Two short sequences have positive effects on the human p27Kip1 gene transcription

Abstract The cyclin-dependent kinase (Cdk) inhibitor p27 Kip1 plays an important role in the progression from G1 to S phase in the cell cycle. To study the activities of its promoter and other regulatory elements, we have cloned and characterized the 5′-flanking region of the human p27 Kip1 gene. This region, about 3 kb in length, is GC-rich and shares homology with that of the mouse p27 Kip1 gene. Transcription start points (tsp) determined by the oligo-capping method are mapped in two regions, the cluster I (−479 to −403) and cluster II (−280 to −273). The cluster I was the primary functional site in transcription initiation. The luciferase activities of serial deletion mutants indicated that two short sequences (−581 to −557 and −556 to −526) had positive effects on transcription. The gel shift assay showed that factors in HeLa nuclear extract bound to these sequences. Sp1 was the major binding factor to the sequence of −556 to −526, wheres yet unidentified positive factors bound to the sequence of −581 to −557.