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Gene. 2005 Aug 15;356:193-202.

The proximal promoter of the human cathepsin G gene conferring myeloid-specific expression includes C/EBP, c-myb and PU.1 binding sites.

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  • 1Division of Hematology and Transfusion Medicine, Lund University, BMC, C14, S-221 84 Lund, Sweden.

Abstract

Cathepsin G is a hematopoietic serine protease stored in the azurophil granules of neutrophil granulocytes. The mRNA of cathepsin G is transiently expressed during the promyelocyte stage of neutrophil maturation. The protease plays several roles in inflammatory actions of neutrophils, such as bactericidal effects. A human cathepsin G gene fragment of 6 kb directs a promyelocyte-specific expression in transgenic mice, indicating the presence of necessary cis-acting elements. However, neither the precise architecture of the promoter, nor the trans-acting factors responsible for its activation, have been characterized. In the present work, 2.6 kb upstream of the translation start site of the human cathepsin G gene was cloned. When transfected to monoblast-like U937 or to acute promyelocytic leukemia NB4 cells, both expressing endogenous cathepsin G, the initial 360 bp upstream of the translation start were sufficient to direct a strong expression of a luciferase reporter gene. No expression was observed in erythroid K562 control cells. Further deletions revealed three major regulatory regions containing the consensus binding-sites for the transcription factors C/EBP, c-myb and PU.1. Moreover, a GC-rich region, similar to a cis-element in the proteinase 3 promoter, was identified. Direct binding of the trans-factors C/EBPalpha, C/EBPepsilon, c-myb and PU.1 to the promoter was shown by chromatin immunoprecipitation. The functional significance of the cis-elements was verified by site-directed mutagenesis. Mutations of the putative PU.1 site moderately decreased the activity of the promoter in monoblastic U937 cells, but not in promyelocytic NB4 cells. Separate mutations of the putative C/EBP binding site, c-myb-binding site or the GC-rich element resulted in a dramatically reduced transcriptional activity in both cell lines, suggesting cooperation between corresponding trans-factors.

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