Notch1 specifically upregulates expression of the cytokine interferon‐γ in peripheral T cells through activation of NF‐κB. However, how Notch mediates NF‐κB activation remains unclear. Here, we examined the temporal relationship between Notch signaling and NF‐κB induction during T‐cell activation. NF‐κB activation occurs within minutes of T‐cell receptor (TCR) engagement and this activation is sustained for at least 48 h following TCR signaling. We used γ‐secretase inhibitor (GSI) to prevent the cleavage and subsequent activation of Notch family members. We demonstrate that GSI blocked the later, sustained NF‐κB activation, but did not affect the initial activation of NF‐κB. Using biochemical approaches, as well as confocal microscopy, we show that the intracellular domain of Notch1 (N1IC) directly interacts with NF‐κB and competes with IκBα, leading to retention of NF‐κB in the nucleus. Additionally, we show that N1IC can directly regulate IFN‐γ expression through complexes formed on the IFN‐γ promoter. Taken together, these data suggest that there are two ‘waves’ of NF‐κB activation: an initial, Notch‐independent phase, and a later, sustained activation of NF‐κB, which is Notch dependent.

SUMMARY How innate lymphoid cells (ILCs) in the thymus and gut become specialized effectors is unclear. The prototypic innate-like γδ T cells (Tγδ17) are a major source of interleukin-17 (IL-17). We demonstrate that Tγδ17 cells are programmed by a gene regulatory network consisting of a quartet of High Mobility Group box (HMG) transcription factors, SOX4, SOX13, TCF1 and LEF1, and not by conventional TCR signaling. SOX4 and SOX13 directly regulated the two requisite Tγδ17 cell-specific genes, Rorc and Blk, whereas TCF1 and LEF1 countered the SOX proteins and induced genes of alternate effector subsets. The T cell lineage specification factor TCF1 was also indispensable for the generation of IL-22 producing gut NKp46+ ILCs and restrained cytokine production by Lymphoid Tissue inducer-like effectors. These results indicate that similar gene network architecture programs innate sources of IL-17, independent of anatomical origins.

The maturation of naive CD8+ T cells into effector CTLs is a critical feature of a functional adaptive immune system. Development of CTLs depends, in part, upon the expression of the transcriptional regulator eomesodermin (EOMES), which is thought to regulate expression of two key effector molecules, perforin and granzyme B. Although EOMES is important for effector CTL development, the precise mechanisms regulating CD8+ effector cell maturation remains poorly understood. In this study, we show that Notch1 regulates the expression of EOMES, perforin, and granzyme B through direct binding to the promoters of these crucial effector molecules. By abrogating Notch signaling, both biochemically as well as genetically, we conclude that Notch activity mediates CTL activity through direct regulation of EOMES, perforin, and granzyme B.

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The Tec family tyrosine kinase, Itk, regulates signaling downstream of the T cell receptor (TCR). The absence of Itk in CD4+ T cells results in impaired Th2 responses along with defects in maturation, cytokine production and survival of iNKT cells. Paradoxically, Itk−/− mice have spontaneously elevated serum IgE levels, resulting from an expansion of the Vγ1.1+Vδ6.3+ subset of γδ T cells, known as γδ NKT cells. Comparisons between γδ NKT cells and αβ iNKT cells showed convergence in the pattern of cell surface marker expression, cytokine profiles, and gene expression, suggesting that these two subsets of NKT cells undergo similar differentiation programs. Hepatic γδ NKT cells have an invariant TCR and are derived predominantly from fetal progenitors that expand in the thymus during the first weeks of life. The adult thymus contains these invariant γδ NKT cells plus a heterogeneous population of Vγ1.1+Vδ6.3+ T cells with diverse CDR3 sequences. This latter population, normally excluded from the liver, escapes the thymus and homes to the liver when Itk is absent. In addition, Itk−/− γδ NKT cells persistently express high levels of Zbtb16 (PLZF) and Il4, genes that are normally down-regulated in the most mature subsets of NKT cells. These data indicate that Itk signaling is required to prevent the expansion of γδ NKT cells in the adult thymus, to block their emigration, and to promote terminal NKT cell maturation.

Background Among the complexities of skeletal muscle differentiation is a temporal distinction in the onset of expression of different lineage-specific genes. The lineage-determining factor MyoD is bound to myogenic genes at the onset of differentiation whether gene activation is immediate or delayed. How temporal regulation of differentiation-specific genes is established remains unclear. Results Using embryonic tissue, we addressed the molecular differences in the organization of the myogenin and muscle creatine kinase (MCK) gene promoters by examining regulatory factor binding as a function of both time and spatial organization during somitogenesis. At the myogenin promoter, binding of the homeodomain factor Pbx1 coincided with H3 hyperacetylation and was followed by binding of co-activators that modulate chromatin structure. MyoD and myogenin binding occurred subsequently, demonstrating that Pbx1 facilitates chromatin remodeling and modification prior to myogenic regulatory factor binding. At the same time, the MCK promoter was bound by HDAC2 and MyoD, and activating histone marks were largely absent. The association of HDAC2 and MyoD was confirmed by co-immunoprecipitation, proximity ligation assay (PLA), and sequential ChIP. Conclusion MyoD differentially promotes activated and repressed chromatin structures at myogenic genes early after the onset of skeletal muscle differentiation in the developing mouse embryo.

T cell stimulation requires the input and integration of external signals. Signaling through the T cell receptor (TCR) is known to induce formation of the membrane-tethered CBM complex, comprising CARMA1, BCL10, and MALT1, which is required for TCR-mediated NF-κB activation. TCR signaling has been shown to activate NOTCH proteins, transmembrane receptors also implicated in NF-κB activation. However, the link between TCR-mediated NOTCH signaling and early events leading to induction of NF-κB activity remains unclear. In this report, we demonstrate a novel cytosolic function for NOTCH1 and show that it is essential to CBM complex formation. Using a model of skin allograft rejection, we show in vivo that NOTCH1 acts in the same functional pathway as PKCθ, a T cell-specific kinase important for CBM assembly and classical NF-κB activation. We further demonstrate in vitro NOTCH1 associates physically with PKCθ and CARMA1 in the cytosol. Unexpectedly, when NOTCH1 expression was abrogated using RNAi approaches, interactions between CARMA1, BCL10, and MALT1 were lost. This failure in CBM assembly reduced inhibitor of kappa B alpha phosphorylation and diminished NF-κB–DNA binding. Finally, using a luciferase gene reporter assay, we show the intracellular domain of NOTCH1 can initiate robust NF-κB activity in stimulated T cells, even when NOTCH1 is excluded from the nucleus through modifications that restrict it to the cytoplasm or hold it tethered to the membrane. Collectively, these observations provide evidence that NOTCH1 may facilitate early events during T cell activation by nucleating the CBM complex and initiating NF-κB signaling.

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells (1-3). This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions. We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events. In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.5(4,5). Myogenin is a regulatory factor required for skeletal muscle differentiation (6-9). The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development (6,10), the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos