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The proximal interferon-stimulated response elements are essential for interferon responsiveness: a promoter analysis
T Ronni1, S Matikainen, A Lehtonen
1Department of Virology, National Public Health Institute, Helsinki, Finland.
Abstract:
Interferon (IFN)-inducible human MxA protein mediates resistance against influenza and several other RNA viruses. The MxA gene is under the control of type I IFN and, in certain cell types, is also directly activated by viruses. Here we show that in human macrophages, MxA mRNA levels are upregulated by very low doses of IFN-alpha in a dose-dependent manner. A similar, albeit much weaker, dose-dependent induction was seen with IFN-gamma. The induction was rapid and independent of protein synthesis. Interleukin-6 (IL-6) or tumor necrosis factor-alpha (TNF-alpha) did not influence MxA mRNA levels alone or in combination with IFNs, in spite of the presence of putative response elements of these cytokines in the MxA promoter. We show that the promoter of the MxA gene contains two functional IFN-stimulated response elements (ISRE) near the transcription start site and one homologous ISRE-like element, which is apparently nonfunctional, further upstream. The two proximal ISRE sites are essential for IFN-alpha-induced transcription and appear to be binding sites for IFN-stimulated gene factor 3 complex. In addition, EMSA and DNAse I footprinting analysis demonstrated that Spl binds with high affinity to a region encompassing nucleotides -25 and -50 and, thus, may provide means of interaction with the basal transcriptional machinery.
Insights
Human MxA protein confers resistance to RNA viruses. Its gene expression in macrophages is rapidly induced by low doses of Interferon-alpha (IFN-alpha), independent of protein synthesis, highlighting key regulatory elements.
Area of Science:
- Immunology
- Molecular Biology
- Virology
Background:
- The human MxA protein is a key mediator of cellular resistance against various RNA viruses, including influenza.
- MxA gene expression is typically regulated by type I interferons (IFNs) and can be directly induced by viral infection in certain cell types.
Purpose of the Study:
- To investigate the regulation of MxA gene expression in human macrophages.
- To identify the specific regulatory elements and signaling pathways involved in MxA induction by interferons.
Main Methods:
- Quantitative analysis of MxA mRNA levels in human macrophages following stimulation with different doses of IFN-alpha and IFN-gamma.
- Assessment of the role of Interleukin-6 (IL-6) and Tumor Necrosis Factor-alpha (TNF-alpha) in MxA induction.
- Analysis of the MxA promoter region using techniques such as Electrophoretic Mobility Shift Assay (EMSA) and DNase I footprinting to identify transcription factor binding sites.
Main Results:
- MxA mRNA levels in human macrophages are significantly upregulated by very low doses of IFN-alpha in a dose-dependent manner.
- IFN-gamma also induced MxA expression, but to a much weaker extent.
- The induction of MxA by IFNs was rapid and did not require de novo protein synthesis.
- IL-6 and TNF-alpha did not affect MxA mRNA levels, even in combination with IFNs.
- The MxA promoter contains two functional IFN-stimulated response elements (ISRE) crucial for IFN-alpha-induced transcription, which bind the IFN-stimulated gene factor 3 complex.
- The transcription factor Spl binds to a specific region of the MxA promoter, potentially interacting with the basal transcriptional machinery.
Conclusions:
- The study elucidates the intricate transcriptional regulation of the MxA gene in human macrophages, emphasizing the critical role of IFN-alpha.
- The findings identify specific ISRE sites and the transcription factor Spl as key players in mediating MxA induction, providing insights into antiviral defense mechanisms.