Regulation of the human cyclin C gene via multiple vitamin D3-responsive regions in its promoter

Abstract

The candidate human tumor suppressor gene cyclin C is a primary target of the anti-proliferative hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], but binding sites for the 1alpha,25(OH)2D3 receptor (VDR), so-called 1alpha,25(OH)2D3 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 1alpha,25(OH)2D3 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 1alpha,25(OH)2D3, but four independent promoter regions showed a consistent, 1alpha,25(OH)2D3-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 1alpha,25(OH)2D3-responding genes.

Figure 1

Comparison of cyclin C and CYP24 mRNA expression. Real-time quantitative PCR was used to determine the ratio of the basal levels of cyclin C and CYP24 mRNA relative to the control gene ARP0 in MCF-7 and MDA-MB453 human breast cancer and in LNCaP and PC-3 human prostate cancer cells (A). A logarithmic scale is employed on the y-axis to better present the data. (B) In the same four cell lines, the induction of cyclin C and CYP24 mRNA after 2 h treatment with 10 nM 1α,25(OH)₂D₃ was measured. (C) The time course of cyclin C mRNA expression in response to 10 nM 1α,25(OH)₂D₃ was determined in MCF-7 cells. Data points (A and C) and columns (B) indicate the means of at least three independent cell treatments and the bars represent the standard error of mean. The standard deviations in (A) are too small to be visible in relationship with the data points.

Figure 2

Chromatin activity of the human cyclin C promoter. Chromatin was extracted from MCF-7 cells that had been treated for indicated time periods with 10 nM 1α,25(OH)₂D₃. ChIP assays using an antibody against AcH4 were performed. The histone 4 acetylation states of 23 overlapping regions representing the first 8.4 kb of the human cyclin C promoter was monitored for the five treatment times in comparison with the activity of the proximal promoter of the CYP24 gene (promoter region 1). (A) Representative agarose gels are shown. (B) The basal activity of the promoter regions (time point 0 min) was determined by quantifying the ethidium bromide-stained PCR products on a FLA-3000 reader in relationship with the chromatin input. Columns indicate the means of at least six independent cell treatments and the bars represent standard error of mean.

Figure 3

Ligand-modulated VDR and RXR binding to the human cyclin C promoter. Chromatin was extracted from MCF-7 cells that had been treated for indicated time periods with 10 nM 1α,25(OH)₂D₃. ChIP assays using an antibody against VDR (A) and RXR (B) were performed. The VDR and RXR association of the 23 regions of the human cyclin C promoter was monitored for the six treatment times in comparison with that of the proximal promoter of the CYP24 gene (promoter region 1). Representative agarose gels are shown.

Figure 4

In silico and in vitro screening for VDREs in the human cyclin C promoter. (A) In silico promoter analysis indicated nine putative VDREs (RE1–RE9) found within seven regions of the human cyclin C promoter. The two hexameric half sites of each VDRE core sequence are shown in boldface and their relative orientation and number of spacing nucleotides are indicated. (B) Gel-shift experiments were performed with in vitro translated human VDR and human RXR alone or in combination and in the presence of ³²P-labeled REs. Protein–DNA complexes were resolved from free probe through non-denaturing 8% polyacrylamide gels. Representative gels are shown. The relative amount of VDR–RXR heterodimer complex formation was quantified on a FLA-3000 reader in relationship with the DR3-type VDRE of the proximal promoter of the CYP24 gene. NS indicates non-specific complexes. (C) The order and the results of the screening procedure are schematically depicted in a flow chart.

Figure 5

Functionality of VDREs. Reporter gene assays were performed with extracts from MCF-7 cells that were transiently transfected with a luciferase reporter construct containing two copies of RE2, RE5, RE7, RE8, the DR3-type VDRE of the rat ANF gene and the DR4-type RE of the rat pit-1 gene (A) or 390 bp fragment of the human cyclin C promoter containing RE2 and a comparable fragment of the human CYP24 promoter including the DR3-type VDRE (B) and an expression vector for human VDR. Cells were treated for 16 h with either solvent or 100 nM 1α,25(OH)₂D₃. Relative luciferase activity is shown and fold inductions are indicated above the columns. Columns represent means of at least three experiments and bars indicate standard deviations.

Figure 6

VDR complexes in 1α,25(OH)₂D₃-responsive promoter regions. Chromatin was extracted from MCF-7 cells that had been treated for indicated time periods with 10 nM 1α,25(OH)₂D₃. Re-ChIP experiments were performed with a first immuno-precipitation with anti-VDR antibody and a second precipitation with anti-RXR, anti-NCoA3, anti-MED1 or anti-phosphorylated Pol II antibodies. The association of VDR and its partner proteins was monitored on the human cyclin C promoter regions 6 (A), 11 (B) (negative control), 16 (C), 20 (D), 23 (E) and region 1 of the CYP24 promoter (F) (positive control). Representative agarose gels are shown.