The proximal promoter of the human cathepsin G gene conferring myeloid-specific expression includes C/EBP, c-myb and PU.1 binding sites.
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.
Lennartsson
Andreas
A
Division of Hematology and Transfusion Medicine, Lund University, BMC, C14, S-221 84 Lund, Sweden.
Garwicz
Daniel
D
Lindmark
Anders
A
Gullberg
Urban
U
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D056649:Cathepsin G; D002403:Cathepsins / Q000235:genetics*; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000378:metabolism*; D020862:GC Rich Sequence / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020014:K562 Cells; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D022423:Myeloid Cells / Q000378:metabolism* / Q000473:pathology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D020598:Proto-Oncogene Proteins c-myb / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D012697:Serine Endopeptidases / Q000235:genetics*; D015534:Trans-Activators / Q000378:metabolism; D024363:Transcription Initiation Site; D014162:Transfection; D020298:U937 Cells; D000099232:Proto-Oncogene Protein Spi-1
D022762:CCAAT-Enhancer-Binding Proteins; D056649:Cathepsin G; D002403:Cathepsins; D004268:DNA-Binding Proteins; D008156:Luciferases; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1; C534094:CTSG protein, human; C534096:Ctsg protein, mouse