Brd4 is an atypical kinase that regulates transcription

Division Of Basic Sciences - Nci

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Abstract

Understanding how the bromodomain 4 (BRD4) exerts its pleiotropic functions requires a detailed understanding of its structure, both in terms of functional domains and 3D organization. The N-terminal half of BRD4 contains the highly ordered bromodomains and ET domain, whose X-ray structures have been resolved. We recently characterized the N-terminal B motif as responsible for BRD4 dimerization and the kinase domain within the BD2-B-BID domain. Additional N-terminal elements - A motif and SEED domains - are largely uncharacterized, both structurally and functionally. The C-terminal half of BRD4 is predicted to be intrinsically disordered; it is responsible for forming phase separated condensates, both in vitro and in vivo. Our studies on BRD4 structure led to the finding that the full-length molecule exists as a dimer, both in vitro and in vivo, which is nucleated by the central B motif. We have now demonstrated that BRD4 exists as an anti-parallel dimer that is anchored by the leucine-zipper segment of the B motif. To further characterize the 3D structure of the dimer, we have been attempting to apply cryoEM analyses, either alone or in the presence of its various substrates. Due to the highly disordered C-terminal domain of BRD4, we encountered challenges that initially precluded our being able to generate a set of data that converged on a single structure. We hypothesized that the flexibility of the BRD4 structure enables it to interact with multiple substrates which bind to different motifs within the molecule and that the C-terminal domain of BRD4 adopts discrete conformations as a function of binding its substrates or post-translational modifications. More recently, the addition of MYC appears to able to stabilize the molecule into a single conformation, which we are pursuing. To further pursue the structural characterization of BRD4, we have undertaken a screen of a natural products library, in collaboration with Barry O'Keefe (NCI), for molecular binders that either stabilize or destabilize BRD4 structure, as assessed by differential scanning fluorimetry (DSF). To date, we have identified approximately two dozen small molecules that stably interact with BRD4. In preliminary experiments, we find that BRD4 stabilizers enhance its kinase activity. Unfortunately, none of these molecules bound with sufficient affinity to be used in cryoEM studies. In parallel with our physical characterization of BRD4, we have studied the mechanisms the regulate BRD4's pleiotropic functions. As noted above, BRD4 exhibits dual functionality: chromatin-bound BRD4 regulates remodeling through its acetyltransferase (HAT) activity, while promoter-associated BRD4 regulates transcription through its kinase activity. Notably, chromatin-bound BRD4 lacks kinase activity, and RNA Pol II-bound BRD4 exhibits no HAT activity. This provides one mechanism underlying BRD4's functional switch: BRD4's enzymatic activity is regulated by its physical interactions. In response to diverse stimuli, JNK-mediated phosphorylation of human BRD4 at Thr1186 and Thr1212 triggers its transient release from chromatin, disrupting its HAT activity and potentiating its kinase activity. Released BRD4 directly interacts with and phosphorylates Pol II, PTEFb, and c-MYC, thereby promoting transcription of target genes involved in immune and inflammatory responses. JNK-mediated BRD4 functional switching induces CD8 expression in thymocytes and epithelial-to-mesenchymal transition (EMT) in prostate cancer cells. Unlike other transcriptional regulators, BRD4 largely remains bound to chromosomes during early mitosis. We have discovered that BRD4 also regulates mitosis through its direct interaction with and phosphorylation of Aurora B kinase, an essential regulator of mitosis. BRD4 binding to Aurora B inhibits its kinase activity, preventing autophosphorylation and phosphorylation of the key mitotic targets histone H3 and MCAK, the mitotic centromere associated kinesin. This inhibition is relieved during metaphase when JNK is activated and phosphorylates BRD4, triggering its transient release from chromatin. Importantly, Aurora B activity during mitosis inversely correlates with BRD4 binding and directly correlates with JNK activation and BRD4 release. Our findings thus reveal a regulatory mechanism whereby Aurora B activity is directly controlled by BRD4, which in turn is regulated by JNK. Aurora B is a serine/threonine kinase that plays crucial roles during mitosis and cytokinesis, ensuring accurate chromosome segregation. Although Aurora B is expressed throughout the cell cycle, its kinase activity is primarily active during mitosis. The mechanisms regulating its kinase activity have not been extensively characterized. Our current studies demonstrate that Aurora B kinase activity is regulated by the bromodomain protein, BRD4, which interacts with Aurora B to inhibit its activity. This inhibition of Aurora B kinase activity is relieved during metaphase following JNK-mediated phosphorylation of BRD4 which results in the transient release of BRD4 from chromatin and the disruption of its interaction with Aurora B. During anaphase, Aurora B binds to the mitotic spindle whereas BRD4 reassociates with chromatin and is no longer co-localized with Aurora B. The current results reveal that BRD4 plays an active role during mitosis by modulating mitotic progress through its direct regulation of the Aurora B kinase. BRD4 has been previously reported to regulate the levels of Aurora B through transcriptional and chromatin regulation. The current findings showing post-transcriptional regulation of Aurora B by BRD4 identify an additional level of control by BRD4, extending BRD4's known pleiotropic functions. Although JNK activity has been linked to the expression of Aurora B and the activation of its kinase through upstream mechanisms in vivo, the activation mechanism was unknown. Our results reveal a mechanism whereby JNK relieves the suppression of Aurora B kinase activity through its phosphorylation of BRD4, which releases it from chromatin and disrupts its interaction with Aurora B.