Introduction: Cardiac fibrosis is mediated by the activation of fibroblasts to myofibroblasts, a cell state transition which involves the coordination of expression of hundreds of genes. Previous studies have demonstrated that inhibition of bromodomain and extra-terminal domain (BET) proteins attenuates fibrosis. We recently discovered a novel role of bromodomaincontaining protein 4 (BRD4), a BET family member, in maintaining genome folding by stabilizing the cohesin complex (necessary for genome-genome interactions).

Hypothesis: We hypothesize that BRD4-mediated genome folding is critical for maintaining the enhancer-promoter interactions at genes required for fibroblast activation (Fig 1.)

Aims/Goals: The aims of the project are: 1. Determine if knockdown of BRD4 disturbs the enhancer-promoter loop formation at the Meox1 locus during fibroblast activation. 2. Determine if BRD4 and/or NIPBL mediates expression of Meox1 during fibroblast activation. 3. Determine if genetic deletion of BRD4 and/or NIPBL modulates fibrosis in vivo during fibroblast activation.

Project Design/Methods: A cardiac fibroblast cell line was generated in which a degron epitope tag was appended biallelically to Brd4 to enable acute BRD4 degradation. Fibroblasts were activated via addition of TGFβ. Meox1, a critical transcription factor mediating a broad fibrotic gene program, was used as a model locus to investigate chromatin looping. The proximity between the Meox1 enhancer and promoter, Meox1 expression, and protein occupancy at the locus were determined using DNA fluorescence in situ hybridization, RT-qPCR, and chromatin immunoprecipitation (ChIP)-qPCR, respectively.

Results/Product: The proximity between the Meox1 enhancer and promoter and Meox1 expression increased upon TGFβ-induced activation. BRD4 occupancy was enriched at the enhancer of Meox1 upon activation, and BRD4 depletion reduced Meox1 expression and the co-localization of the Meox1 enhancer and promoter. BRD4 physically interacts with the cohesin agonist NIPBL, and we found that co-depletion of BRD4 and a cohesin antagonist normalizes the Meox1 enhancer and promoter proximity and Meox1 expression levels. Ongoing studies include genome-wide occupancy studies and expanding our findings to in vivo models of cardiac fibrosis.

Discussion/Conclusion: Our studies provide a mechanistic understanding of the functional relevance of genome organization during fibroblast activation. These studies will also expand our knowledge on how genome folding regulates cell plasticity and inform therapeutic approaches for targeting pathologic fibrotic remodeling.