Sp1 and NF-Y synergistically mediate the effect of vitamin D-3 in the p27(Kip1) gene promoter that lacks vitamin D response elements

Vitamin D-3 promotes myeloid leukemic cell lines to differentiate terminall y into monocytes/macrophages. It has been reported that overexpression of t he cdk inhibitor p27(Kip1) results in the differentiation of the myelomonoc ytic U937 cell line and that this gene is the target of vitamin D-3. To ide ntify the sequences required for the positive regulation of p27(Kip1) trans cription by man p27(Kip1) 3.6-kilobase 5'-flanking region of the human was examined by transiently transfecting luciferase reporter constructs into U9 37 cells. The transcriptional activity of this construct was activated by v itamin D-3. Deletion and mutational analysis revealed that both a GGGCGG se quence (-545/-539) and a CCAAT sequence (-525/-520) were necessary to induc e p27(Kip1) gene expression. Importantly, the region containing both of the se elements conferred positive responsiveness to vitamin D-3 to a heterolog ous promoter. Gel shift assays showed that Spl binds to the GGGCGG sequence and that NF-Y binds to the CCAAT sequence. Consistent with the roles of th ese transcription factors, treatment with vitamin DQ Stimulated the DNA bin ding activities of these factors to each element and induced the change of one NF-Y subunit. We conclude that vitamin D-3 stimulates transcription of the p27(Kip1) gene by a novel mechanism involving Spl and NF-Y, but not the vitamin D receptor, during the early stages of U937 cell differentiation.