Interleukin (IL)-17-producing T helper (Th17) cells are crucial for host defense against extracellular microbes and pathogenesis of autoimmune diseases. Here we show that the AP-1 transcription factor JunB is required for Th17 cell development. Junb-deficient CD4+ T cells are able to develop in vitro into various helper T subsets except Th17. The RNA-seq transcriptome analysis reveals that JunB is crucial for the Th17-specific gene expression program. Junb-deficient mice are completely resistant to experimental autoimmune encephalomyelitis, a Th17-mediated inflammatory disease, and naive T helper cells from such mice fail to differentiate into Th17 cells. JunB appears to activate Th17 signature genes by forming a heterodimer with BATF, another AP-1 factor essential for Th17 differentiation. The mechanism whereby JunB controls Th17 cell development likely involves activation of the genes for the Th17 lineage-specifying orphan receptors RORγt and RORα and reduced expression of Foxp3, a transcription factor known to antagonize RORγt function.
The nuclear protein IjBf activates transcription of a subset of NF-jB-dependent innate immune genes such as Lcn2 encoding the antibacterial protein lipocalin-2. IjBf functions as a coactivator via its interaction with NF-jB p50, which contains a DNA-binding Rel-homology domain but lacks a transcriptional activation domain. However cis-regulatory elements involved in IjBf function have remained unknown. Here, we show that, although IjBf by itself is unable to associate with the Lcn2 promoter, IjBf interacts with the promoter via p50 binding to the NF-jB-binding site (jB site) and the interaction also requires the pyrimidinerich site (CCCCTC) that localizes seven bases downstream of the jB site. The pyrimidine-rich site is also essential for IjBf-mediated activation of the Lcn2 gene. Introduction of both sites into an IjBf-independent gene culminates in IjBf-p50-DNA complex formation and transcriptional activation. Furthermore, spacing between the two sites is crucial for both IjBf-DNA interaction and IjBf-mediated gene activation. Thus, the pyrimidine-rich IjBf-responsive site plays an essential role in productive interaction of IjBf with the p50-DNA complex.
The nuclear protein IB, comprising the N-terminal transactivation domain and the C-terminal ankyrin repeat (ANK) domain composed of seven ANK motifs, activates transcription of a subset of nuclear factor-B (NF-B)-dependent innate immune genes such as Lcn2 encoding the antibacterial protein lipocalin-2. Lcn2 activation requires formation of a complex containing IB and NF-B p50, a transcription factor that harbors the DNA-binding Rel homology region but lacks a transactivation domain, on the promoter with the canonical NF-Bbinding site (B site) and its downstream cytosine-rich element. Here we show that IB productively interacts with p50 via Asp-451 in the N terminus of ANK1, a residue that is evolutionarily conserved among IB and the related nuclear IB proteins Bcl-3 and IB NS . Threonine substitution for Asp-451 abrogates direct association with the B-site-binding protein p50, complex formation with the Lcn2 promoter DNA, and activation of Lcn2 transcription. The basic residues Lys-717 and Lys-719 in the C-terminal region of ANK7 contribute to IB binding to the Lcn2 promoter, probably via interaction with the cytosinerich element required for Lcn2 activation; glutamate substitution for both lysines results in a loss of transcriptionally active complex formation without affecting direct contact of IB with p50. Both termini of the ANK domain in Bcl-3 and IB NS function in a manner similar to that of IB to interact with promoter DNA, indicating a common mechanism in which the nuclear IBs form a regulatory complex with NF-B and promoter DNA via the invariant aspartate in ANK1 and the conserved basic residues in ANK7. Nuclear factor-B (NF-B)2 plays central roles in host defense and inflammation as a homo-or heterodimer of NF-B/Rel family proteins by controlling the expression of genes for pro-inflammatory cytokines, chemokines, and antibacterial proteins (1-4). The mammalian NF-B family is composed of five structurally related polypeptides: p50, p52, p105 (the precursor of p50), p100 (the precursor of p52), p65 (also known as RelA), RelB, and c-Rel. They share the Rel homology region, which mediates dimerization, nuclear translocation, binding to specific DNA sequences known as NF-B-binding elements (B sites), and association with one of the IB family proteins (1-4). Among the members of the family, p65, RelB, and c-Rel have an ability to activate transcription by themselves via the C-terminal trans-activation domain, which is absent in the smaller p50 and p52 proteins. In resting cells, NF-B dimers are retained in the cytoplasm by associating with a member of the prototypical/cytoplasmic IB proteins including IB␣, IB, and IB⑀ (1-4). Cell activation with appropriate stimuli such as bacterial LPS leads to phosphorylation-induced degradation of cytoplasmic IBs and resultant liberation of NF-B dimers (1, 5, 6). The released NF-B dimers subsequently translocate to the nucleus and thus induce the expression of primary response genes via binding to B sites on their promoter/enhancer regions (1-4).The primarily induced gen...
Transmembrane glycoproteins, synthesized at the endoplasmic reticulum (ER), generally reach the Golgi apparatus in COPII-coated vesicles en route to the cell surface. Here, we show that the bona fide nonglycoprotein Nox5, a transmembrane superoxide-producing NADPH oxidase, is transported to the cell surface in a manner resistant to co-expression of Sar1 (H79G), a GTP-fixed mutant of the small GTPase Sar1, which blocks COPII vesicle fission from the ER. In contrast, Sar1 (H79G) effectively inhibits ER-to-Golgi transport of glycoproteins including the Nox5-related oxidase Nox2. The trafficking of Nox2, but not that of Nox5, is highly sensitive to over-expression of syntaxin 5 (Stx5), a t-SNARE required for COPII ER-to-Golgi transport. Thus, Nox2 and Nox5 mainly traffic via the Sar1/Stx5-dependent and -independent pathways, respectively. Both participate in Nox1 trafficking, as Nox1 advances to the cell surface in two differentially N-glycosylated forms, one complex and one high mannose, in a Sar1/Stx5-dependent and -independent manner, respectively. Nox2 and Nox5 also can use both pathways: a glycosylation-defective mutant Nox2 is weakly recruited to the plasma membrane in a less Sar1-dependent manner; N-glycosylated Nox5 mutants reach the cell surface in part as the complex form Sar1-dependently, albeit mainly as the high-mannose form in a Sar1-independent manner.
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