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. 2010 Jul;121(1-2):136-41.
doi: 10.1016/j.jsbmb.2010.02.011. Epub 2010 Feb 18.

Genome-wide analysis of the VDR/RXR cistrome in osteoblast cells provides new mechanistic insight into the actions of the vitamin D hormone

Mark B Meyer  1 , Paul D Goetsch, J Wesley Pike
Affiliations

Genome-wide analysis of the VDR/RXR cistrome in osteoblast cells provides new mechanistic insight into the actions of the vitamin D hormone

Mark B Meyer et al. J Steroid Biochem Mol Biol. 2010 Jul.

Abstract

The vitamin D receptor (VDR) mediates the actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in target cells and tissues by orchestrating the expression of gene networks responsible for vitamin D-induced phenotypes. The molecular mechanisms of these regulatory systems have been studied for decades under the principle that transcriptional regulation occurs near the transcriptional start site of the gene. However, this now appears to be an outdated view of transcriptional control. In this study, we examined the genome-wide chromatin immunoprecipitation on microarray (ChIP-chip) across pre-osteoblastic cells for VDR, retinoid X receptor (RXR), RNA polymerase II, and histone H4 acetylation (H4ac). We uncovered potential regulatory mechanisms for genes important to osteoblast biology as well as skeletal formation under the control of 1,25(OH)2D3. We found that VDR, along with RXR and H4ac, binds to distal regions 43% of the time; and within gene introns and exons 44%, leaving only 13% of activation at traditional promoter regions. Here, we briefly summarize our findings for all the VDR/RXR cis-acting transcriptional elements (VDR/RXR cistrome) in pre-osteoblastic cells, MC3T3-E1, provide a few examples of this dynamic control by VDR and 1,25(OH)2D3, and demonstrate that distal transcriptional control contributes to the majority of vitamin D3-mediated transcription.

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Figures

Figure 1
Figure 1
Summary of genome-wide ChIP-chip and gene expression studies reveal the VDR/RXR cistrome. A, Schematic venn diagram representations of the data displayed in the table below summarizing the number of peaks found with vehicle treatment (Veh/Input) and with 1,25(OH)2D3 treatment (1,25/Input) as well as Vehicle only, 1,25 only, and an overlap of regions from ChIP-chip data for VDR, RXR, RNA polymerase II (RNA Pol II) and histone H4 acetylation (H4ac). MC3T3-E1 cells treated for 3 hours prior to ChIP with vehicle or 100nM 1,25(OH)2D3. B, Overlapping peaks were tabulated for VDR/RXR/RNA Pol II (left column), VDR/RXR/RNA Pol II/H4ac (center column) and VDR/RXR/H4ac (right column). Peaks were mapped to their surrounding genes and categorized into intragenic (Intron or Exon), 5′near (within 5kb upstream of the 5′ end of gene), 3′near (within 5kb downstream of 3′ end of gene) or Distal (any region not within the gene or within 5kb of the gene at either end). C, the peaks were mapped to their closest surrounding gene and listed as correlated genes from ChIP-chip data. D, gene expression analysis was performed on MC3T3-E1 cells for 24 hours with either vehicle or 100nM 1,25(OH)2D3. These genes were then cross referenced with the genes that were associated with the ChIP-chip peaks and displayed as either up- or down-regulated genes.
Figure 2
Figure 2
ChIP-chip data for genes important for skeletal biology reveal novel enhancers at the promoter as well as distal to the TSS. A, the genomic location for the Spp1 (Opn) gene are shown for chromosome 5 with genomic base pairs given in kilobases (k). ChIP-chip data are listed for each antibody in the basal (Veh/Input) or activated (1,25/Input) state and are displayed as log 2 ratios (log2R). Antibodies used are VDR (blue), RXR (green), RNA Pol II (orange), H4ac (brown). Statistically significant peaks were called and are highlighted in red as described in Materials and Methods. B, the same analysis is shown for the Cyp24a1 gene. Putative regulatory regions discovered are highlighted with their position relative to the transcriptional start site of the gene.
Figure 3
Figure 3
Transcriptional control can occur within intragenic regions at introns and exons. A, the genomic location for the Rarβ gene are shown for chromosome 5 with genomic base pairs given in kilobases (k). ChIP-chip data are listed for each antibody in the basal (Veh/Input) or activated (1,25/Input) state and are displayed as log 2 ratios (log2R). Antibodies used are VDR (blue), RXR (green), RNA Pol II (orange), H4ac (brown). Statistically significant peaks were called and are highlighted in red as described in Materials and Methods. B, the same analysis is shown for the Prcka (Protein Kinase Cα) gene. Putative regulatory regions discovered are highlighted with their position relative to the transcriptional start site of the gene.
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