Molecular cloning and characterization of the human p19 INK4d gene promoter

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Abstract
P19INK4d, 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 p19INK4d gene, we characterized the 5′‐flanking region of the human p19INK4d 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 p19INK4d 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 p19INK4d 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 p19INK4d gene is under the control of TATA‐less promoter and the Sp1 binding site is involved in the transcription.