Nonconventional initiation complex assembly by STAT and NF-kappaB transcription factors regulates nitric oxide synthase expression.
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.
Affiliation: Max F. Perutz Laboratories, Department of Genetics, Microbiology and Immunobiology, University of Vienna, Dr. Bohr-Gasse 9/4, A1030 Vienna, Austria.Show MeSH
Mentions: Pol II can be bound to transcription start sites in a poised state (Adelman et al., 2009; Koch et al., 2008; Margaritis and Holstege, 2008). Alternatively, the enzyme is recruited in response to the stimulus of gene activation (Adelman et al., 2009). To determine which situation applies to the macrophage Nos2 gene, we analyzed pol II association by ChIP. As shown in Figure 5A, infection with L. monocytogenes strongly increased pol II binding, suggesting that it occurs by regulated recruitment. Surprisingly, treatment with IFN-I alone also stimulated binding of pol II (Figure 5B). Association was somewhat, but not much, weaker than after the additional presence of hkL. In contrast to IFN-I, hkL alone did not stimulate pol II binding (Figure 5C). This result indicates (1) that the histone acetylation caused by NF-κB is not an absolute requirement for pol II binding and (2) that there is a mechanistic difference between ISGF3 and NF-κB in their mode of activating the Nos2 promoter. IFN and STAT-dependent recruitment of pol II predicts that binding of TFIID and its TBP subunit displays the same requirement. Figures 5D and 5E indeed show that both pol II and TBP binding was completely abrogated when Stat1−/−, Stat2−/−, or Irf9−/− macrophages were infected with L. monocytogenes.
Affiliation: Max F. Perutz Laboratories, Department of Genetics, Microbiology and Immunobiology, University of Vienna, Dr. Bohr-Gasse 9/4, A1030 Vienna, Austria.