The Dual Bromodomain Protein Brd2 Controls Primary B Cell Mitogenesis and Cell Cycle in Mice Reconstituted with Lentivirus-Transduced HSCs

Transcriptional control of cellular proliferation and differentiation is critically important in hematopoiesis; specifically, the role of chromatin-dependent regulatory processes in this context is poorly understood. The human BRD2 proto-oncogene encodes a double bromodomain protein that binds to acetylated histone H4 in chromatin and is located within the MHC class II locus, suggesting Brd2 plays a role in immunity. However, BRD2 shares no sequence similarity with other MHC genes, nor is Brd2 involved in antigen processing, but rather it plays a role in mitogenic signal transduction. We have previously found that whole-body knockout of Brd2 is lethal to mice. However, when Brd2 was expressed constitutively in the B cells of transgenic mice, Brd2 binds E2F proteins, histone acetylases and Swi/Snf complexes, and co-activates cyclin A leading to B cell lymphoma and leukemia. Importantly, elevated levels of Brd2 have been reported in primary malignant B cells from human and mouse. We therefore hypothesize that Brd2 multiprotein complexes, working through chromatin modification, are crucial in the control of the cell cycle and in the mitogen responsiveness and proliferation of the B cell compartment. To study the effects of Brd2 in B cell development and proliferation, we performed bone marrow transplants of hematopoietic stem cells in a chimeric mouse model. Hematopoietic stem cells were sorted from CD45.1 donor mice with the characteristic ‘side population’ profile by flow cytometry and transduced with lentivirus containing vectors for Brd2 overexpression, shRNA knockdown, or control vectors. Recipient CD45.2 mice were lethally irradiated and a functional immune system was successfully reconstituted with donor cells and CD45.2 competitor BM cells. Mice were immunophenotyped and functional B cell mitogenic capacity was examined by BrdU incorporation into LPS-stimulated B cells. We found that in the spleen, Brd2 expression dramatically expands the CD45.1 (but not CD45.2) B cell compartment at the expense of T cells and renders B cells mitogenically hypersensitive. Compared with control, there was an increase in BrdU incorporation at 24 and 48 hours (29.8% v. 43.5% at T=24 h; 56.9% v. 66.7% at T=48 h). Preliminary results also suggest that B cell development was skewed in the bone marrow and periphery towards B1a phenotype. Moreover, downregulation of Brd2 via shRNA blocked cyclin A transcription and completely arrested B cell development and proliferation. Taken together, these data suggest that Brd2, through epigenetic regulation of the cell cycle, plays an important role in B-lymphopoiesis, proliferation, and stimulation.