Fig 1: Binding sites of HNF3ß in the CPS1 promoter. (A), Nucleotide sequence of -70 nt to +73 nt region of CPS1 promoter is shown. The translation initiation site (+1) is indicated by the arrow. The putative HNF3ß binding sites in the nucleotide region -38 nt~-27 nt (site 1) and -21 nt~-10 nt (site 2) are underlined. In the binding site 1, TT is mutation into AC, named mut1. Similarly, TA is mutation into AG named mut2 in the binding site 2. (B), Effect of mutation of the HNF3ß binding sites on the activity of the CPS1 promoter. HNF3ß binding sites were subjected to site-directed mutagenesis. A total of 1 µg of mutants together with 100 ng of pRL-TK was cotransfected into HepG2 cells. pGL4Basic served as the negative control. Luciferase activities were measured 48 hrs after transfection. The relative luciferase units (RLU) were obtained (right) by comparison with the wild-type of pGL4-70, which was set to 1. (C), Western blot analysis of CPS1 and HNF3ß expression in the various cell lines; 30 µg of cellular proteins was used in the Western blot. ß-Actin serves as an internal control. (D), EMSA of HNF3ß. Competition assays with unlabelled cold probe at a concentration of 50 (lane 3)- or 100 (lane 5)-fold molar excess over that of the biotin-labelled probe (lane 2). In the lane 6, supershift assays were carried out with 4 µg specific antibody raised against HNF3ß and the quality of poly (dI-dC) is 0.8 µg. An arrowhead indicates the shift band (SB, left) and the supershift band (SS, right). (E), ChIP assay. Chromatin from HepG2 cells was immunoprecipitated with the anti-HNF3ß. The total extracted DNA (Input DNA, 5%) prior to immunoprecipitation and the immunoprecipitated samples were PCR-amplified using primers specific to a region that spanned -70 nt to +73 nt (containing the HNF3ß binding sites) of the CPS1 promoter. The normal rabbit IgG or no antibody control was also performed for control purpose. Band signals were quantified using the densitometric software, and the relative intensities to the input which was set to 1.00 were calculated. ND: None detected (*P < 0.05).
Fig 2: Transcription regulation of CPS1 promoter by HNF3ß. (A), Overexpression of HNF3ß enhances CPS1 promoter activities. HepG2 and BEL-7404 cells were cotransfected with 0.2 µg of pGL4-70 and increasing amounts (0, 0.5, 1, 1.5 or 2.0 µg) of expression vectors (pFLAG-HNF3ß or empty vector pFLAG-CMV-2); 10 ng of pRL-TK was used to normalize the transfection efficiency. Cells were harvested 48 hrs after transfection. The relative luciferase units (RLU) were obtained by comparison with the pFLAG-CMV-2, which was set to 1. Each transfection was performed in duplicate, and the data were expressed as the mean ± SD of three separate experiments. (B), Knockdown of endogenous HNF3ß decreased CPS1 promoter activity. HepG2 and BEL-7404 cells were cotransfected with HNF3ß siRNA and 0.2 µg of pGL4-70. At 48 hrs after transfection, the relative luciferase units (RLU) were obtained by comparison with the negative control (NC), which was set to 1. Each transfection was performed in duplicate, and the data were expressed as the mean ± SD of three separate experiments. *P < 0.05 versus NC. (C) and (D), Western blot analysis in BEL-7404 cells after overexpression of HNF3ß or interference of HNF3ß siRNA. (C): BEL-7404 cells were transfected with 0.5 µg of pFLAG-HNF3ß or pFLAG-CMV-2 (empty vector); (D): BEL-7404 cells were treated with 100 pmol of HNF3ß siRNA or negative control (NC). Cells were harvested 48 hrs after transfection; 30 µg of cellular proteins was used in the Western blot and ß-actin served as a loading control. (E) and (F), Influences of HNF3ß or HNF3ß siRNA on CPS1 gene mRNA level in HepG2 cells. Overexpression of HNF3ß increased CPS1 gene transcription, HepG2 cells were transfected with 0.5 µg of pFLAG-HNF3ß or empty control pFLAG-CMV-2 (E); knockdown of endogenous HNF3ß decreased CPS1 gene transcription, HepG2 cells were treated with 100 pmol of HNF3ß siRNA or negative control (NC) (F). Cells were harvested 48 hrs after transfection, 3 µg of the total RNA was used to detect the CPS1 mRNA level by real-time RT-PCR (*P < 0.05).
Fig 3: The regulatory effect of HNF3ß on CPS1 promoter transcription. (A) and (B), The plasmid pGL4-2086 (1 µg) was transfected transiently with LETFs (HNF1a, HNF3ß, HNF4a, HNF6, C/EBPa and C/EBPß) (500 ng), respectively, into the HepG2 and BEL-7404 cells. Luciferase activities were measured 48 hrs after transfection, and the plasmid pGL4-2086 served as the negative control. (C), Activity analysis of CPS1 promoter. BEL-7404 cells were transfected with 1 µg each of the CPS1 promoter constructs (reporter plasmid); 100 ng of the renilla luciferase expression vector pRL-TK was used for normalization, and the promoterless vector pGL4Basic served as the negative control. Luciferase activities were measured 48 hrs after transfection. (D), 1 µg each of the CPS1 promoter constructs was cotransfected with 500 ng HNF3ß, respectively, into the BEL-7404 cells. In each recombinant plasmid, fluorescent activity of non transfected with HNF3ß served as the negative control. Each transfection was performed in duplicate, and the data were expressed as the mean ± SD of three separate experiments (*P < 0.05).
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