title: Oct-1 cooperates with the TATA binding initiation complex to control rapid transcription of human iNOS.
abstract: Expression of the human inducible nitric oxide synthase (hiNOS) is generally undetectable in resting cells, but stimulation by a variety of signals including cytokines induces transcription in most cell types. The tight transcriptional regulation of the enzyme is a complex mechanism many aspects of which remain unknown. Here, we describe an octamer (Oct) element in hiNOS proximal promoter, located close to the TATA box. This site constitutively binds Oct-1 and its deletion abrogates cytokine-induced transcription, showing that it is indispensable though not sufficient for transcription. Increasing the distance between Oct and the TATA box by inserting inert DNA sequence inhibits transcription, and footprinting of this region shows no other protein binding in resting cells, suggesting an interaction between the two complexes. Chromatin immunoprecipitation assays detect the presence of Oct-1, RNA polymerase II and trimethyl K4 histone H3 on the proximal promoter in resting cells, confirming that the gene is primed for transcription before stimulation. RT-PCR of various fragments along the hiNOS gene shows that transcription is initiated in resting cells and this is inhibited by interference with Oct-1 binding to the proximal site of the promoter. We propose that, through interaction with the initiation complex, Oct-1 regulates hiNOS transcription by priming the gene for the rapid response required in an immune response.
authors.affiliation: EPHE Laboratory, Faculty of Medicine, University of Bourgogne, Dijon, France.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Reveneau
authors.forename: Thodoris G
authors.initials: TG
authors.lastname: Petrakis
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Goldring
authors.forename: Aurélie
authors.initials: A
authors.lastname: Chantôme
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Jeannin
authors.forename: Alena
authors.initials: A
authors.lastname: Pance
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D045325:HCT116 Cells; D006801:Humans; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D052247:Nitric Oxide Synthase Type II / Q000235:genetics*; D050811:Octamer Transcription Factor-1 / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D016385:TATA Box; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers; D050811:Octamer Transcription Factor-1; C494165:POU2F1 protein, human; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II