Sp1 and Sp3 are involved in up-regulation of human deoxyribonuclease II transcription during differentiation of HL-60 cells.
Expression of DNase II in macrophages is potentially crucially important in the removal of unwanted DNA. We have previously shown that DNase II expression is up-regulated at the transcriptional level during the phorbol 12-myristate-13-acetate (PMA)-induced differentiation of HL-60 and THP-1 cells. In this study, we investigated the cis-regulatory elements and transcription factors involved in this process in HL-60 cells. cis-Regulatory elements in the DNase II promoter were located by 5' deletion and site-directed mutagenesis of promoter-luciferase constructs and transient transfection of HL-60 cells. Furthermore, the binding proteins were identified by electrophoretic mobility shift assay (EMSA) in the presence of specific antibodies. In the DNase II promoter, 249 base pairs upstream of the transcription start site were essential for maximal promoter activity in both untreated and PMA-treated HL-60 cells and, within this region, three Sp1 and Sp3 binding sites were identified as essential for transcriptional regulation and PMA induction. Western blot analysis showed that PMA treatment resulted in increased levels of Sp1 and Sp3 proteins. Furthermore, cotransfection analysis in Drosophila SL2 cells showed that Sp1 was more potent than Sp3 in activating the DNase II promoter. We therefore conclude that Sp1 and/or Sp3 are involved in the up-regulation of DNase II expression during the differentiation of HL-60 cells.
Chou
San-Fang
SF
Department of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Chen
Hui-Ling
HL
Lu
Shao-Chun
SC
D001483:Base Sequence; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D016335:Zinc Fingers
D017931:DNA Primers; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D008156:Luciferases; D004706:Endodeoxyribonucleases; C019329:deoxyribonuclease II; D013755:Tetradecanoylphorbol Acetate