Molecular characterization of the cyclin-dependent kinase inhibitor p27 promoter.
p27Kip1 is a member of the family of cyclin-dependent kinase inhibitors (CKIs), which play critical roles in the regulation of cell cycle. To study the transcriptional regulation that controls the expression of p27, we have isolated the p27 promoter, defined its transcription initiation site, and performed various analyses for sequences upstream to 3 kb. Transient transfection assays using fusion reporters containing progressively truncated p27 promoter fragments showed that a region of 170 bp upstream of the start site is sufficient for maximal transcription activity. Detailed sequence analysis of this 170 bp region identified several GC-rich segments, putative sites of the transcription factor Sp1. Footprinting experiments revealed two Sp1-protected boxes, named BoxI and BoxII, which are located at positions -133 to -117 and -87 to -72, respectively. Binding of Sp1 to the two boxes was further demonstrated by gel mobility shift assays and supershift assays. Co-transfection studies in Drosophila Schneider line 2 cells showed that Sp1 indeed activates the p27 promoter constructs that harbor one or both of the GC-rich sequences. Furthermore, the GC-rich sequences could confer Sp1-dependent transactivation to a heterologous prolactin minimal promoter. Mutations in the GC-rich sequences abolished both binding and transactivation by Sp1. Taken together, our data strongly show that the p27 promoter is activated by the ubiquitously expressed transcription factor Sp1, which may provide a molecular mechanism for the constitutive nature of p27 transcription.
Zhang
Y
Y
Regulatory Biology Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, Singapore.
Lin
S C
SC
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins; D002460:Cell Line; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018983:DNA Footprinting; D004330:Drosophila; D004791:Enzyme Inhibitors; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics*; D014162:Transfection; D025521:Tumor Suppressor Proteins
C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004247:DNA; D018844:Cyclin-Dependent Kinases