The BET Protein BRD2 Cooperates with CTCF to Enforce a Transcriptional Boundary in Erythroid Cells

Pharmacologic inhibitors of the bromodomain and extraterminal motif (BET) family of proteins have shown promise in the treatment of hematologic and other malignancies and are being developed for clinical use. However, BET inhibitors do not discriminate between the family members BRD2, BRD3, and BRD4, and thus the mechanistic basis for their therapeutic efficacy is not well understood. In addition, BRD2 and BRD4 are individually required for the activation of genes driven by the erythroid transcription factor GATA1 (Stonestrom et al., Blood 2015), yet how BRD2 in particular contributes to this process has not been studied. We examined BRD2 occupancy genome-wide in erythroid cells and find that BRD2 colocalizes extensively with the architectural/insulator protein CCCTC-binding factor (CTCF). We define a functional hierarchy whereby CTCF is required for BRD2 to occupy co-bound sites, while CTCF binding is BRD2-independent. Using CRISPR/Cas9-based genome editing we identify a boundary element occupied by CTCF and BRD2 that is adjacent to a GATA1-driven enhancer and ensures appropriate transcriptional regulation at the locus. Employing single-molecule RNA FISH we show that either site-specific CTCF disruption or BRD2 depletion leads to increased correlation in mature mRNA levels between the genes flanking this boundary, suggesting that they become inappropriately coregulated. Taken together these findings indicate that BRD2 collaborates with CTCF to constrain the activity of an erythroid enhancer and reveal a potential new role for BET proteins in chromatin domain boundary function. Disclosures No relevant conflicts of interest to declare.