Regulation of the human cyclin C gene via multiple vitamin D-3-responsive regions in its promoter

The candidate human tumor suppressor gene cyclin C is a primary target of the anti-proliferative hormone 1 alpha,25-dihydroxyvitamin D-3 [1 alpha,25(OH)(2)D-3], but binding sites for the 1 alpha,25(OH)(2)D-3 receptor (VDR), so-called 1 alpha,25(OH)(2)D-3 response elements (VDREs), have not yet been identified in the promoter of this gene. We screened various cancer cell lines by quantitative PCR and found that the 1 alpha,25(OH)(2)D-3 inducibility of cyclin C mRNA expression, in relationship with the 24-hydroxylase (CYP24) gene, was best in MCF-7 human breast cancer cells. To characterize the molecular mechanisms, we analyzed 8.4 kb of the cyclin C promoter by using chromatin immunoprecipitation assays (ChIP) with antibodies against acetylated histone 4, VDR and its partner receptor, retinoid X receptor (RXR). The histone 4 acetylation status of all 23 investigated regions of the cyclin C promoter did not change significantly in response to 1 alpha,25(OH)(2)D-3, but four independent promoter regions showed a consistent, 1 alpha,25(OH)(2)D-3-dependent association with VDR and RXR over a time period of 240 min. Combined in silico/in vitro screening identified in each of these promoter regions a VDRE and reporter gene assays confirmed their functionality. Moreover, re-ChIP assays monitored simultaneous association of VDR with RXR, coactivator, mediator and RNA polymerase II proteins on these regions. Since cyclin C protein is associated with those mediator complexes that display transcriptional repressive properties, this study contributes to the understanding of the downregulation of a number of secondary 1 alpha,25(OH)(2)D-3-responding genes.