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The BET Protein BRD2 Cooperates with CTCF to Enforce Transcriptional and Architectural Boundaries
Sarah C Hsu1, Thomas G Gilgenast2, Caroline R Bartman1
1Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Abstract:
Bromodomain and extraterminal motif (BET) proteins are pharmacologic targets for the treatment of diverse diseases, yet the roles of individual BET family members remain unclear. We find that BRD2, but not BRD4, co-localizes with the architectural/insulator protein CCCTC-binding factor (CTCF) genome-wide. CTCF recruits BRD2 to co-bound sites whereas BRD2 is dispensable for CTCF occupancy. Disruption of a CTCF/BRD2-occupied element positioned between two unrelated genes enables regulatory influence to spread from one gene to another, suggesting that CTCF and BRD2 form a transcriptional boundary. Accordingly, single-molecule mRNA fluorescence in situ hybridization (FISH) reveals that, upon site-specific CTCF disruption or BRD2 depletion, expression of the two genes becomes increasingly correlated. HiC shows that BRD2 depletion weakens boundaries co-occupied by CTCF and BRD2, but not those that lack BRD2. These findings indicate that BRD2 supports boundary activity, and they raise the possibility that pharmacologic BET inhibitors can influence gene expression in part by perturbing domain boundary function.
Insights
Bromodomain and extraterminal motif (BET) protein BRD2, not BRD4, forms transcriptional boundaries with CTCF. Disrupting this boundary alters gene expression correlation, indicating BRD2’s role in gene regulation.
Area of Science:
- Genomics
- Molecular Biology
- Epigenetics
Background:
- Bromodomain and extraterminal motif (BET) proteins are key drug targets, but individual family member functions are not fully understood.
- Understanding the specific roles of BET proteins like BRD2 and BRD4 is crucial for developing targeted therapies.
- CCCTC-binding factor (CTCF) is a known architectural protein involved in genome organization.
Purpose of the Study:
- To elucidate the distinct roles of individual BET proteins, specifically BRD2 and BRD4, in gene regulation.
- To investigate the functional relationship between BRD2 and the architectural protein CTCF.
- To determine if BET proteins contribute to the formation and function of transcriptional boundaries.
Main Methods:
- Genome-wide co-localization analysis of BRD2 and BRD4 with CTCF.
- Functional assays involving disruption of CTCF/BRD2-occupied elements.
- Single-molecule mRNA fluorescence in situ hybridization (FISH) to assess gene expression correlation.
- Hi-C experiments to evaluate the impact of BRD2 depletion on chromatin architecture.
Main Results:
- BRD2, but not BRD4, was found to co-localize with CTCF genome-wide.
- CTCF recruits BRD2 to co-bound sites, and BRD2 is essential for maintaining transcriptional boundary function.
- Disruption of CTCF/BRD2 boundaries led to increased correlation in the expression of adjacent genes.
- BRD2 depletion weakened CTCF/BRD2 co-occupied boundaries, while boundaries lacking BRD2 remained unaffected.
Conclusions:
- BRD2 plays a significant role in establishing and maintaining transcriptional boundaries in conjunction with CTCF.
- The findings suggest that BET inhibitors may impact gene expression by disrupting these domain boundary functions.
- This study clarifies the specific role of BRD2 within the BET protein family and its contribution to genome organization.