title: Hepatocyte nuclear factor 4α regulates the expression of the murine pyruvate carboxylase gene through the HNF4-specific binding motif in its proximal promoter.
abstract: Pyruvate carboxylase (PC) is the first regulatory enzyme of gluconeogenesis. Here we report that the proximal promoter of the murine PC gene contains three binding sites for hepatocyte nuclear factor 4α (HNF4α). These sites include the classical direct repeat 1 (DR1) (-386/-374), non-perfect DR1 (-118/-106) and HNF4α-specific binding motif (H4-SBM) (-26/-14). Under basal conditions, mutation of the non-perfect DR1 decreased promoter activity by 50%, whereas mutation of neither the DR1 nor the H4-SBM had any effect. In marked contrast, only mutation of the H4-SBM decreased HNF4α-transactivation of the promoter activity by 65%. EMSA revealed that HNF4α binds to the DR1site and H4-SBM with similar affinity while it binds poorly to the non-perfect DR1. Interestingly, this non-perfect DR1 also coincides with two E-boxes. Mutation of the non-perfect DR1 together with the nearby E-box reduced USF1- but not USF2-transactivation of promoter activity, suggesting that USF1 partly contributes to the basal activity of the promoter. Substitution of the H4-SBM with the DR1 marginally reduced the basal promoter activity but did not eliminate HNF4α-transactivation, suggesting that HNF4α can exert its effect via DR1 within this promoter context. ChIP-assay confirmed that HNF4α is associated with the H4-SBM. Suppression of HNF4α expression in AML12 cells down-regulated PC mRNA and PC protein by 60% and 50%, respectively, confirming that PC is a target of HNF4α. We also propose a model for differential regulation of P1 promoter of PC gene in adipose tissue and liver.
authors.affiliation: Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors.forename: Tanit
authors.initials: T
authors.lastname: Chavalit
authors.forename: Pinnara
authors.initials: P
authors.lastname: Rojvirat
authors.forename: Sureeporn
authors.initials: S
authors.lastname: Muangsawat
authors.forename: Sarawut
authors.initials: S
authors.lastname: Jitrapakdee
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic; D051557:Hepatocyte Nuclear Factor 4 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011766:Pyruvate Carboxylase / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051720:Upstream Stimulatory Factors / Q000235:genetics*
chemical_list: C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051720:Upstream Stimulatory Factors; D011766:Pyruvate Carboxylase
keywords: Biotin-containing enzyme; ChIP; DR1; EMSA; FBPase; G6Pase; Gluconeogenesis; HIP1; HNF4α; Hepatocyte nuclear factor 4alpha; MODY; NR; P1; P2; PC; PEPCK; PPARγ; PPRE; Pyruvate carboxylase; Transcription; USF; Upstream stimulatory factor; chromatin immunoprecipitation; direct repeat 1; distal promoter; electrophoretic mobility shift assay; fructose-1,6-bisphosphatase; glucose-6-phosphatase; hepatocyte nuclear factor 4 alpha; housekeeping initiator protein 1; maturity onset diabetes of the young; nuclear receptor; peroxisome proliferator activated receptor gamma; peroxisome proliferator activated receptor-responsive element; phosphoenolpyruvate carboxykinase; proximal promoter; pyruvate carboxylase; upstream stimulatory factor