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Regulatory elements responsible for inducible expression of the granulocyte colony-stimulating factor gene in
1Osaka Bioscience Institute, Japan.
Abstract:
Granulocyte colony-stimulating factor (G-CSF) plays an essential role in granulopoiesis during bacterial infection. Macrophages produce G-CSF in response to bacterial endotoxins such as lipopolysaccharide (LPS). To elucidate the mechanism of the induction of G-CSF gene in macrophages or macrophage-monocytes, we have examined regulatory cis elements in the promoter of mouse G-CSF gene. Analyses of linker-scanning and internal deletion mutants of the G-CSF promoter by the chloramphenicol acetyltransferase assay have indicated that at least three regulatory elements are indispensable for the LPS-induced expression of the G-CSF gene in macrophages. When one of the three elements was reiterated and placed upstream of the TATA box of the G-CSF promoter, it mediated inducibility as a tissue-specific and orientation-independent enhancer. Although this element contains a conserved NF-kappa B-like binding site, the gel retardation assay and DNA footprint analysis with nuclear extracts from macrophage cell lines demonstrated that nuclear proteins bind to the DNA sequence downstream of the NF-kappa B-like element, but not to the conserved element itself. The DNA sequence of the binding site was found to have some similarities to the LPS-responsive element which was recently identified in the promoter of the mouse class II major histocompatibility gene.
Insights
Granulocyte colony-stimulating factor (G-CSF) gene expression in macrophages is induced by lipopolysaccharide (LPS). Researchers identified key regulatory elements in the G-CSF promoter essential for this LPS-induced response.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Granulocyte colony-stimulating factor (G-CSF) is crucial for granulopoiesis, particularly during bacterial infections.
- Macrophages produce G-CSF in response to bacterial endotoxins like lipopolysaccharide (LPS).
Purpose of the Study:
- To investigate the regulatory cis-elements controlling the induction of the mouse G-CSF gene in macrophages.
- To elucidate the molecular mechanisms underlying LPS-mediated G-CSF gene expression.
Main Methods:
- Analysis of G-CSF promoter mutants using chloramphenicol acetyltransferase (CAT) assays.
- Gel retardation assays and DNA footprinting with nuclear extracts from macrophage cell lines.
Main Results:
- At least three regulatory elements in the G-CSF promoter are essential for LPS-induced expression.
- A specific element, when reiterated, functions as a tissue-specific, orientation-independent enhancer.
- Nuclear proteins bind downstream of an NF-kappa B-like site, not to the site itself, suggesting a novel binding interaction.
- The identified binding site shares similarities with the LPS-responsive element of the mouse class II major histocompatibility gene.
Conclusions:
- The study identifies critical regulatory elements and binding sites involved in LPS-induced G-CSF gene expression in macrophages.
- These findings contribute to understanding the innate immune response to bacterial endotoxins at the molecular level.