Involvement of specific proteins (Sp1/Sp3) and nuclear factor Y in basal transcription of the distal promoter of the rat pyruvate carboxylase gene in beta-cells.
Pyruvate carboxylase plays diverse roles in different biosynthetic pathways, including glucose-induced insulin secretion in pancreatic beta-cells. We have localized the control region of the P2 promoter by generating a series of 5'-nested deletion constructs, and both 25- and 9-bp internal deletion constructs, as well as by performing site-directed mutagenesis. Transient transfections of these constructs into INS-1 cells identified a CCAAT box and a GC box that are located at -65/-61 and -48/-41, respectively, as the important determinants. Disruption of the GC box resulted in a 4-fold reduction of the reporter activity, while disruption of the proximal CCAAT box (-65/-61) but not the distal CCAAT box (-95/-91) increased the reporter activity by 3-fold. Simultaneous disruptions of both the GC box and the CCAAT box reduced the reporter activity to a level that was close to that of the single GC box mutation. Electrophoretic mobility shift assays (EMSAs) and supershift EMSAs using nuclear extract from INS-1 cells demonstrated that Sp1 and Sp3 bind a GC box while the nuclear factor Y was shown to bind the proximal but not the distal CCAAT box.
Sunyakumthorn
Piyanate
P
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
Boonsaen
Thirajit
T
Boonsaeng
Vichai
V
Wallace
John C
JC
Jitrapakdee
Sarawut
S
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001710:Biotin / Q000737:chemistry; D023081:CCAAT-Binding Factor / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017930:Genes, Reporter; D005947:Glucose / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D007515:Islets of Langerhans / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011766:Pyruvate Carboxylase / Q000378:metabolism; D051381:Rats; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D023081:CCAAT-Binding Factor; D004268:DNA-Binding Proteins; D007328:Insulin; D016329:Sp1 Transcription Factor; C497123:Sp3 protein, rat; D014157:Transcription Factors; C402244:nuclear factor Y; D051705:Sp3 Transcription Factor; D001710:Biotin; D004247:DNA; D008156:Luciferases; D011766:Pyruvate Carboxylase; D005947:Glucose