Project description:To investigate gene targets of the E-proteins HEB and E2A during the CD4+CD8+ double positive (DP) stage of T cell development. We examined E-protein function by simultaneous removal of both HEB (Tcf12) and E2A (Tcfe2a) genes at the DP stage. This was done by crossing mice containing HEB floxed and E2A floxed alleles to a CD4Cre background (Tcf12f/fTcfe2af/fCD4Cre mice). Microarray analysis was used to compare gene expression in HEB and E2A double deficient DP thymocytes (Cre+) to Cre- control DP thymocytes. Keywords: genetic modification
Project description:Analysis of gene expression in double positive (DP) thymoctes from 3w old LCK Hdac3 conditional KO mice. In this data set we sorted GFP double positive (DP) thymocytes from 3w old mice to identify possible signaling and transcripitonal programs that might explain the DP to single positive block in cell maturation
Project description:H3K27Ac ChIP-seq in wild type and cohesin-deficient thymocytes Rad21 was deleted in CD4+ CD8+ double positive (DP) thymocytes by crossing a Rad21 floxed allele with a Cd4-driven Cre transgene. DP positive thymocytes were FACS-sorted from control and Rad21-/- littermates, which were then used to perform chromatin immunoprecipitation for histone H3 acetylated on lysine 27 (H3K27Ac).
Project description:CD4+ and CD8+ double-positive (DP) thymocytes are at a critical stage during the T cell development in thymus. DP cells rearrange the T cell receptor gene Tcra to generate T cell receptors with TCR. Then DP cells differentiate into CD4 or CD8 single-positive (SP) thymocytes, Regulatory T cells, or invariant nature kill T cells (iNKT) according to the TCR signal. Chromatin organizer SATB1 is highly expressed in DP cells and plays an essential role in regulating Tcra rearrangement and differentiation of DP cells. Here we explored the mechanism of SATB1 orchestrating gene expression in DP cells. Single-cell RNA sequencing assay of SATB1-deficient thymocytes showed that the cell identity of DP thymocytes was changed, and the genes specifically highly expressed in DP cells were down-regulated. ChIP-seq and ATAC-seq data showed the similar tendency. The super-enhancers regulate the expressions of the DP-specific genes, and the SATB1 deficiency reduced the super-enhancer activity. Hi-C data showed that interactions in super-enhancers and between super-enhancers and promoters decreased in SATB1 deficient thymocytes. We further explored the regulation mechanism of two SATB1-regulating genes, ETS2 and Bcl6, in DP cells and found that the knockout of the super-enhancers of these two genes impaired the development of DP cells. Our research reveals that SATB1 globally regulates super-enhancers of DP cells and promotes the establishment of DP cell identity, which helps understand the role of SATB1 in thymocyte development.
Project description:The lifespan of double-positive (DP) thymocytes is critical for intrathymic development and shaping the peripheral T cell repertoire. However, the molecular mechanisms that control DP thymocyte survival remain poorly understood. Paxbp1 is a conserved nuclear protein that has been reported to play important roles in cell growth and development. Its high expression in T cells suggests a possible role in T cell development. Here, we observed that deletion of Paxbp1 resulted in thymic atrophy in mice lacking Paxbp1 in the early stages of T cell development. Conditional loss of Paxbp1 resulted in fewer CD4+CD8+ DP T cells, CD4 and CD8 single positive (SP) T cells in the thymus, and fewer T cells in the periphery. Meanwhile, Paxbp1 deficiency had limited effects on the CD4-CD8- double negative (DN) or immature single-positive (ISP) cell populations. Instead, we observed a significant increase in the susceptibility of Paxbp1-deficient DP thymocytes to apoptosis. Consistent with this, RNA-Seq analysis revealed a significant enrichment of the apoptotic pathway within differentially expressed genes in Paxbp1-deficient DP cells compared to control DP cells. Together, our results suggest a new function for Paxbp1, which is an important mediator of DP thymocyte survival and critical for proper thymic development.
Project description:Both TCF-1 and its coactivator β-catenin are known to be required for supporting normal double positive (DP) thymocyte survival through upregulating Bcl-xL. However, the downstream factors mediating this effect remained unknown. We used microarray to compare the global expression difference among WT, TCF-1-deficient, and β-catenin transgenic thymocytes to search for the genes that are down-regulated and up-regulated in TCF-1-deficient and β-catenin transgenic thymocytes, respectively.
Project description:Double-positive (DP) thymocytes respond to intrathymic TCR signals by undergoing positive selection and lineage differentiation into single-positive (SP) mature cells. Concomitant with these wellcharacterized events is the acquisition of a mature T cell gene expression program characterized by the induction of effector molecules IL-7Ra, S1P1 and CCR7, but the underlying mechanism remains elusive. We report here that transcription repressor Gfi1 orchestrates the fidelity of DP gene expression program and developmental maturation into SP cells. Loss of Gfi1 resulted in premature induction of effector genes and transcription factors Foxo1 and Klf2 in DP thymocytes, and accumulation of post-selection intermediate populations and accelerated transition into SP cells. Strikingly, partial loss of Foxo1 function, but not restored survival fitness, rectified the dysregulated gene expression and thymocyte maturation in Gfi1-deficient mice. Our results establish Gfi1-Foxo1 axis and the transcriptional circuitry that actively maintain DP identity and shape the proper generation of mature T cells