Bromodomain-containing protein 4 (BRD4) is a member of the bromo- and extraterminal (BET) domain-containing family of epigenetic readers which is under intensive investigation as a target for anti-tumor therapy. BRD4 plays a central role in promoting the expression of select subsets of genes including many driven by oncogenic transcription factors and signaling pathways. However, the role of BRD4 and the effects of BET inhibitors in non-transformed cells remain mostly unclear. We demonstrate that BRD4 is required for the maintenance of a basal epithelial phenotype by regulating the expression of epithelial-specific genes including TP63 and Grainy Head-like transcription factor-3 (GRHL3) in non-transformed basal-like mammary epithelial cells. Moreover, BRD4 occupancy correlates with enhancer activity and enhancer RNA (eRNA) transcription. Motif analyses of cell context-specific BRD4-enriched regions predicted the involvement of FOXO transcription factors. Consistently, activation of FOXO1 function via inhibition of EGFR-AKT signaling promoted the expression of TP63 and GRHL3. Moreover, activation of Src kinase signaling and FOXO1 inhibition decreased the expression of FOXO/BRD4 target genes. Together, our findings support a function for BRD4 in promoting basal mammary cell epithelial differentiation, at least in part, by regulating FOXO factor function on enhancers to activate TP63 and GRHL3 expression.

Bromodomain-containing protein 4 (BRD4) is an important epigenetic reader which promotes gene transcription to modulate cell-specific functions and is under intensive investigation for its potential as an anti-tumor therapeutic target. However, the role of BRD4 in non-transformed cells remains unclear. Using RNA sequencing and chromatin immunoprecipitation-sequencing analyses in normal basal-like breast epithelial cells, we demonstrate that BRD4 is required for the expression of epithelial-specific genes and suppression of stem cell-like properties by binding to the distal regions of epithelial-related genes. Moreover, BRD4 occupancy correlates with enhancer activity and enhancer RNA (eRNA) transcription of these genes. Interestingly, we show that BRD4 perturbation regulates the expression of p63 and Grainy Head-like transcription factor-3, GRHL3, whose depletion partially mimics BRD4 inhibition and impairs a differentiated phenotype. By binding to the distal regions of p63 and GRHL3, BRD4 promotes RNA polymerase-II occupancy and thus affects eRNA transcription. Motif analyses on cell-specific BRD4 binding sites predict the involvement of FOXO transcription factors. Consistently, inhibition of EGFR-AKT signalling activates FOXO1 function and promotes the expression of p63 and GRHL3. Activation of Src kinase signaling decreases RNA polymerase-II occupancy on BRD4 target genes and enhancers. Altogether, these findings provide evidence that BRD4 promotes a differentiated epithelial phenotype in non-transformed mammary cells at least in part through the activation of p63 and GRHL3 expression by associating with FOXO factors. Citation Format: Sankari Nagarajan, Upasana Bedi, Anusha Budida, Wanhua Xie, Feda Hisham Moh’d Hamdan, Vivek Kumar Mishra, Zeynab Najafova, Malik Alawi, Daniela Indenbirken, Stephan Knapp, Cheng-Ming Chiang, Adam Grundhoff, Christina H. Scheel, Vijayalakshmi Kari, Florian Wegwitz, Steven A. Johnsen. BRD4-FOXO axis maintains differentiated mammary epithelial phenotype by regulating p63 and GRHL3 expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1982.