Histone-Binding Protein RBBP4 Is Necessary to Promote Neurogenesis in the Developing Mouse Neocortical Progenitors

Chromatin regulation plays a crucial role in neocortical neurogenesis, and mutations in chromatin modifiers are linked to neurodevelopmental disorders. RBBP4 is a core subunit of several chromatin-modifying complexes; however, its functional role and genome-wide occupancy profile in the neocortical primordium are unknown. To address this, we performed RBBP4 knockdown using CRISPR/Cas9 on neocortical progenitors derived from mice of both sexes at embryonic age 12.5 during deep-layer neurogenesis. Our study demonstrates that downregulation of RBBP4 in the E12.5 neocortical progenitors reduced neuronal output, specifically affecting CTIP2-expressing neurons. We demonstrate that RBBP4 plays an essential role in regulating neocortical progenitor proliferation. However, overexpression of RBBP4 alone was not sufficient to regulate neuronal fate.Genome-wide occupancy analysis revealed that RBBP4 primarily binds to distal regulatory elements, and neuron differentiation is a significant GO biological pathway of RBBP4-bound genes. Interestingly, we found that RBBP4 binds to <i>Cdon</i>, a receptor protein in the Shh signaling pathway, and knockdown of <i>Cdon</i> phenocopies RBBP4 knockdown resulting in a significant reduction in neurogenesis, particularly CTIP2-expressing neurons. CDON overexpression could rescue the phenotype caused upon loss of RBBP4 in the neocortex, thereby suggesting the functional link between RBBP4 and its target gene CDON. Our results shed light on the cellular role of RBBP4 and identify CDON as a novel regulator of deep-layer neurogenesis in the neocortical progenitors. Our findings are significant in the context of understanding how dysregulated chromatin regulation impacts cellular mechanisms in neurodevelopmental disorders.<b>Significance Statement</b> Chromatin modifier RBBP4 regulates chromatin structure and, thereby, gene expression. It is expressed in the dorsal telencephalon progenitors during deep-layer neurogenesis. In this study, we unveil a novel role for RBBP4 in regulating deep-layer neurogenesis in the neocortical progenitors. Our research underscores RBBP4's critical role in governing progenitor proliferation and neuronal subtype specification in the neocortex while identifying its genome-wide binding occupancy profile. Moreover, we identify Cdon as a novel binding target of RBBP4, also involved in regulating deep-layer neurogenesis. These findings illuminate the mechanisms by which chromatin modifiers influence neocortical development, offering insights into how mutations in chromatin modifiers could impact cortical development and contribute to neurodevelopmental disorders.