In vivo function of BRD4

Division Of Basic Sciences - Nci

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Abstract

The dependence on both robust proliferation and expression of lineage specific genes during thymic differentiation suggested that BRD4 may play a critical role in this process. Indeed, our studies have established a detailed gene expression profile of DN, ISP and DP pre-selection thymocytes and determined that BRD4 affects their patterns of gene expression, proliferation and differentiation. We identified the ISP as a cell type that is molecularly distinct from either the DN or DP subpopulations. Furthermore, by conditionally deleting BRD4 at various stages of thymic differentiation, we established that BRD4 selectively targets gene expression in the ISP cells: deletion of BRD4 in ISP down-regulates cell cycle and metabolic pathways, leading to a block in the transition to the DP stage. BRD4 is not necessary either for proliferation at the DN stage or for the subsequent maturation of conventional CD4 and CD8 single positive thymocytes from the DP stage. These studies lead to two unexpected conclusions: 1) that the ISP subpopulation is not a hybrid transitional state but a molecularly distinct subpopulation and 2) that ISP differentiation is selectively dependent on BRD4. We are continuing to explore the role of Bromodomain 4 (BRD4) in thymic development. In our previous studies, described above, we examined the intrinsic effect of conditionally deleting BRD4 in thymocytes. However, 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 and then 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 FoxN1, a transcription factor that belongs to the forkhead/winged-helix family of transcription factors. 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 large decrease in the proportion of mTECs with a concomitant increase in the proportion of cTECs without a change in total cell number and aberrant thymic architecture. scRNA analysis of WT BRD4 has identified over 20 cell clusters that represent each of the different stages of mTEC and cTEC maturation. As noted in the total cell analysis, cTECs are not affected in number or gene expression profile by the deletion of BRD4. In dramatic contrast, deletion of BRD4 blocks maturation of mTEC at two distinct stages: the bipotent precursor and the AIREhiMHCIIhi stage. Cells that are able to escape those blockages are able to develop normally. These findings indicate that the generation of mTECs is critically dependent on two distinct stages in mTEC development. In contrast, cTEC development is BRD4-independent and a default pathway in the absence of BRD4. Surprisingly, among thymocytes in the thymus where BRD4 has been deleted selectively in the epithelial compartment, there is a significant loss in the frequency and cell number of both CD8+ T cells and Tregs. Studies characterizing the effect of BRD4 loss on chromatin architecture and, since BRD4 is known to regulate alternative splicing, on alternative splicing are currently being analyzed. The report of these studies is being prepared.