Molecular cloning and characterization of the human p19(INK4d) gene promoter.
p19(INK4d), a member of the INK4 family of cyclin-dependent kinase (CDK) inhibitors, negatively regulates the cyclin D-CDK4/6 complexes, which promote G1/S transition by phosphorylating the retinoblastoma tumor-suppressor gene product. To investigate the mechanism of transcriptional regulation of the p19(INK4d) gene, we characterized the 5'-flanking region of the human p19(INK4d) gene. The cap-site hunting method revealed that the transcription starts at -16 nucleotide (nt) upstream of the initiation codon. The 5'-flanking region of the human p19(INK4d) gene was ligated to a luciferase reporter gene and possessed functional promoter activity. Luciferase assay with a series of truncated 5'-flanking regions indicated that the region from -81 to -2 nt could drive the transcription of the p19(INK4d) gene. Several Sp1 and activating protein 2 binding sites are located within the region from -81 to -2 nt. Mutation of the second Sp1 binding site from -33 to -25 nt decreased the promoter activity. Collectively, it was demonstrated that the human p19(INK4d) gene is under the control of TATA-less promoter and the Sp1 binding site is involved in the transcription.
Matsuzaki
Youichirou
Y
Department of Preventive Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, Japan.
Miyazawa
Kazuhiro
K
Yokota
Tomoya
T
Hitomi
Toshiaki
T
Yamagishi
Hisakazu
H
Sakai
Toshiyuki
T
D024506:5' Flanking Region / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D050764:Cyclin-Dependent Kinase Inhibitor p19; D004268:DNA-Binding Proteins / Q000235:genetics; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D017421:Sequence Analysis; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000235:genetics*; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000235:genetics; D024363:Transcription Initiation Site; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
C493974:CDKN2D protein, human; D018797:Cell Cycle Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050764:Cyclin-Dependent Kinase Inhibitor p19; D004268:DNA-Binding Proteins; D016329:Sp1 Transcription Factor; D050656:Transcription Factor AP-2; D014157:Transcription Factors