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. 2010 Jul 23;33(1):25-34.
doi: 10.1016/j.immuni.2010.07.001.

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

Matthias Farlik  1 , Benjamin Reutterer, Christian Schindler, Florian Greten, Claus Vogl, Mathias Müller, Thomas Decker
Affiliations

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

Matthias Farlik et al. Immunity. .

Abstract

Transcriptional regulation of the Nos2 gene encoding inducible nitric oxide synthase (iNOS) requires type I interferon (IFN-I) signaling and additional signals emanating from pattern recognition receptors. Here we showed sequential and cooperative contributions of the transcription factors ISGF3 (a complex containing STAT1, STAT2, and IRF9 subunits) and NF-kappaB to the transcriptional induction of the Nos2 gene in macrophages infected with the intracellular bacterial pathogen Listeria monocytogenes. 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.

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Figures

Figure 1
Figure 1
Kinetics of iNOS Induction Determined by q-PCR (A) Exposure of bone marrow-derived macrophages to living L. monocytogenes (LL) or to cotreatment with heat-killed Listeria (hkL) and IFN-β. (B) Bone marrow-derived macrophages were treated with hkL, IFN-β, or a combination of both. Error bars represent standard deviations from triplicate samples. The experiments were repeated at least three times.
Figure 2
Figure 2
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.
Figure 3
Figure 3
Binding of STATs and NF-κB to the Nos2 Promoter (A) Schematic drawing of the IFN response region and the NF-κB sites (NF-κB BS) in the Nos2 promoter (Kleinert et al., 2003). Binding of STATs and NF-κB to the Nos2 promoter in response to signals stimulated by exposure to L. monocytogenes. (B) Bone marrow-derived macrophages were stimulated with hkL and IFN-β and the cells were processed for ChIP at the indicated time points. Antibodies used for ChIP are shown on the left, P.I. indicates controls performed with preimmune sera. The precipitates were amplified with primers flanking the proximal (NF-κB) or distal (STAT1, IRF) promoter regions as depicted in (A) and analyzed by gel electrophoresis. (C) Bone marrow-derived macrophages were infected with viable L. monocytogenes and processed as described in (B). (D) Bone marrow-derived macrophages were either treated with IFN-β or infected with living L. monocytogenes (LL) for the times indicated and processed for ChIP-Re-ChIP. Antibodies used for ChIP and Re-ChIP are shown on top of the panels. The precipitates were amplified with primers flanking the distal Nos2 promoter region and analyzed by q-PCR. Error bars represent standard deviations from triplicate samples. The experiments were repeated at least three times.
Figure 4
Figure 4
Histone 4 Acetylation at the Nos2 Promoter Bone marrow-derived macrophages were treated with hkL and IFN-β (A), IFN-β alone (B), or hkL alone (C) as indicated. ChIP was performed with antibodies to acetyl-histone 4 (acH4) and with antibodies to histone 3 (H3). The presence of distal (black) or proximal (white) Nos2 promoter fragments was determined by q-PCR. Data are expressed as increase of acH4 signals normalized to H3 signals to correct for histone eviction. The histograms thus denote the ratio of acetyl-histone 4 binding as a function of total histone 3 (acH4/H3). Error bars represent standard deviations from triplicate samples. All experiments were repeated at least five times.
Figure 5
Figure 5
Recruitment of RNA Polymerase II to the Nos2 Promoter by L. monocytogenes-Derived Signals Bone marrow-derived macrophages from wild-type mice (A–E) or Stat1−/−, Stat2−/−, and Irf9−/− mice (D, E) were infected with living L. monocytogenes (LL [A, D, E]), with IFN-β alone (B), heat-killed Listeria alone (hkL [C]), or with a combination of IFN-β and hkL (B, C) for the times indicated. The cells were processed for ChIP with antibodies against pol II (A–D) or TBP (E). The precipitated DNA was analyzed by q-PCR with primers amplifying the distal (black) and proximal (white) promoter regions. Panels (D) and (E) show a comparison of proximal promoter fragments in ChIP from WT (black), Stat1−/− (red), Stat2−/− (yellow), and Irf9−/− (green) macrophages. Error bars represent standard deviations from triplicate samples. The experiments were repeated at least three times.
Figure 6
Figure 6
TFIIH-CDK7 Recruitment to the Nos2 Promoter and S5 Phosphorylation of the RNA Polymerase II CTD by L. monocytogenes-Derived Signals; Analysis of Nos2 Promoter Priming by hkL (A–H) Bone marrow-derived macrophages from WT mice (A–H), Ikbkb−/− mice (E, F), or Rela−/− mice (G, H) were infected with living L. monocytogenes (LL [D–H]), with IFN-β alone (A, B), with hkL alone (C), or with a combination of IFN-β and hkL (A–C) for the times indicated. The cells were processed for ChIP with antibodies against S5-phosphorylated pol II (A), CDK7 (B–E, G), or the TFIIH subunit p62 (F, H). The precipitated DNA was analyzed by q-PCR with primers amplifying the distal (black) and proximal (white) promoter regions. Panels (E)–(H) show a comparison of proximal promoter fragments in ChIP from WT (black), Ikbkb−/− (red, E, F), and Rela−/− (orange, G, H) macrophages. (I) Bone marrow-derived macrophages were pretreated with hkL for 2 hr or left without pretreatment followed by extensive washing of the cells. The cells were then left without treatment for different periods of time (indicated as hours gap). Thereafter cells were stimulated with hkL + IFN-β, IFN-β alone, or hkL alone for 4 hr. iNOS mRNA expression was determined by q-PCR. Error bars represent standard deviations from triplicate samples. The experiments were repeated at least three times.
Figure 7
Figure 7
RNA Pol II and CDK7 Recruitment to the Proximal Promoter Regions of the IFN-Inducible Mx2 Gene and the Gene Encoding IκB; Analysis of Mx2 and Nfkbia Promoter Priming by hkL (A–D) Bone marrow-derived macrophages from wild-type mice were treated with hkL + IFN-β, hkL alone, or IFN-β alone for the times indicated. The cells were processed for ChIP with antibodies against pol II (A, C) or CDK7 (B, D). The precipitated DNA was analyzed by q-PCR with primers amplifying the proximal promoter regions of the Mx2 gene (A, B) and the Nfkbia gene (the gene encoding IκBα) (C, D). (E and F) Bone marrow-derived macrophages were pretreated with hkL for 2 hr or left without pretreatment followed by extensive washing of the cells. The cells were then left without treatment for different periods of time (indicated as hours gap). Thereafter cells were stimulated with hkL + IFN-β, IFN-β alone, or hkL alone for 4 hr. Mx2 (E) and IκBα (F) mRNA expression was determined by q-PCR. Error bars represent standard deviations from triplicate samples. The experiments were repeated at least three times.
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