Cells were treated for 16 h with either solvent or 100 nM 1,25(OH)2D3. period of 240 min. Combined 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 1,25(OH)2D3-responding genes. INTRODUCTION The biologically most active vitamin D metabolite, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], is essential for mineral homeostasis and skeletal integrity (1), but also has important roles in the control of cell growth and differentiation in normal and malignant tissues (2). 1,25(OH)2D3 levels are tightly controlled by the monooxygenase vitamin D 24-hydroxylase (CYP24), which metabolizes the active hormone. The gene is also the most responsive primary 1,25(OH)2D3 target gene and shows at the mRNA level up to 1000-fold inducibility by the hormone (3). Most other known primary 1,25(OH)2D3 target genes are much less responsive and often show an inducibility of 2-fold or less after short-term treatment with 1,25(OH)2D3 (4,5). One of these genes is (6). Cyclin C belongs to the cyclin protein superfamily, whose members control cell cycle transitions through activation of cyclin-dependent kinases (CDKs). Human and cyclin C proteins share a high degree of homology (72% identity), which suggests an important role for this gene PF-06424439 methanesulfonate product that is reflected in its conservation in diverse animal species (7). Interestingly, the cyclin CCCDK8 complex was found to be associated with the RNA polymerase II (Pol II) basal transcriptional machinery (8), and is considered as a functional part of those mediator protein (MED) complexes that are involved in gene repression (9). This observation suggests a general role for cyclin C in reducing the transcriptional activity of a cell. Another role of cyclin C, in complex with CDK3, seems to be the regulation of the G0 to G1 transition of the cell cycle through specific phosphorylation of the PF-06424439 methanesulfonate retinoblastoma protein pRb (10). Moreover, the fact that the gene, being located in chromosome 6q21, is deleted in a subset of acute lymphoblastic leukemias, suggests its involvement in tumorigenesis (11). The 1,25(OH)2D3 receptor (VDR) is the only nuclear protein that binds 1,25(OH)2D3 with high affinity (gene (23). However, the DR3-type VDRE of the rat gene (24) PF-06424439 methanesulfonate is the only VDR binding site that is presently understood in its promoter context, where chromatin organization and flanking binding sites for other transcription factors, such as Runx2 and YY1, are taken into consideration (25). The major protein constituents of chromatin are histones, and the covalent modifications of lysines at their N-terminal tails neutralize their positive charge and thus their attraction for the negatively charged DNA is diminished (26). This influences the packaging grade of the chromatin and regulates the access of transcription factors to their potential SAPKK3 binding sites. More than 10 specific modifications of histones are known, but the acetylation of the lysine at position 8 of histone 4 correlates strongly with the activation of chromatin on a promoter preceding the initiation of transcription (27). Therefore, in most cases, the histones associated with active regions of promoters have a higher degree of acetylation at certain positions than in repressed or silent regions. To date, most studies on transcriptional regulation have been concentrated on isolated promoter regions or proximal promoters, where binding sites of nuclear receptors and other transcription factors have been localized (28). We have previously identified the human gene as a primary 1,25(OH)2D3 target (6). Since neither VDREs nor chromatin packaging of the promoter of this gene was known, we analyzed, in MCF-7 human breast cancer cells, 8.4 kb of the human promoter by using chromatin immunoprecipitation assay (ChIP) with antibodies against acetylated histone 4 (AcH4), VDR and RXR. Interestingly, 1,25(OH)2D3 treatment did not change the acetylation status of histone 4 on any region of the promoter. In contrast to this finding, up to five promoter regions showed a consistent, 1,25(OH)2D3-dependent association with VDR and RXR over time and in four of these regions re-ChIP assays confirmed the simultaneous association of VDR with RXR, NCoA3, MED1 and Pol II. Furthermore, screening, gel-shift and reporter gene assays identified in each of these four regions a DR3- or DR4-type VDRE. MATERIALS AND METHODS Cell culture MCF-7 and MDA-MB453 human breast cancer cells and LNCaP and PC-3 human prostate cancer cell were grown in phenol red-free DMEM and RPMI, respectively, supplemented with 5% charcoal-treated fetal.