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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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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.
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fig6: 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.

Mentions: Pol II, once stably bound to the initiation site, must be phosphorylated at its CTD to associate with proteins required for promoter clearance, capping of the mRNA, and elongation (Chapman et al., 2008; Hirose and Ohkuma, 2007). Serine 5 (S5) of the CTD amino acid heptarepeat becomes phosphorylated first, followed by S2, to proceed to productive elongation. With NF-κB playing only a minor role in pol II recruitment, we wondered whether it might play a role in distinct steps of transcriptional initiation. We investigated CTD phosphorylation at S5 by using phosphospecific antibodies for ChIP. S5-phosphorylated pol II was precipitated from the Nos2 initiation site only after treatment with both hkL and IFN-I, but not after treatment with IFN-I alone (Figure 6A). This confirms our notion that NF-κB might be involved in regulating CTD phosphorylation. CTD S5 kinase activity is associated with the general transcription factor TFIIH. TFIIH usually joins the initiation complex only after pol II binding. It is a multiprotein transcription factor containing the CTD S5 kinase CDK7 and a number of additional subunits including p62 (Egly, 2001). As in the case of S5-phosphorylated pol II, CDK7 was associated with the Nos2 initiation site after stimulation with hkL and IFN-I, but not after treatment with IFN-I alone (Figure 6B). In contrast to IFN-I, hkL treatment alone produced as much CDK7 binding as the combined IFN-I-hkL treatment (Figure 6C). The kinetics of CDK7 binding as induced by L. monocytogenes demonstrated association with the Nos2 promoter at 2 hr postinfection (Figure 6D). At this time, NF-κB is associated with Nos2 chromatin, but no or very little ISGF3 is present (Figure 3C). Binding of CDK7 as well as that of TFIIH p62 was abrogated by both NF-κB p65 and IKKβ deficiency (Figures 6E–6H). Together, these data confirm the hypothesis that a TFIIH complex is recruited by NF-κB, providing kinase activity for the pol II CTD at S5. Comparing the kinetics of NF-κB and TFIIH binding in the course of infection suggested that TFIIH remains bound at the promoter even after dissociation of NF-κB (Figures 3, 6G, and 6H; Figure S2). We tested the possibility that NF-κB, by depositing TFIIH, primes the Nos2 promoter for subsequent ISGF3 activity, thus providing a “transcriptional memory” effect. To this end, macrophages were given a 2 hr pulse of hkL treatment, a period sufficient for CDK7 recruitment (Figure 6D). The pulsed cells were left without further stimulation for various intervals, followed by a 4 hr treatment with either IFN-β alone, hkL alone, or a combination of IFN-β and hkL. The data show that for at least 24 hr, the level achieved by IFN-β treatment of pulsed cells exceeded the level achieved by IFN-β treatment of unpulsed cells (Figure 6I). This result is in agreement with the notion of a transcriptional memory or priming effect of NF-κB-recruited CDK7.


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)

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.
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fig6: 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.
Mentions: Pol II, once stably bound to the initiation site, must be phosphorylated at its CTD to associate with proteins required for promoter clearance, capping of the mRNA, and elongation (Chapman et al., 2008; Hirose and Ohkuma, 2007). Serine 5 (S5) of the CTD amino acid heptarepeat becomes phosphorylated first, followed by S2, to proceed to productive elongation. With NF-κB playing only a minor role in pol II recruitment, we wondered whether it might play a role in distinct steps of transcriptional initiation. We investigated CTD phosphorylation at S5 by using phosphospecific antibodies for ChIP. S5-phosphorylated pol II was precipitated from the Nos2 initiation site only after treatment with both hkL and IFN-I, but not after treatment with IFN-I alone (Figure 6A). This confirms our notion that NF-κB might be involved in regulating CTD phosphorylation. CTD S5 kinase activity is associated with the general transcription factor TFIIH. TFIIH usually joins the initiation complex only after pol II binding. It is a multiprotein transcription factor containing the CTD S5 kinase CDK7 and a number of additional subunits including p62 (Egly, 2001). As in the case of S5-phosphorylated pol II, CDK7 was associated with the Nos2 initiation site after stimulation with hkL and IFN-I, but not after treatment with IFN-I alone (Figure 6B). In contrast to IFN-I, hkL treatment alone produced as much CDK7 binding as the combined IFN-I-hkL treatment (Figure 6C). The kinetics of CDK7 binding as induced by L. monocytogenes demonstrated association with the Nos2 promoter at 2 hr postinfection (Figure 6D). At this time, NF-κB is associated with Nos2 chromatin, but no or very little ISGF3 is present (Figure 3C). Binding of CDK7 as well as that of TFIIH p62 was abrogated by both NF-κB p65 and IKKβ deficiency (Figures 6E–6H). Together, these data confirm the hypothesis that a TFIIH complex is recruited by NF-κB, providing kinase activity for the pol II CTD at S5. Comparing the kinetics of NF-κB and TFIIH binding in the course of infection suggested that TFIIH remains bound at the promoter even after dissociation of NF-κB (Figures 3, 6G, and 6H; Figure S2). We tested the possibility that NF-κB, by depositing TFIIH, primes the Nos2 promoter for subsequent ISGF3 activity, thus providing a “transcriptional memory” effect. To this end, macrophages were given a 2 hr pulse of hkL treatment, a period sufficient for CDK7 recruitment (Figure 6D). The pulsed cells were left without further stimulation for various intervals, followed by a 4 hr treatment with either IFN-β alone, hkL alone, or a combination of IFN-β and hkL. The data show that for at least 24 hr, the level achieved by IFN-β treatment of pulsed cells exceeded the level achieved by IFN-β treatment of unpulsed cells (Figure 6I). This result is in agreement with the notion of a transcriptional memory or priming effect of NF-κB-recruited CDK7.

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