Project description:We report the application of single-molecule-based sequencing technology for mapping the Egr2 transcriptional program in developing thymic NKT. We found that Egr2 controls the induction of genes required for NKT development. Overall design: Examination of developing NKT cells and thymocytes receiving a strong TCR signal in vivo by injecting 500ug anti-TCRb antibody.
Project description:We report the application of single-molecule-based sequencing technology for mapping the Egr2 transcriptional program in developing thymic NKT. We found that Egr2 controls the induction of genes required for NKT development. Examination of developing NKT cells and thymocytes receiving a strong TCR signal in vivo by injecting 500ug anti-TCRb antibody.
Project description:Interactions driven by the T cell antigen receptor (TCR) determine the lineage fate of CD4(+)CD8(+) thymocytes, but the molecular mechanisms that induce the lineage-determining transcription factors are unknown. Here we found that TCR-induced transcription factors Egr2 and Egr1 had higher and more-prolonged expression in precursors of the natural killer T (NKT) than in cells of conventional lineages. Chromatin immunoprecipitation followed by deep sequencing showed that Egr2 directly bound and activated the promoter of Zbtb16, which encodes the NKT lineage-specific transcription factor PLZF. Egr2 also bound the promoter of Il2rb, which encodes the interleukin 2 (IL-2) receptor ?-chain, and controlled the responsiveness to IL-15, which signals the terminal differentiation of the NKT lineage. Thus, we propose that persistent higher expression of Egr2 specifies the early and late stages of NKT lineage differentiation, providing a discriminating mechanism that enables TCR signaling to 'instruct' a thymic lineage.
Project description:?? NKT cells are neonatal-derived ?? T lymphocytes that are grouped together with invariant NKT cells based on their shared innate-like developmental program characterized by the transcription factor PLZF (promyelocytic leukemia zinc finger). Previous studies have demonstrated that the population size of ?? NKT cells is tightly controlled by Id3-mediated inhibition of E-protein activity in mice. However, how E proteins promote ?? NKT cell development and expansion remains to be determined. In this study, we report that the transcription factor Egr2, which also activates PLZF expression in invariant NKT cells, is essential for regulating ?? NKT cell expansion. We observed a higher expression of Egr family genes in ?? NKT cells compared with the conventional ?? T cell population. Loss of function of Id3 caused an expansion of ?? NKT cells, which is accompanied by further upregulation of Egr family genes as well as PLZF. Deletion of Egr2 in Id3-deficient ?? NKT cells prevented cell expansion and blocked PLZF upregulation. We further show that this Egr2-mediated ?? NKT cell expansion is dependent on c-Myc. c-Myc knockdown attenuated the proliferation of Id3-deficient ?? NKT cells, whereas c-Myc overexpression enhanced the proliferation of Id3/Egr2-double-deficient ?? NKT cells. Therefore, our data reveal a regulatory circuit involving Egr2-Id3-E2A, which normally restricts the population size of ?? NKT cells by adjusting Egr2 dosage and c-Myc expression.
Project description:The influence of signals transmitted by the phosphatase calcineurin and the transcription factor NFAT on the development and function of natural killer T (NKT) cells is unclear. In this report, we demonstrate that the transcription factor early growth response 2 (Egr2), a target gene of NFAT, was specifically required for the ontogeny of NKT cells but not that of conventional CD4(+) or CD8(+) T cells. NKT cells developed normally in the absence of Egr1 or Egr3, which suggests that Egr2 is a specific regulator of NKT cell differentiation. We found that Egr2 was important in the selection, survival and maturation of NKT cells. Our findings emphasize the importance of the calcineurin-NFAT-Egr2 pathway in the development of the NKT lymphocyte lineage.
Project description:We report the identification of DN NKT cells developed from DN stage thymocytes. We analyzed the gene expression profiles of NKT cells that have developed from DN stage thymocytes isolated from DP-specific E8III-Cre transgenic Rag2-floxed mouse strain, and NKT cells developed from DP thymocytes sorted as YFP-reporter positive NKT cells that were isolated from E8III-Cre transgenic Rosa26-loxP-STOP-loxP mouse. Overall design: Next generation sequencing of total transcriptomes using total RNA isolated from FACS sorted ex vivo thymic and liver NKT cells. DN-origin NKT cells were obtained from DP-specific E8III-Cre transgenic Rag2-floxed mice, and DP-origin NKT cells were obtained from E8III-Cre transgenic Rosa26-loxP-STOP-loxP mouse. The sorting gate was determined as: 7AAD-CD45R-CD4-CD8-CD1d dimer+TCRb+ cells.
Project description:Activation of germline promoters is central to V(D)J recombinational accessibility, driving chromatin remodeling, nucleosome repositioning, and transcriptional read-through of associated DNA. We have previously shown that of the two TCR? locus (Tcrb) D segments, D?1 is flanked by an upstream promoter that directs its transcription and recombinational accessibility. In contrast, transcription within the DJ?2 segment cluster is initially restricted to the J segments and only redirected upstream of D?2 after D-to-J joining. The repression of upstream promoter activity prior to Tcrb assembly correlates with evidence that suggests DJ?2 recombination is less efficient than that of DJ?1. Because inefficient DJ?2 assembly offers the potential for V-to-DJ?2 recombination to rescue frameshifted V-to-DJ?1 joints, we wished to determine how D?2 promoter activity is modulated upon Tcrb recombination. In this study, we show that repression of the otherwise transcriptionally primed 5'D?2 promoter requires binding of upstream stimulatory factor (USF)-1 to a noncanonical E-box within the D?2 12-recombination signal sequence spacer prior to Tcrb recombination. USF binding is lost from both rearranged and germline D?2 sites in DNA-dependent protein kinase, catalytic subunit-competent thymocytes. Finally, genotoxic dsDNA breaks lead to rapid loss of USF binding and gain of transcriptionally primed 5'D?2 promoter activity in a DNA-dependent protein kinase, catalytic subunit-dependent manner. Together, these data suggest a mechanism by which V(D)J recombination may feed back to regulate local D?2 recombinational accessibility during thymocyte development.
Project description:A TCR? enhancer, known as the E? enhancer, plays a critical role in V(D)J recombination and transcription of the Tcrb gene. However, the coordinated action of trans-acting factors in the activation of E? during T cell development remains uncharacterized. Here, we characterized the roles of Runx complexes in the regulation of the E? function. A single mutation at one of the two Runx binding motifs within the E? severely impaired Tcrb activation at the initiation phase in immature thymocytes. However, TCR? expression level in mature thymocytes that developed under such a single Runx site mutation was similar to that of the control. In contrast, mutations at two Runx motifs eliminated E? activity, demonstrating that Runx complex binding is essential to initiate E? activation. In cells expressing Tcrb harboring rearranged V(D)J structure, Runx complexes are dispensable to maintain TCR? expression, whereas E? itself is continuously required for TCR? expression. These findings imply that Runx complexes are essential for E? activation at the initiation phase, but are not necessary for maintaining E? activity at later developmental stages. Collectively, our results indicate that the requirements of trans-acting factor for E? activity are differentially regulated, depending on the developmental stage and cellular activation status.
Project description:Using biotinylation-based ChIP-seq and microarray analysis of both NKT and PLZF-transgenic thymocytes, we identified several layers of regulation of the innate-like NKT effector program. The transcription factor PLZF is induced during the development of innate and innate-like lymphocytes to direct their acquisition of a T helper effector program, but the molecular mechanisms involved are poorly understood. Using biotinylation-based ChIP-seq and microarray analysis of both NKT and PLZF-transgenic thymocytes, we identified several layers of regulation of the innate-like NKT effector program: first, PLZF bound and regulated genes encoding cytokine receptors as well as homing and adhesion receptors; second, PLZF bound and activated T helper-specific transcription factor genes that in turn control T helper specific programs; finally, PLZF bound and suppressed the transcription of Bach2, a potent general repressor of effector differentiation in naive T cells. These findings reveal the architecture of the transcriptional program recruited by PLZF and elucidate how a single transcription factor can drive the developmental acquisition of a broad effector program. Overall design: Examination of PLZF binding sites in both NKT and PLZF-transgenic thymocytes
Project description:The expression of CD5 increases progressively as thymocytes mature. We have shown that CD5 expression is controlled by a tissue-specific regulatory promoter located upstream of the CD5 translation start sites. Deletion of this regulatory promoter, which contains three potential transcription factor binding sites (CCAAT, kappa E2, and ets) reduces the promoter activity to basal level. Of these sites, only ets proved essential for CD5 expression in T cell lines. Here, we introduce a role for the E47 transcription factor and the CD5 promoter kappa E2 site in regulating CD5 expression during thymocyte development. Using T cell lines, we show that (i) mutation of the kappa E2 site in the CD5 regulatory promoter results in a significant elevation of CD5 promoter activity; (ii) the E47 transcription factor binds to the kappa E2 site; and (iii) overexpression of E47 inhibits CD5 expression. We then show, in high-dimensional fluorescence-activated cell sorting studies with primary thymocytes at successive developmental stages, that (i) intracellular E47 levels decrease as surface CD5 expression increases; (ii) E47 expression is down-regulated and CD5 expression is correspondingly up-regulated in DN3 thymocytes in RAG-2-deficient mice injected with anti-CD3 to mimic pre-T cell receptor stimulation; and (iii) E47 expression is down-regulated and CD5 expression is up-regulated when double positive thymocytes are stimulated in vitro with anti-CD3. Based on these data, we propose that E47 negatively regulates CD5 expression by interacting with the kappa E2 site in the CD5 regulatory promoter and that decreases in E47 in response to developmental signals are critical to the progressive increase in CD5 expression as thymocytes mature.