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 (-5.3kb) 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.
Highlights
The expression of inducible nitric oxide synthase can be activated by immunologic and inflammatory stimuli such as cytokines or lipopolysaccharide (LPS) in different types of cells and tissues [1], [2]
We observed that human inducible nitric oxide synthase (hiNOS) gene was strongly induced with the cytokine mixture (CM) of TNF-α, IL-1β and IFN-γ
When the enhancer region at -5 kb to -6 kb was deleted from the hiNOS promoter (Pr 8–Del), all CM-induced activity was lost, and the ability of p300 to super-induce hiNOS promoter activity was abrogated (Fig 2A). These findings indicate that the cytokine-induced enhancer region at -5 kb to -6 kb is required for p300-mediated increase in CM-induced hiNOS promoter activity
Summary
The expression of inducible nitric oxide synthase (iNOS) can be activated by immunologic and inflammatory stimuli such as cytokines or lipopolysaccharide (LPS) in different types of cells and tissues [1], [2]. The expression of human inducible nitric oxide synthase (hiNOS) gene in primary human hepatocytes was originally identified by stimulating with a cytokine mixture (CM) of TNF-α, IL-1β, IFN-γ, and LPS [3]. The human iNOS gene was cloned from LPS and CM-stimulated primary human hepatocytes [4]. The molecular regulation of the human iNOS gene is complex and is mostly regulated at transcriptional level. PLOS ONE | DOI:10.1371/journal.pone.0146640 January 11, 2016
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
