Double bromodomain proteins as regulators of lymphopoiesis and inflammation
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Histone post-translational modifications are essential for the regulation of gene expression. These modifications are recognized by chromatin "readers" that control gene co-activation and co-repression through intrinsic or recruited enzymatic activity. Deregulated function of chromatin-binding proteins is associated with immune diseases and cancer. Bromodomains are highly conserved protein structures that bind acetylated lysines of nucleosomal histones. Among the diverse group of all bromodomain-containing proteins, the members of BET (bromodomain extra terminal) family- Brd2, Brd3, Brd4 and Brdt - are mutually homologous and feature tandem bromodomains that bind acetylated histones H3 and H4. BET proteins recruit diverse transcriptional machinery to gene promoters, including chromatin "writers", "erasers" and transcription elongation complexes. Lack of model systems hampers molecular understanding of BET protein function; because eukaryotic BET proteins and their orthologs are essential, knockouts are lethal. However, we previously reported a Brd2 whole-body knockdown. These 'brd2 lo' mice show reduced inflammation, while Brd2 transgenic mice develop an aggressive B-cell lymphoma. Based on these data, we hypothesized that BET proteins regulate proliferation of immune cells and inflammatory responses. We transduced hematopoietic stem cells with Brd2-expressing lentivirus, engrafted the cells in irradiated mice and found that Brd2 expression expands donor-derived B cells and promotes B-cell mitogenic responsiveness in the periphery through cyclin A upregulation, whereas Brd2 knockdown in hematopoietic cells ablates hematopoiesis. In macrophages, Brd2 knockdown diminishes inflammatory responses; the same effect was observed with Brd3 and Brd4. We found that BET proteins physically associate with promoter chromatin of cytokine genes. This association is disrupted by JQ1, a novel small molecule inhibitor of BET protein function, which competes with acetylated lysine for binding the bromodomain pocket of BET proteins. As expected, JQ1 displaces BET proteins from cytokine genes, thereby ablating cytokine production in vitro. Likewise, in an in vivo model of endotoxemia, JQ1 dramatically reduces pro-inflammatory cytokines in serum and protects mice from death. Considered together, these findings reveal how Brd2 expression skews hematopoiesis and couples inflammatory signal transduction to chromatin status. Targeting BET proteins with small molecule therapeutics may benefit patients with hematologic malignancies and inflammatory diseases.
Thesis (Ph.D.)--Boston University