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.
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