In vivo function of BRD4

Division Of Basic Sciences - Nci

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Abstract

BRD4 is being widely investigated as a therapeutic target in inflammatory diseases, as well as a variety of solid and hematological cancers (). It is a chromatin-binding protein with both kinase and acetyl transferase activities that links chromatin structure and transcription and plays an active role in regulating early embryonic development. BRD4 contributes to gene expression in multiple ways, as a scaffold that delivers transcription factors and nucleates superenhancers, as a chromatin remodeler, as a transcription factor that phosphorylates the Pol II CTD. Because deletion of BRD4 is embryonic lethal, the role of BRD4 in normal differentiation has not been extensively investigated in vivo. Using conditional BRD4 knock-out mice, we have shown that consistent with its role as a chromatin remodeler, BRD4-/- thymocytes have significantly reduced levels of acetylated histones necessary for chromatin decompaction and transcription, suggesting the BRD4 plays an active role in thymocyte development. An examination of the in vivo requirement for BRD4 during thymocyte development showed that the ISP stage, which represents a discrete subpopulation with a gene expression profile distinct from either the DN precursors or the DP successors, is selectively inhibited by BRD4 deletion, although BRD4 is expressed throughout all stages of thymocyte differentiation. The maturation of thymocytes is accompanied by large changes in gene expression profiles which are reflected in the patterns of cell surface markers and cell cycle genes. Whereas the DN thymocytes express neither CD4 nor CD8 and are highly proliferative, the ISPs express CD8 and undergo only a single round of proliferation. Accordingly, the transition from DN to ISP is accompanied by large changes in both immune and cell cycle pathways. Interestingly, despite the differences in proliferation, both cell populations express c-Myc at approximately equal levels. Thus, Myc expression alone does not determine the extent or frequency of proliferation. Although the maturation from ISP to DP is accomplished during a single round of cell cycle, it is accompanied by the differential expression of a large number of genes in immune and metabolic pathways. Unlike either the DN or ISP populations, the DP thymocytes do not express Myc, consistent with their quiescent phenotype. Of particular note, the ISP expression profile is distinct from both the DN and the DP. Thus, it is not a transitional population, midway between the DN and DP. Rather, the ISPs are a distinct thymocyte subpopulation. The ISP subpopulation is also distinct in its dependence on BRD4. Although BRD4 is expressed throughout thymocyte differentiation - and at equivalent levels - BRD4 depletion selectively affects the ISP subpopulation. In the absence of BRD4, DN thymocytes differentiate relatively normally, although BRD4-/- DN4 thymocytes are smaller than WT and accumulate in somewhat larger numbers. However, BRD4-/- DN4 thymocytes proliferate and undergo TcRbeta rearrangement normally, reflecting the finding that the expression of only 100 genes is affected. Among the pathways affected are those involved in inflammation and jak-stat signaling. Similarly, BRD4 plays a relatively small role in DP or SP thymocytes which are phenotypically normally and regulates expression of only between 300-400 genes in each cell type which fall into immune system and metabolic processes. In sharp contrast to the modest effects of BRD4 deletion in the DN, DP and SP thymocytes, the ISP are dramatically affected. The expression of over 1100 genes - most in cell cycle and metabolic pathways - is affected. This results in BRD4-/- ISP that are smaller than the WT, unable to undergo cell cycle and deficient in metabolic activity. Importantly, a large fraction of the genes uniquely expressed in ISPs are regulated by BRD4. This leads to the unexpected conclusion that BRD4 is a determining factor in ISP differentiation. BRD4 deletion profoundly affects ISP maturation and progression to the DP stage. Since that progression requires a round of cell cycle, this finding is consistent with the observed loss of the cell cycle regulator c-Myc in the absence of BRD4. Based on our findings, we have proposed a model in which the transition from the highly proliferative DN stage to the quiescent DP stage requires a reprogramming through the ISP stage that is regulated by BRD4. The complete conditional deletion of BRD4 did not allow us to distinguish the roles of its two enzymatic activities in thymocyte differentiation. BRD4 has intrinsic kinase and HAT activities, which have been shown to contribute to the regulation of transcription and chromatin organization, respectively. Replacement of wild type BRD4 with mutants of either kinase or HAT activities results in reduced cell cycle and proliferation of cell lines. Indeed, RNA sequence analysis shows dramatic changes in gene expression with either the BRD4 kinase or HAT mutant. Therefore, we have generated transgenic mice that express either BRD4 kinase-deficient or HAT-deficient mutant forms in a vector with a Dox-inducible promoter. These mice have been bred to the BRD4 conditional deletion to allow selective deletion of the endogenous wild type BRD4 and allow us to determine the respective roles of each of these activities in differentiation in vivo and in different tissues. Unfortunately, although induction in cell lines by Dox was vigorous, all attempts to induce expression in transgenic mice failed. Therefore, we have now generated mice in which the endogenous BRD4 gene has been deleted of either the HAT domain or the B domain using CRISPR technology. These mice are currently being analyzed.