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The human inducible NOS (iNOS) promoter transcriptionally regulated by 5′ flanking region extending 16 kb upstream that contains cytokine-responsive DNA motifs. In this study, we further identified a classic inducible enhancer located between --5 and --6 kb in the hiNOS upstream promoter. This 1 kb promoter sequence functions as a cytokine-inducible enhancer in an orientation- and position-independent manner in human lung A549 and liver AKN1 cells. This DNA enhancer also confers cytokine inducibility to the heterologous thymidine kinase (TK) promoter. Chromatin immunoprecipitation (ChIP) analysis was applied, and confirmed cytokine-inducible in vivo DNA-protein interactions within this enhancer region. In vivo functional binding of both NF-κB (p65/p50) and Stat-lα at the --5.8 kb human iNOS promoter site was significantly increased in A549 cells after cytokine stimulation, while only Stat-1α bound at the --5.2 kb site. These results identify the --5 to --6 kb promoter region as a classic transcriptional enhancer for the human iNOS gene and provide definitive in vivo evidence of specific NF-κB and Stat-1 nuclear protein binding that mediates transcription of the hiNOS gene under cytokine stimulation. [ABSTRACT FROM AUTHOR]

Served as one of general transcription coactivators, p300 plays an important role in mediating gene transcriptional activation. Our previous work demonstrated that human iNOS expression can be highly induced with the cytokine mixture (CM) of TNFα + IL-1β + IFNγ. In this study, we explored the potential function of p300 in the regulation of human iNOS gene expression. Our initial results showed that overexpression of p300 by transient transfection in human A549 lung cells significantly increased the basal and cytokine induced human iNOS promoter activities. Interestingly, p300 activated cytokine induced human iNOS transcription which was completely abrogated by the deletion of an enhancer region between -5 kb and -6 kb in the hiNOS upstream promoter. However, the enhanced basal promoter activity by p300 overexpression still remained high even with the enhancer deletion. Our data suggest that coactivator p300 is capable of up-regulating human iNOS transcription. The transcription machinery responsible for p300 interaction with necessary transcription factors is being further investigated. [ABSTRACT FROM AUTHOR]

Transcriptional regulation of hiNOS gene is highly complex and tightly controlled. It requires an orchestrated flow of positive and negative transcription factors that bind to specific cis-acting upstream response elements. The hiNOS promoter is known to be one of the largest promoters with inducible elements extending up to -16 kb. Oct-1 protein belongs to the POU domain transcription factor family and is constitutively expressed in all dividing cells. Therefore, it's essential for proliferation, differentiation and other key cell processes. Oct-1 binding to DNA may either stimulate or inhibit transcription of the target gene. In this work, the octamer sequence ATGCAAAT at -10.2 kb of the hiNOS promoter was identified as high-affinity Oct-1 binding by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (CHIP) assay. Cytokine-induced hiNOS promoter activity was significantly reduced by Oct-1 siRNA targeting. Mutation of the Oct-1 motif decreased cytokine-induced hiNOS promoter activity by 40 % and Oct-1 silencing also reduced the promoter activity of mutant hiNOS at -10.2 Oct-1. Taken together, we found a functional Oct-1 motif regulating hiNOS gene expression at -10.2 kb in the hiNOS promoter. [ABSTRACT FROM AUTHOR]