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 plays an active role in regulating early embryonic development. 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. Differentiation of thymocytes is dependent on their interactions with distinct epithelial compartments within the thymus. Thymocyte progenitors emigrate from the bone marrow to the thymus where they enter the thymic cortex as CD4-CD8- double negative (DN) thymocytes where they undergo maturation to become TCR-expressing CD4+CD8+ double positive (DP) thymocytes. Productive interaction with cortical epithelial cells (cTECs) results in positive selection and lineage choice to generate CD4+ or CD8+ single positive thymocytes that express chemokine receptors. Medullary epithelial cells (mTEC) express chemokine ligands that attract the SP thymocytes into the medulla. Within the medulla, mTECs express an array of self-antigen peptides that negatively select autoreactive thymocytes, leading to their apoptosis or generation of Tregs. Although cTECs and mTECs are distinguished by a series of cell surface markers and transcription factors, they arise from a common progenitor and both express the FoxN1, a transcription factor that belongs to the forkhead/winged-helix family of transcription factors, which are characterized by the presence of a forkhead domain that is involved in DNA binding. To further characterize the role of BRD4 in thymic differentiation, BRD4 was conditionally deleted in the thymic epithelium by FoxN1-Cre, which results in the deletion of BRD4 in both the cTEC and mTEC compartments. Loss of BRD4 results in a striking epithelial phenotype: a massive increase in cTECs with a concomitant decrease in mTECs and aberrant thymic architecture. Among thymoctyes, there is a significant loss in the frequency and cell number of both CD8+ T cells and Tregs. Studies are currently in progress to characterize the effect of BRD4 loss on RNA expression profiles in cTECs, mTECs and the different thymocyte populations using scRNA-seq. The above studies have documented the effects of conditionally deleting the entire BRD4 molecule on thymic development. However, they have not addressed the roles of the individual HAT and kinase activities in vivo. Therefore, using CRISPR technology, we have deleted either the HAT domain or the kinase domain of BRD4. We will first determine whether either enzymatic activity is necessary for embryonic development, as previous studies from others have reported that germline deletion is embryonic lethal. Preliminary results indicate that while heterozygotes of either deletion are viable, deletion of either the HAT domain or the kinase domain is embryonic lethal. Timed pregnancies will be performed to determine the stage at which development is arrested and the associated molecular defects; conditional deletions will be generated to further characterize adult-tissue defects. These mice are being bred to the BRD4fl/fl mice to allow conditional deletion in the thymus and other organs.