Histone hyperacetylation disrupts core gene regulatory architecture in rhabdomyosarcoma

Core regulatory transcription factors (CR TFs) orchestrate the placement of super-enhancers (SEs) to activate transcription of cell-identity specifying gene networks, and are critical in promoting cancer. Here, we define the core regulatory circuitry of rhabdomyosarcoma (RMS) and identify critical CR TF dependencies. These CR TFs build SEs that have the largest levels of histone acetylation, yet paradoxically SEs also harbor the highest amounts of histone deacetylases (HDACs). We find that hyperacetylation selectively halts CR TF transcription. To investigate the architectural determinants of this phenotype, we developed Absolute Quantification of Architecture (AQuA) HiChIP, revealing erosion of native SE contacts, and aberrant spreading of contacts involving histone acetylation. Hyperacetylation removes RNA Pol2 from core regulatory genetic elements, and eliminates RNA-Pol2 but not BRD4 phase condensates. This study identifies a SE-specific requirement for balancing histone modification states to maintain SE architecture and CR TF transcription.