiation. CUT&Tag for CHD7, CTCF, H3K4me3, EP300, and secondary antibody only (no primary antibody) control from proliferating iMOPs (immortablized mutipotent otic progenitors) and iMOP-derived neurons.