Fig 2.

HBP1 suppresses DNMT1 promoter activity in a DNA-binding-dependent manner. (A) Schematic diagram of the DNMT1 promoter. Shown is the HBP1 affinity site within the DNMT1 promoter at positions −115 to −134 from the transcriptional start site (Luc-DNMT1 [top]). Luc-ΔDNMT1 is a mutant DNMT1 promoter with a deletion in the HBP1 affinity site (bottom). (B) Schematic diagram of wild-type HBP1 and associated mutants. (C) Expression of exogenous HBP1 decreases DNMT1 protein level. HEK293T cells were transfected with HA-HBP1 or HA-pmHMG. Western blotting was performed on protein lysates using anti-HA antisera, anti-DNMT1, or anti-GAPDH. (D) Relative activities of HBP1 and associated mutant on the native DNMT1 promoter (Luc-DNMT1) or the mutant DNMT1 promoter (Luc-ΔDNMT1). HEK293T cells were cotransfected with 0.1 μg of the indicated reporters and 0.6 μg of HBP1 or mutant expression plasmids. The luciferase activities were expressed as the means ± standard errors of the means from four experiments. Statistical differences were analyzed using the t test; P < 0.01. (E) HBP1 suppresses DNMT1 promoter activity in a dose-dependent manner. HEK293T cells were cotransfected with 0.1 μg of Luc-DNMT1 and different doses (0 to 2.0 μg) of HBP1. The luciferase activities were expressed as the means ± standard errors of the means from four experiments. Statistical differences were analyzed using the t test; P < 0.01. (F) The integrity of affinity site is indispensable for HBP1 suppressing DNMT1 promoter in vivo. Shown is a schematic diagram of the native DNMT1 promoter and its mutant promoter. The mutant promoter of Luc-mDNMT1 contains two point mutations in HBP1 affinity site (TT to GG) in the background of native Luc-DNMT1 promoter (left). The relative activities of HBP1 on the native (Luc-DNMT1) and mutant (Luc-mDNMT1) DNMT1 promoters are shown (right).