title: Up-Regulation of Human Inducible Nitric Oxide Synthase by p300 Transcriptional Complex.
abstract: p300, a ubiquitous transcription coactivator, plays an important role in gene activation. Our previous work demonstrated that human inducible nitric oxide synthase (hiNOS) expression can be highly induced with the cytokine mixture (CM) of TNF-α + IL-1β + IFN-γ. In this study, we investigated the functional role of p300 in the regulation of hiNOS gene expression. Our initial data showed that overexpression of p300 significantly increased the basal and cytokine-induced hiNOS promoter activities in A549 cells. Interestingly, p300 activated cytokine-induced hiNOS transcriptional activity was completely abrogated by deleting the upstream hiNOS enhancer at -5 kb to -6 kb in the promoter. Furthermore, p300 over-expression increased cytokine-induced transcriptional activity on a heterologous minimal TK promoter with the same hiNOS enhancer. Site-directed mutagenesis of the hiNOS AP-1 motifs revealed that an intact upstream (-5.3 kb) AP-1 binding site was critical for p300 mediated cytokine-induced hiNOS transcription. Furthermore, our ChIP analysis demonstrated that p300 was binding to Jun D and Fra-2 proteins at -5.3 kb AP-1 binding site in vivo. Lastly, our 3C assay was able to detect a long DNA loop between the hiNOS enhancer and core promoter site, and ChIP loop assay confirmed that p300 binds to AP-1 and RNA pol II proteins. Overall, our results suggest that coactivator p300 mediates cytokine-induced hiNOS transactivation by forming a distant DNA loop between its enhancer and core promoter region.
authors.affiliation: Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, 15261, United States of America.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Guo
authors.affiliation: Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, 15261, United States of America.
authors.forename: Liang
authors.initials: L
authors.lastname: Zheng
authors.affiliation: Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, 15261, United States of America.
authors.forename: Xinghua
authors.initials: X
authors.lastname: Liao
authors.affiliation: Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, 15261, United States of America.
authors.forename: David
authors.initials: D
authors.lastname: Geller
mesh_terms: D001665:Binding Sites; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D016207:Cytokines / Q000378:metabolism; D050881:E1A-Associated p300 Protein / Q000378:metabolism*; D051921:Fos-Related Antigen-2 / Q000378:metabolism; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D015854:Up-Regulation; D050880:p300-CBP Transcription Factors / Q000378:metabolism
chemical_list: D016207:Cytokines; C497872:FOSL2 protein, human; D051921:Fos-Related Antigen-2; C573415:IFNG protein, human; C508585:IL1B protein, human; D053583:Interleukin-1beta; C559371:JunD protein, human; D016328:NF-kappa B; D016755:Proto-Oncogene Proteins c-jun; D011994:Recombinant Proteins; C439524:TNF protein, human; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; D050881:E1A-Associated p300 Protein; C494360:EP300 protein, human; D050880:p300-CBP Transcription Factors