Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells [ChIP-seq]

Recent sequencing-based experiments mapping ensemble interaction frequency among regulatory elements in cancer cells support the existence of complex topological assemblies of enhancers and promoters known as promoter-enhancer hubs or cliques. Yet, the prevalence of promoter-enhancer hubs in individual cells, factors regulating their dynamics and assembly, as well as their role in transcriptional dysregulation in cancer remain unclear. Here, we systematically integrated functional genomics, transcription factor screening, and optical mapping of promoter-enhancer interactions to identify key promoter-enhancer hubs, examine heterogeneity of their assembly, determine their regulators, and elucidate their role in gene expression control in individual triple negative breast cancer (TNBC) cells. Optical mapping of individual SOX9 and MYC alleles revealed the existence of frequent multiway interactions among gene promoters and enhancers within promoter-enhancer hubs. Our single-allele studies further demonstrated that lineage-determining SOX9 and signaling-dependent NOTCH1 transcription factors compact MYC and SOX9 promoter-enhancer hubs, respectively. Together, our findings suggest that promoter-enhancer hubs are dynamic and heterogeneous topological assemblies controlled by oncogenic transcription factors potentially in a cancer subtype-restricted manner to facilitate aberrant gene expression. Chromatin immynoprecipitation DNA-sequencing (ChIP-seq) to evaluate FLAG (Flag-dCas9-KRAB) colocalization and H3K27ac and H3K9me3 levels after inactivation of TNBC-restricted SOX9 enhancers; MB157 stably expressing Flag-tagged dCas9-KRAB were transfected with control sgRNA or sgRNA targeting SOX9.EC1, SOX9.EC2, and SOX9.EC3 for five days