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Molecular cloning and characterization of the human p27Kip1 gene promoter
S Minami1, N Ohtani-Fujita, E Igata
1Department of Preventive Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Japan.
Abstract:
p27Kip1 is an inhibitor of multiple cyclin-dependent kinases (cdk), and can arrest the cell-cycle progression by inhibiting the phosphorylation of the retinoblastoma gene family products. Tumor formation in p27Kip1 knockout mice clearly shows that p27Kip1 plays an important role in inhibiting tumor formation and progression. To investigate the mechanism of transcriptional p27Kip1 gene expression, we isolated the genomic DNA fragment of the 5' flanking region of the human p27Kip1 gene and characterized its promoter region. The human p27Kip1 promoter is TATA-less, and the sequence is highly homologous to the murine p27Kip1 promoter sequence. In the promoter assay, deletion from -774 to -435 relative to the initiating codon resulted in a 15-20-fold reduction of the p27Kip1 promoter activity, suggesting that the elements for basal promoter activity exist in this highly conserved 340 bp region, where putative CTF and ATF sites are conserved.
Insights
The p27Kip1 protein inhibits cell cycle progression and tumor formation. Its gene promoter contains essential elements for activity within a conserved 340 bp region.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- p27Kip1 functions as a cyclin-dependent kinase (CDK) inhibitor, regulating cell cycle progression.
- p27Kip1 plays a critical role in tumor suppression, as evidenced by tumor formation in knockout mice.
Purpose of the Study:
- To investigate the transcriptional regulation of the human p27Kip1 gene.
- To characterize the promoter region of the human p27Kip1 gene.
Main Methods:
- Isolation and characterization of the 5' flanking genomic DNA fragment of the human p27Kip1 gene.
- Promoter assays involving deletion analysis of the 5' flanking region.
Main Results:
- The human p27Kip1 promoter is TATA-less and highly homologous to the murine promoter.
- Deletion of the region from -774 to -435 significantly reduced p27Kip1 promoter activity (15-20 fold).
- This conserved 340 bp region contains putative conserved CTF and ATF binding sites essential for basal promoter activity.
Conclusions:
- The 5' flanking region of the human p27Kip1 gene contains critical elements for its transcriptional regulation.
- A conserved 340 bp region is crucial for basal promoter activity, suggesting its importance in p27Kip1 gene expression.