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Nonconventional initiation complex assembly by STAT and NF-kappaB transcription factors regulates nitric oxide synthase expression.

Farlik M, Reutterer B, Schindler C, Greten F, Vogl C, Müller M, Decker T - Immunity (2010)

Bottom Line: NF-kappaB preceded ISGF3 at the Nos2 promoter and generated a transcriptional memory effect by depositing basal transcription factor TFIIH with the associated CDK7 kinase for serine 5 phosphorylation of the RNA polymerase II (pol II) carboxyterminal domain (CTD).Subsequent to TFIIH deposition by NF-kappaB, ISGF3 attracted the pol II enzyme and phosphorylation at CTD S5 occurred.Thus, STATs and NF-kappaB cooperate through pol II promoter recruitment and the phosphorylation of its CTD, respectively, as a prerequisite for productive elongation of iNOS mRNA.

View Article: PubMed Central - PubMed

Affiliation: Max F. Perutz Laboratories, Department of Genetics, Microbiology and Immunobiology, University of Vienna, Dr. Bohr-Gasse 9/4, A1030 Vienna, Austria.

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iNOS mRNA Induction by L. monocytogenes Requires Stat1, Stat2, IRF9, and NF-κB Signaling(A) Bone marrow-derived macrophages of WT, Stat1−/−, and Rela−/− mice were infected with living L. monocytogenes (LL) for the times indicated. IFN-β was additionally present to compensate for potential defects in IFN-I production. iNOS mRNA expression was determined by q-PCR.(B) Bone marrow-derived macrophages with the indicated genotypes were infected with living L. monocytogenes (LL) for 6 hr or a combination of LL and IFN-β (Ikbkb−/− + IFN-β; Rela−/− + IFNβ; Irf3−/− + IFN-β) for 4 hr. iNOS mRNA expression was determined by q-PCR.To be able to compare data between individual experiments, genotype-specific expression is shown as percent induction found in wild-type macrophages. Error bars represent standard deviations from triplicate samples. The mentioned experiments were repeated at least three times.
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fig2: iNOS mRNA Induction by L. monocytogenes Requires Stat1, Stat2, IRF9, and NF-κB Signaling(A) Bone marrow-derived macrophages of WT, Stat1−/−, and Rela−/− mice were infected with living L. monocytogenes (LL) for the times indicated. IFN-β was additionally present to compensate for potential defects in IFN-I production. iNOS mRNA expression was determined by q-PCR.(B) Bone marrow-derived macrophages with the indicated genotypes were infected with living L. monocytogenes (LL) for 6 hr or a combination of LL and IFN-β (Ikbkb−/− + IFN-β; Rela−/− + IFNβ; Irf3−/− + IFN-β) for 4 hr. iNOS mRNA expression was determined by q-PCR.To be able to compare data between individual experiments, genotype-specific expression is shown as percent induction found in wild-type macrophages. Error bars represent standard deviations from triplicate samples. The mentioned experiments were repeated at least three times.

Mentions: To examine transcription factor requirements for transcriptional induction of the Nos2 gene, we used bone marrow-derived macrophages from either wild-type or gene-targeted mice and infected them with L. monocytogenes (Figure 2). As expected, Nos2 expression required signaling through both the IFN and NF-κB pathways as deletion of either the Stat1 or Rela (NF-κB p65) genes strongly suppressed iNOS mRNA induction in infected macrophages (Figure 2A). More refined analyses confirmed the importance of the IFN-I receptor (Ifnar1−/− mice) and the NF-κB pathway (Rela−/− and Ikbkb−/− mice, deficient for NF-κB p65 and the IKKβ kinase, respectively) and established the importance of the ISGF3 subunits STAT1, STAT2, and IRF9 (Figure 2B). The diminished Nos2 expression observed upon interference with NF-kB signaling was not due to reduced IFN-I production as shown by the fact that addition of exogenous IFN-β did not rescue this effect. Use of macrophages derived from mice expressing STAT1 mutated at its S727 phosphorylation site (STAT1S727A) showed that phosphorylation of STAT1 at S727, important for full transcriptional induction of some IFN-γ-induced genes (Varinou et al., 2003), was not required for Nos2 expression. This contrasts with the reduced induction of Nos2 by IFN-γ early after treatment in STAT1S727A-expressing macrophages (Varinou et al., 2003). In further distinction from the IFN-γ response, the decrease resulting from IRF1 deficiency was marginal. Two additional members of the IRF family, IRF3 and IRF7, are active in L. monocytogenes-infected macrophages (Stockinger et al., 2009). IRF7 deficiency did not affect Nos2 expression. IRF3 deficiency reduced Nos2 induction, but the defect could be rescued by the addition of IFN-β, suggesting that it resulted from reduced IFN-β synthesis, but not from a direct effect on the Nos2 gene. The data suggest that IFN-I participate in Nos2 regulation during L. monocytogenes infection by deploying the ISGF3 complex, but not the ancillary activity of IRFs. The low levels of iNOS expression seen after treatment up to 6 hr with IFN-β alone (Figure 1) were strongly reduced in mice unable to form ISGF3 (data not shown). Interestingly, this differs from the regulation of Nos2 mRNA during the late stage of the IFN-I response, which has been shown to be independent of STAT1 (Plumlee et al., 2009).


Nonconventional initiation complex assembly by STAT and NF-kappaB transcription factors regulates nitric oxide synthase expression.

Farlik M, Reutterer B, Schindler C, Greten F, Vogl C, Müller M, Decker T - Immunity (2010)

iNOS mRNA Induction by L. monocytogenes Requires Stat1, Stat2, IRF9, and NF-κB Signaling(A) Bone marrow-derived macrophages of WT, Stat1−/−, and Rela−/− mice were infected with living L. monocytogenes (LL) for the times indicated. IFN-β was additionally present to compensate for potential defects in IFN-I production. iNOS mRNA expression was determined by q-PCR.(B) Bone marrow-derived macrophages with the indicated genotypes were infected with living L. monocytogenes (LL) for 6 hr or a combination of LL and IFN-β (Ikbkb−/− + IFN-β; Rela−/− + IFNβ; Irf3−/− + IFN-β) for 4 hr. iNOS mRNA expression was determined by q-PCR.To be able to compare data between individual experiments, genotype-specific expression is shown as percent induction found in wild-type macrophages. Error bars represent standard deviations from triplicate samples. The mentioned experiments were repeated at least three times.
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fig2: iNOS mRNA Induction by L. monocytogenes Requires Stat1, Stat2, IRF9, and NF-κB Signaling(A) Bone marrow-derived macrophages of WT, Stat1−/−, and Rela−/− mice were infected with living L. monocytogenes (LL) for the times indicated. IFN-β was additionally present to compensate for potential defects in IFN-I production. iNOS mRNA expression was determined by q-PCR.(B) Bone marrow-derived macrophages with the indicated genotypes were infected with living L. monocytogenes (LL) for 6 hr or a combination of LL and IFN-β (Ikbkb−/− + IFN-β; Rela−/− + IFNβ; Irf3−/− + IFN-β) for 4 hr. iNOS mRNA expression was determined by q-PCR.To be able to compare data between individual experiments, genotype-specific expression is shown as percent induction found in wild-type macrophages. Error bars represent standard deviations from triplicate samples. The mentioned experiments were repeated at least three times.
Mentions: To examine transcription factor requirements for transcriptional induction of the Nos2 gene, we used bone marrow-derived macrophages from either wild-type or gene-targeted mice and infected them with L. monocytogenes (Figure 2). As expected, Nos2 expression required signaling through both the IFN and NF-κB pathways as deletion of either the Stat1 or Rela (NF-κB p65) genes strongly suppressed iNOS mRNA induction in infected macrophages (Figure 2A). More refined analyses confirmed the importance of the IFN-I receptor (Ifnar1−/− mice) and the NF-κB pathway (Rela−/− and Ikbkb−/− mice, deficient for NF-κB p65 and the IKKβ kinase, respectively) and established the importance of the ISGF3 subunits STAT1, STAT2, and IRF9 (Figure 2B). The diminished Nos2 expression observed upon interference with NF-kB signaling was not due to reduced IFN-I production as shown by the fact that addition of exogenous IFN-β did not rescue this effect. Use of macrophages derived from mice expressing STAT1 mutated at its S727 phosphorylation site (STAT1S727A) showed that phosphorylation of STAT1 at S727, important for full transcriptional induction of some IFN-γ-induced genes (Varinou et al., 2003), was not required for Nos2 expression. This contrasts with the reduced induction of Nos2 by IFN-γ early after treatment in STAT1S727A-expressing macrophages (Varinou et al., 2003). In further distinction from the IFN-γ response, the decrease resulting from IRF1 deficiency was marginal. Two additional members of the IRF family, IRF3 and IRF7, are active in L. monocytogenes-infected macrophages (Stockinger et al., 2009). IRF7 deficiency did not affect Nos2 expression. IRF3 deficiency reduced Nos2 induction, but the defect could be rescued by the addition of IFN-β, suggesting that it resulted from reduced IFN-β synthesis, but not from a direct effect on the Nos2 gene. The data suggest that IFN-I participate in Nos2 regulation during L. monocytogenes infection by deploying the ISGF3 complex, but not the ancillary activity of IRFs. The low levels of iNOS expression seen after treatment up to 6 hr with IFN-β alone (Figure 1) were strongly reduced in mice unable to form ISGF3 (data not shown). Interestingly, this differs from the regulation of Nos2 mRNA during the late stage of the IFN-I response, which has been shown to be independent of STAT1 (Plumlee et al., 2009).

Bottom Line: NF-kappaB preceded ISGF3 at the Nos2 promoter and generated a transcriptional memory effect by depositing basal transcription factor TFIIH with the associated CDK7 kinase for serine 5 phosphorylation of the RNA polymerase II (pol II) carboxyterminal domain (CTD).Subsequent to TFIIH deposition by NF-kappaB, ISGF3 attracted the pol II enzyme and phosphorylation at CTD S5 occurred.Thus, STATs and NF-kappaB cooperate through pol II promoter recruitment and the phosphorylation of its CTD, respectively, as a prerequisite for productive elongation of iNOS mRNA.

View Article: PubMed Central - PubMed

Affiliation: Max F. Perutz Laboratories, Department of Genetics, Microbiology and Immunobiology, University of Vienna, Dr. Bohr-Gasse 9/4, A1030 Vienna, Austria.

Show MeSH