title: A far-upstream Oct-1 motif regulates cytokine-induced transcription of the human inducible nitric oxide synthase gene.
abstract: Transcriptional regulation of the human inducible nitric oxide synthase (hiNOS) gene is highly complex and requires an orchestrated flow of positive and negative transcription factors that bind to specific cis-acting upstream response elements. Very little specific information exists about the far-upstream region of the hiNOS gene. Oct-1 protein belongs to the Pit-Oct-Unc domain transcription factor family and is constitutively expressed in all dividing cells. It is essential for proliferation, differentiation, and other key cell processes. However, the role of Oct-1 in regulating hiNOS gene expression has not been reported. In this work, the octamer sequence 5'-ATGCAAAT-3' at -10.2 kb in the hiNOS promoter was identified as high-affinity Oct-1 binding by electrophoretic mobility shift assay in vitro and chromatin immunoprecipitation assay in vivo. Mutation of Oct-1 motif at -10.2 kb in the hiNOS promoter decreased cytokine-induced hiNOS promoter activity by 40%. Cytokine-induced hiNOS promoter activity was also significantly reduced by Oct-1 small interfering RNA targeting. Overexpression of Oct-1 increased cytokine-induced hiNOS protein expression in primary human hepatocytes. Furthermore, the Oct-1 motif at -10.2 kb of the hiNOS promoter conferred increased transcriptional activity to the heterologous thymidine kinase promoter irrespective of cytokine induction. Taken together, this work identifies a far-upstream functional Oct-1 enhancer motif at -10.2 kb in the hiNOS promoter that regulates cytokine-induced hiNOS gene transcription and further underscores tight control mechanisms regulating the expression of the hiNOS gene.
authors.affiliation: Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors.forename: Kyung Soo
authors.initials: KS
authors.lastname: Park
authors.forename: Zhong
authors.initials: Z
authors.lastname: Guo
authors.forename: Lifang
authors.initials: L
authors.lastname: Shao
authors.forename: Qiang
authors.initials: Q
authors.lastname: Du
authors.forename: David A
authors.initials: DA
authors.lastname: Geller
mesh_terms: D020816:Amino Acid Motifs; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D016207:Cytokines / Q000494:pharmacology*; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D009154:Mutation; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis / Q000235:genetics*; D050811:Octamer Transcription Factor-1 / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics; D015533:Transcriptional Activation
chemical_list: D016207:Cytokines; D050811:Octamer Transcription Factor-1; D034741:RNA, Small Interfering; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; D013937:Thymidine Kinase