A downstream intergenic cluster of regulatory enhancers contributes to the induction of CYP24A1 expression by 1alpha,25-dihydroxyvitamin D3

Abstract

CYP24A1 expression is up-regulated by 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) via a vitamin D receptor (VDR)/retinoid X receptor (RXR) heterodimer that binds to two vitamin D response elements (VDREs) located near the proximal promoter. Interestingly, although 1,25(OH)(2)D(3) induced VDR/RXR binding to the VDRE-containing proximal promoter, the VDR/RXR heterodimer also localized to a cluster of at least four potential enhancers located in intergenic regions 50-69 kb downstream of the human CYP24A1 gene and 35-45 kb downstream of the mouse Cyp24a1 gene as revealed by ChIP-chip and ChIP-seq analyses. To address whether this downstream region and potential VDREs located within mediated CYP24A1 induction, we constructed recombinant wild-type and mutant bacterial artificial chromosome clones that spanned mouse and human loci and contained luciferase reporters inserted into their 3'-untranslated regions. The activity of these clones in stably transfected cells revealed that both the proximal and the putative downstream elements contributed to CYP24A1 up-regulation by 1,25(OH)(2)D(3). Further analysis using transfected enhancer fragments led to the identification of contributing regulatory elements in several of these downstream regions. Additional studies of coregulator recruitment using ChIP-chip analysis revealed both similarities and differences between the region located proximal to and those located downstream of the promoter. Recruitment of these coregulators was likely responsible for the increase in RNA polymerase II and histone H4 acetylation, which was also observed in response to 1,25(OH)(2)D(3) at the enhancer sites across the locus. We conclude that a more complex mechanism is responsible for the striking CYP24A1 up-regulation induced by the vitamin D hormone in target cells.

FIGURE 1.

ChIP-chip and ChIP-seq analysis at the CYP24A1 transcriptional locus. Schematic diagram of the human CYP24A1 locus displayed with nucleotide bases indicated in megabases (Mb) on chromosome 20 (hg18). Genes displayed are CYP24A1 (reverse strand) and PFDN4 (forward strand). Exons are indicated in black. Human LS180 cells were treated with 10⁻⁷ m 1,25(OH)₂D₃ (1,25) or ethanol vehicle (Veh) for 3 h prior to ChIP assay. These samples were further analyzed by microarrays (ChIP-chip), and the data are displayed as the log₂ ratios of the fluorescence obtained following cohybridization of DNA precipitated with either VDR (A) or RXR (B) antibodies in both the vehicle versus input (Veh/Input) and 1,25(OH)₂D₃ versus input (1,25/Input) conditions. Statistically significant peaks were called from the enriched regions of ChIP-chip data using algorithms described under “Experimental Procedures” (false discovery rate, FDR < 0.05) and are indicated in red. The transcriptional start site (TSS) is indicated by a gray box. Novel regulatory regions at +41, +50, +60, +66, and +69 kb downstream of the TSS are indicated in blue. C, ChIP-seq was performed with the same VDR ChIP samples as in A, and the data are displayed as tag density (tag den). All regions shown are statistically significant above threshold and are enriched for VDR binding in both vehicle- and 1,25(OH)₂D₃-treated conditions. The data are representative of at least three similar ChIP-chip analyses and verified through direct qPCR-based ChIP analyses.

FIGURE 2.

BAC clone reporter analysis links transcriptional activity of distal segments to CYP24A1 gene. A, schematic diagrams of BAC clone reporter vectors created using recombineering technologies around the CYP24A1 gene locus. Each BAC clone encompasses 190 kb of genomic sequence, including the entire CYP24A1 gene as well as 43 kb upstream (past PFDN4 TSS) and 128 kb downstream (into BCAS1). A series of constructs were created to accompany the wild-type (wt) construct that contains a luciferase reporter gene along with a mammalian selectable marker (IRES cassette) inserted into the 3′-UTR of the CYP24A1 gene. Known promoter VDREs were mutated (Pro ΔVDREs), the +50-kb region alone was deleted (Δ+50kb), the +66-kb region alone was deleted (Δ+66kb), and all downstream regions with VDR activity were deleted (Δ+71–37kb) as well as a final double deletion of the promoter and downstream elements (ΔVDREs Δ+71–37kb). B, these constructs (A) were stably integrated into MC3T3-E1 cells and assayed for their luciferase activities following treatment with ethanol vehicle (V) or increasing concentrations of 1,25(OH)₂D₃ (10⁻¹⁰ to 10⁻⁸ m). Each point represents the relative light unit (RLU) average normalized to total protein ± S.E. for a triplicate set of assays. *, p < 0.05 as compared with vehicle treatment. The data are representative of at least three independent collections of stable cells that were evaluated for each construct.

FIGURE 3.

Distal VDR transcriptional regulation is positionally conserved in mouse Cyp24a1. A, mouse MC3T3-E1 cells were treated with ethanol vehicle (Veh) or 10⁻⁷ m 1,25(OH)₂D₃ for 3 h prior to ChIP assay. The ChIP DNA was further analyzed by ChIP-chip analysis and displayed as log₂ ratios of fluorescence obtained from cohybridization of DNA from VDR precipitated in the vehicle versus input (Veh/Input) and 1,25(OH)₂D₃ versus input (1,25/Input) conditions. Gene landmarks and data evaluation are displayed as in Fig. 2. An infrequently used 5′ exon of the Pfdn4 gene overlaps the 5′-end of the mouse Cyp24a1 gene. The data are representative of at least three similar ChIP-chip analyses and verified through direct qPCR-based ChIP analyses. B, the Cyp24a1 locus from the mouse genome (mm8) was compared with that of the human genome (hg18) via VISTA analysis. The plot displays sequence identity greater than 50% to 100% identity on the y axis. The average sequence identity throughout the entire locus was calculated to be 70%. Sequence identity between the +37-kb region of the mouse gene and +66-kb region of the human gene is indicated. C, BAC reporter clones for the mouse Cyp24a1 gene. The luciferase reporter cassette (IRES) with mammalian selection marker was inserted into the 3′-UTR of the Cyp24a1 gene in a BAC clone containing 138 kb of genomic sequence. Additional constructs as indicated contained mutations in the two known VDREs at the promoter (Pro ΔVDREs), and deletion of regions +35 to +39 (Δ+39–35) or a combination of both (ΔVDREsΔ+39–35). D, constructs were stably integrated into MC3T3-E1 cells and assayed for their luciferase activities following treatment with ethanol vehicle (V) or increasing concentrations of 1,25(OH)₂D₃ (10⁻¹⁰ to 10⁻⁸ m). Each point represents the RLU average normalized to total protein ± S.E. for a triplicate set of assays. *, p < 0.05 as compared with vehicle treatment. The data are representative of at least three independent collections of stable cells that were evaluated for each construct.

FIGURE 4.

3C assay places the distal binding sites proximal to the CYP24A1 TSS. A, schematic of the CYP24A1 genomic locus. Regions of VDR binding (TSS, +50, +60, +66, and +69) as well as a control region are indicated. BglII restriction sites within the locus are depicted by vertical red bars. DNA fragments generated by BglII digestion and examined are indicated by horizontal red bars. The location of the primers used are indicated by carets. A region utilized as a control (Con) is also depicted. B, LS180 cells were treated for 0, 0.5, 1, or 3 h with 10⁻⁷ m 1,25(OH)₂D₃ and subjected to 3C analysis using BglII as indicated under “Experimental Procedures.” PCR was performed on cross-linked and restricted DNA using the specific TSS primer shown and each individual primer indicated to the left of the PCR data. The positive control primer for ligation dependence is designated Adj.TSS. A BAC control (“B”) was included to show the proper PCR product size. W, water negative control for PCR. The BAC was digested and ligated to form all possible combinations. Negative controls include Con and primers at +104, −14, and −35 kb (not depicted in schematic in A (off-scale)). C, schematic interaction model of +50 through +69 kb distal elements in proximity to the CYP24A1 TSS.

FIGURE 5.

Traditional enhancer-reporter plasmid chimeras identify active VDREs in two human CYP24A1 distal enhancers. A, DNA fragments (0.4–1.5 kb) containing either the CYP24A1 promoter region (Pro) or those that span each of the downstream CYP24A1 regulatory regions (+69, +66, +60, +50, +41(a), and +41(b) kb) were cloned into the pTK-luciferase (tkluc) reporter vector and were evaluated for transcriptional activity in LS180 cells following treatment for 16 h with either ethanol vehicle (V) or 1,25(OH)₂D₃ (10⁻⁹ to 10⁻⁷ m). Each point represents the RLU average normalized to β-gal ± S.E. for a triplicate set of assays. *, p < 0.05 as compared with vehicle treatment. These results are representative of at least three similar experiments. B, analysis of the transcriptional activity of mouse Cyp24a1 distal regions (+45, +42, +39, +35(b), +35(a), and +14 kb). DNA fragments were cloned and examined following transfection into mouse MC3T3-E1 cells as in A. Each point represents the RLU average normalized to β-galactosidase ± S.E. for a triplicate set of assays. *, p < 0.05 as compared with vehicle treatment. C, mutation reveals the identity of functional VDREs in the +66- and +50-kb regions of the human CYP24A1 gene. Triplet mutations were introduced into the half-sites of potential VDREs identified in silico in the context of the +66- and +50-kb fragments as indicated (upper panel). Wild-type (wt) and mutant (mut) constructs were examined for inducibility by 1,25(OH)₂D₃ following transfection into host LS180 cells. Each point represents the RLU average normalized to β-galactosidase ± S.E. for a triplicate set of assays. *, p < 0.05 as compared with vehicle treatment. The data are representative of at least three similar analyses.

FIGURE 6.

Coregulatory molecules are present and/or recruited to both the promoter and the downstream regions of CYP24A1. LS180 cells were treated with ethanol vehicle (Veh) or 10⁻⁷ m 1,25(OH)₂D₃ (1,25) for 3 h and subjected to ChIP assay using antibodies to SRC-1, MED1, NCoR, and SMRT. Samples were further analyzed by microarray (ChIP-chip), and the data are displayed as the log₂ ratios of the fluorescence obtained following cohybridization of DNA from samples immunoprecipitated under both vehicle versus input (Veh/Input) and 1,25(OH)₂D₃ versus input (1,25/Input) conditions. Relevant regions are shown. The data are depicted and analyzed as in Fig. 2. These data are representative of at least three similar ChIP-chip analyses and verified through direct qPCR-based ChIP analyses.

FIGURE 7.

The CYP24A1 gene locus displays a broad histone H4ac and RNA pol II recruitment patterns. LS180 cells were treated with ethanol vehicle or 10⁻⁷ m 1,25(OH)₂D₃ for 3 h and then subjected to ChIP-chip analysis using antibodies to tetra-acetylated histone H4 and RNA pol II. Samples were further analyzed by microarrays (ChIP-chip), and the data are displayed as the log₂ ratios of fluorescence obtained following cohybridization of samples under both vehicle versus input (Veh/Input) and 1,25(OH)₂D₃ versus input (1,25/Input) conditions. Gene landmarks and data evaluation are displayed as in Fig. 2. These data are representative of at least three similar ChIP-chip analyses and verified through direct qPCR-based ChIP analyses.