Characterization of the promoter region of the human PARG gene and its response to PU.1 during differentiation of HL-60 cells.
The metabolism of poly(ADP-ribose) plays important roles in the nuclear function of mammalian cells. Previously, we analyzed expression of the poly(ADP-ribose) glycohydrolase (PARG) gene during HL-60 cell differentiation and found that expression was greatly reduced by 4 h after 12-O-tetradecanoyl-phorbol-13-acetate (TPA) treatment and returned to the initial level within 20 h. In the present study, a 2.1-kb fragment of the 5'-flanking (promoter) region of the human PARG gene was isolated from the HL-60 genome by polymerase chain reaction and ligated into a luciferase-expression vector, pGL3, to generate the pPARG-Luc#2 reporter plasmid. Deletion analysis revealed that a 75-nt sequence is required for basal promoter activity and TPA responsiveness. Mutations in this 75-nt sequence reduced promoter activity and the TPA response of HL-60 cells. TFSEARCH analysis revealed that Ets family binding motifs are located in the 75-nt sequence. Chromatin immunoprecipitation assay, electrophoretic mobility shift assay and competition analysis indicated that PU.1 (Spi-1) binds to the 75-nt sequence. Moreover, co-transfection of HL-60 cells with a PU.1 expression plasmid and pPARG-Luc indicated that PU.1 down-regulate the PARG promoter. These results suggest that PARG gene expression is modulated by PU.1 during TPA-induced differentiation of HL-60 cells.
Uchiumi
Fumiaki
F
Department of Gene Regulation, Faculty of Pharmaceutical Sciences, Tokyo University of Scie, Noda, Chiba, Japan. uchiumi@rs.noda.tus.ac.jp
Sakakibara
Gakushi
G
Sato
Junko
J
Tanuma
Sei-ichi
S
D024506:5' Flanking Region; D001483:Base Sequence; D002454:Cell Differentiation / Q000187:drug effects; D017353:Gene Deletion; D015972:Gene Expression Regulation, Neoplastic; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics*; D018922:HL-60 Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D000099232:Proto-Oncogene Protein Spi-1
D006026:Glycoside Hydrolases; D011518:Proto-Oncogene Proteins; D013755:Tetradecanoylphorbol Acetate; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1; C022407:poly ADP-ribose glycohydrolase