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.