BRD4 represses developmental and neuronal genes through interactions with polycomb complexes

BRD4 is a member of the bromodomain and extraterminal domain containing protein family, primarily known for regulating transcriptional elongation and enhancer activity. Heterozygous loss-of-function mutations in BRD4 cause craniofacial and neurodevelopmental impairments. However, it remains unclear why mutations in this general transcriptional activator lead to specific neurodevelopmental defects. Using an in vitro human embryonic brain development model, we demonstrate that BRD4 functions as a repressor of Polycomb-regulated developmental and neuronal genes. Acute degradation or loss-of-function mutations in BRD4 in human embryonic stem cells and neuronal lineage deregulate immediate-early genes important for learning and memory formation. We show that BRD4 interacts with components of the noncanonical Polycomb repressive complex PRC1.6 and co-occupies PRC1.6-bound and bivalently marked promoters (H3K27me3 and H3K4me3). We further demonstrate that H3K14ac and H3K23ac recruit BRD4 to bivalent chromatin via its second bromodomain (BD2). These interaction and co-occupancy data suggest BRD4 could contribute to recruitment or maintenance of PRC1.6 and EED at specific sets of genes. As a result, BRD4 represses key developmental and neuronal transcription factors, as well as genes required for learning and memory formation. Single-cell RNA-seq and single-cell CUT&Tag analyses in unguided neuronal organoids confirm that BRD4 loss-of-function mutations lead to increased expression of Polycomb-regulated developmental transcription factor families, including ZIC, HOX, PAX, SOX, and POU. Additionally, single-cell chromatin accessibility data reveal that BRD4 mutations increase accessibility at transcription factor motifs normally repressed by BRD4. Neuronal organoids with BRD4 mutations lead to altered neuronal cell fate, particularly increased differentiation towards diencephalic and retinal pigment epithelium, including the appearance of eye-like pigmentation. Together, these findings uncover a critical role for BRD4 in preventing the premature activation of developmental transcription factors, providing new mechanistic insights into the pathogenesis of congenital neurodevelopmental disorders. HighlightsO_LIAcute degradation of BRD4 leads to upregulation of bivalently marked developmental and neuronal genes. C_LIO_LIBRD4 interacts with components of the PRC1.6 complex and EED. C_LIO_LIBRD4 represses PRC1.6-repressed and bivalent genes C_LIO_LIH3K14ac and H3K23ac roles in recruiting BRD4 to bivalent chromatin C_LIO_LIBRD4-BD2 mutations lead to premature upregulation of neuronal genes, altering neuronal cell fate C_LI