title: Functional characterization of the proximal promoter of the murine pyruvate carboxylase gene in hepatocytes: role of multiple gc boxes.
abstract: Pyruvate carboxylase (PC) catalyzes the first committed step in gluconeogenesis in the liver. The murine PC gene possesses two promoters, the proximal (P1) and the distal (P2) which mediate production of distinct tissue-specific mRNA isoforms. By comparing the luciferase activities of 5'-nested deletions of the P1-promoter in the AML12 mouse hepatocyte cell line, the critical cis-acting elements required for maintaining basal transcription were located within the 166 nucleotides proximal to the transcription start site. Three GC boxes were identified within this region and shown by gel shift and ChIP assays to bind Sp1/Sp3. Over-expression of Sp1/Sp3 in AML12 and NIH3T3 cells increased P1-promoter activity, with Sp1 being a stronger activator than Sp3. Mutation of any one of the three GC boxes dramatically reduced basal promoter activity by 60-80% suggesting that all three boxes are equally strong regulatory elements. In AML12 cells, over-expression of Sp1/Sp3 restored the transcriptional activity of GC1 and GC2 but not GC3 mutants to levels similar to that of the WT construct, suggesting that GC3 is particularly critical for Sp1/Sp3-mediated induction. In NIH3T3 cells, however, the three boxes were equally important, indicating that the GC boxes differentially contribute to transcriptional regulation of the P1-promoter in the two cell lines. Mutants harboring two disrupted GC boxes showed a further decrease in promoter activity similar to the triple GC box mutant. Neither Sp1 nor Sp3 was able to fully restore the promoter activities of these mutants to that the WT level.
authors: Molecular Metabolism Research Group, Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
authors: Pinnara
authors: P
authors: Rojvirat
authors: Tanit
authors: T
authors: Chavalit
authors: Sureeporn
authors: S
authors: Muangsawat
authors: Ansaya
authors: A
authors: Thonpho
authors: Sarawut
authors: S
authors: Jitrapakdee
mesh_terms: D000818:Animals; D001483:Base Sequence; D047369:Chromatin Immunoprecipitation; D005347:Fibroblasts / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D011766:Pyruvate Carboxylase / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor / Q000378:metabolism
chemical_list: D020033:Protein Isoforms; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D051705:Sp3 Transcription Factor; D011766:Pyruvate Carboxylase