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Mechanisms of Enhancer-mediated Hormonal Control of Vitamin D Receptor Gene Expression in Target Cells*

  • Seong Min Lee
    Affiliations
    Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706
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  • Mark B. Meyer
    Affiliations
    Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706
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  • Nancy A. Benkusky
    Affiliations
    Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706
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  • Charles A. O'Brien
    Affiliations
    the Department of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
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  • J.Wesley Pike
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry, University of Wisconsin-Madison, Hector F. Deluca Laboratories, Rm. 543D, 433 Babcock Dr., Madison, WI 53706. Tel.: 608-262-8229; Fax: 608-263-9609
    Affiliations
    Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706
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  • Author Footnotes
    * This work was supported by the National Institutes of Health Grant DK-45173 from NIDDK (to J. W. P.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Open AccessPublished:October 25, 2015DOI:https://doi.org/10.1074/jbc.M115.693614
      The biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the vitamin D receptor (VDR), whose expression in bone cells is regulated positively by 1,25(OH)2D3, retinoic acid, and parathyroid hormone through both intergenic and intronic enhancers. In this report, we used ChIP-sequencing analysis to confirm the presence of these Vdr gene enhancers in mesenchyme-derived bone cells and to describe the epigenetic histone landscape that spans the Vdr locus. Using bacterial artificial chromosome-minigene stable cell lines, CRISPR/Cas9 enhancer-deleted daughter cell lines, transient transfection/mutagenesis analyses, and transgenic mice, we confirmed the functionality of these bone cell enhancers in vivo as well as in vitro. We also identified VDR-binding sites across the Vdr gene locus in kidney and intestine using ChIP-sequencing analysis, revealing that only one of the bone cell-type enhancers bound VDR in kidney tissue, and none were occupied by the VDR in the intestine, consistent with weak or absent regulation by the 1,25(OH)2D3 hormone in these tissues, respectively. However, a number of additional sites of VDR binding unique to either kidney or intestine were present further upstream of the Vdr gene, suggesting the potential for alternative regulatory loci. Importantly, virtually all of these regions retained histone signatures consistent with those of enhancers and exhibited unique DNase I hypersensitivity profiles that reflected the potential for chromatin access. These studies define mechanisms associated with hormonal regulation of the Vdr and hint at the differential nature of VDR binding activity at the Vdr gene in different primary target tissues in vivo.

      Introduction

      The vitamin D receptor (VDR)
      The abbreviations used are:
      VDR
      vitamin D receptor
      1,25(OH)2D3
      1,25-dihydroxyvitamin D3
      TSS
      transcription start site
      RXR
      retinoid X receptor
      DHS
      DNase I hypersensitivity site
      BAC
      bacterial artificial chromosome
      TK
      thymidine kinase
      PTH
      parathyroid hormone
      MSC
      mesenchymal stem cell
      BMD
      bone mineral density
      qPCR
      quantitative PCR
      atRA
      all-trans retinoic acid
      VDRE
      vitamin D-response element
      Bt2cAMP
      dibutyryl-cAMP
      ChIP-seq
      ChIP-sequencing
      CRE
      CREB-response element
      RARE
      retinoic acid-response element
      RAR
      retinoic acid receptor
      CREB
      cAMP-regulated enhancer-binding protein
      Fsk
      forskolin.
      is a member of the nuclear receptor family of genes that mediates the biological activities of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in target cells (
      • Mangelsdorf D.J.
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      Regulation of target gene expression by the vitamin D receptor–an update on mechanisms.
      ,
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      Fundamentals of vitamin D hormone-regulated gene expression.
      ). Based upon detection of the VDR protein by immunological methods, the VDR gene is expressed in a wide variety of cell and tissue types both in vitro and in vivo. These tissue types include the intestine, kidney, skeleton, and parathyroid glands, which all play a role in calcium and phosphorus homeostasis (
      • Bouillon R.
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      ,
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      Current understanding of the molecular actions of vitamin D.
      ) and additional tissues/cells whose functions are not directly involved in the regulation of mineral metabolism. At these particular sites of VDR expression, which include the skin (
      • Reichrath J.
      • Schilli M.
      • Kerber A.
      • Bahmer F.A.
      • Czarnetzki B.M.
      • Paus R.
      Hair follicle expression of 1,25-dihydroxyvitamin D3 receptors during the murine hair cycle.
      ,
      • Sakai Y.
      • Demay M.B.
      Evaluation of keratinocyte proliferation and differentiation in vitamin D receptor knockout mice.
      ), immune cells (
      • Provvedini D.M.
      • Tsoukas C.D.
      • Deftos L.J.
      • Manolagas S.C.
      1,25-Dihydroxyvitamin D3 receptors in human leukocytes.
      ,
      • Heine G.
      • Niesner U.
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      • Steinmeyer A.
      • Zügel U.
      • Zuberbier T.
      • Radbruch A.
      • Worm M.
      1,25-dihydroxyvitamin D(3) promotes IL-10 production in human B cells.
      ,
      • Chun R.F.
      • Liu P.T.
      • Modlin R.L.
      • Adams J.S.
      • Hewison M.
      Impact of vitamin D on immune function: lessons learned from genome-wide analysis.
      ), pancreatic β cells (
      • Pike J.
      Receptors for 1,25-dihydroxyvitamin D3 in chick pancreas: a partial physical and functional characterization.
      ,
      • Johnson J.A.
      • Grande J.P.
      • Roche P.C.
      • Kumar R.
      Immunohistochemical localization of the 1,25(OH)2D3 receptor and calbindin D28k in human and rat pancreas.
      ,
      • Cheng Q.
      • Li Y.C.
      • Boucher B.J.
      • Leung P.S.
      A novel role for vitamin D: modulation of expression and function of the local renin-angiotensin system in mouse pancreatic islets.
      ), reproductive tissues (
      • Zarnani A.H.
      • Shahbazi M.
      • Salek-Moghaddam A.
      • Zareie M.
      • Tavakoli M.
      • Ghasemi J.
      • Rezania S.
      • Moravej A.
      • Torkabadi E.
      • Rabbani H.
      • Jeddi-Tehrani M.
      Vitamin D3 receptor is expressed in the endometrium of cycling mice throughout the estrous cycle.
      ,
      • Shahbazi M.
      • Jeddi-Tehrani M.
      • Zareie M.
      • Salek-Moghaddam A.
      • Akhondi M.M.
      • Bahmanpoor M.
      • Sadeghi M.R.
      • Zarnani A.H.
      Expression profiling of vitamin D receptor in placenta, decidua and ovary of pregnant mice.
      ,
      • Mahmoudi A.R.
      • Zarnani A.H.
      • Jeddi-Tehrani M.
      • Katouzian L.
      • Tavakoli M.
      • Soltanghoraei H.
      • Mirzadegan E.
      Distribution of vitamin D receptor and 1α-hydroxylase in male mouse reproductive tract.
      ), placenta (
      • Kovacs C.S.
      • Chafe L.L.
      • Woodland M.L.
      • McDonald K.R.
      • Fudge N.J.
      • Wookey P.J.
      Calcitropic gene expression suggests a role for the intraplacental yolk sac in maternal-fetal calcium exchange.
      ,
      • Pike J.W.
      • Goozé L.L.
      • Haussler M.R.
      Biochemical evidence for 1,25-dihydroxyvitamin D receptor macromolecules in parathyroid, pancreatic, pituitary, and placental tissues.
      ), and others, numerous roles for the VDR have been identified, all associated with the ability of the VDR to regulate the expression of genes that govern specialized cell functions. The presence of the VDR has also been suggested in tissues such as liver (
      • Ding N.
      • Yu R.T.
      • Subramaniam N.
      • Sherman M.H.
      • Wilson C.
      • Rao R.
      • Leblanc M.
      • Coulter S.
      • He M.
      • Scott C.
      • Lau S.L.
      • Atkins A.R.
      • Barish G.D.
      • Gunton J.E.
      • Liddle C.
      • et al.
      A vitamin D receptor/SMAD genomic circuit gates hepatic fibrotic response.
      ), muscle (
      • Girgis C.M.
      • Mokbel N.
      • Cha K.M.
      • Houweling P.J.
      • Abboud M.
      • Fraser D.R.
      • Mason R.S.
      • Clifton-Bligh R.J.
      • Gunton J.E.
      The vitamin D receptor (VDR) is expressed in skeletal muscle of male mice and modulates 25-hydroxyvitamin D (25OHD) uptake in myofibers.
      ), and in specific neurons within the CNS (
      • Cui X.
      • Pelekanos M.
      • Liu P.Y.
      • Burne T.H.
      • McGrath J.J.
      • Eyles D.W.
      The vitamin D receptor in dopamine neurons; its presence in human substantia nigra and its ontogenesis in rat midbrain.
      ), frequently prompting the assertion that the VDR is ubiquitously expressed. As the VDR in these tissues is generally detected by PCR analysis that is much more sensitive than techniques that detect the protein, absolute expression of VDR itself often times remains unconfirmed. In these instances, it is uncertain whether VDR expression is simply very low overall or whether expression is restricted to a limited set of cell types within the tissue. As a result, some complex tissues and organs are frequently considered to represent direct targets of vitamin D action, although this is based largely upon the biological activity of the hormone within the tissue that in many cases could be indirect due to changes in the levels of systemic hormones other than 1,25(OH)2D3 or to alterations in blood calcium and/or phosphate levels. Despite these uncertainties, however, the cellular expression of the VDR gene is generally widespread, and its regulation at the cell-specific level is likely diverse.
      The mouse Vdr gene is located on chromosome 15 and is composed of ten exons, two of which represent the 5′ UTR. The gene spans ∼54 kb and is bounded by two active CCCTC-binding factor sites (
      • Zella L.A.
      • Meyer M.B.
      • Nerenz R.D.
      • Lee S.M.
      • Martowicz M.L.
      • Pike J.W.
      Multifunctional enhancers regulate mouse and human vitamin D receptor gene transcription.
      ); the downstream site is located immediately 3′ of the final exon, and the upstream site is located in the intergenic region some 35 kb upstream of the Vdr gene transcription start site (TSS) and immediately preceding the promoter region of neighboring Tmem106c. Based on the ability of occupied CCCTC-binding factor sites to restrict epigenetic histone activity to the region contained within, we recently prepared a mouse VDR bacterial artificial chromosome (BAC) transgenic mouse that spanned this segment of DNA and retained these boundary elements, and demonstrated its capacity to recapitulate expression of the endogenous Vdr gene in all the tissues examined (
      • Lee S.M.
      • Bishop K.A.
      • Goellner J.J.
      • O'Brien C.A.
      • Pike J.W.
      Mouse and human BAC transgenes recapitulate tissue-specific expression of the vitamin D receptor in mice and rescue the VDR-null phenotype.
      ). This BAC transgene was also able to rescue the complex biological phenotype of the VDR null mouse when crossed into the latter genetic background. Importantly, a related segment of the human VDR gene, which is organized in a fashion similar to that of the mouse, was also able to direct appropriate tissue-specific expression of the VDR in normal mice and to rescue the phenotype of the VDR null mouse as well (
      • Lee S.M.
      • Bishop K.A.
      • Goellner J.J.
      • O'Brien C.A.
      • Pike J.W.
      Mouse and human BAC transgenes recapitulate tissue-specific expression of the vitamin D receptor in mice and rescue the VDR-null phenotype.
      ). We conclude from these studies that the two transgenes retained all of the genetic information necessary and sufficient for appropriate basal and tissue-specific expression of these VDR proteins in the mouse.
      The VDR gene is regulated in a tissue-specific manner by a variety of hormones that include 1,25(OH)2D3 as well as a number of transcription factors that are activated via cell-selective sets of signaling pathways (
      • Zella L.A.
      • Kim S.
      • Shevde N.K.
      • Pike J.W.
      Enhancers located within two introns of the vitamin D receptor gene mediate transcriptional autoregulation by 1,25-dihydroxyvitamin D3.
      ,
      • Costa E.M.
      • Hirst M.A.
      • Feldman D.
      Regulation of 1,25-dihydroxyvitamin D3 receptors by vitamin D analogs in cultured mammalian cells.
      ,
      • Strom M.
      • Sandgren M.E.
      • Brown T.A.
      • DeLuca H.F.
      1,25-Dihydroxyvitamin D3 up-regulates the 1,25-dihydroxyvitamin D3 receptor in vivo. Proc.
      ). In many cases, either a developmental or physiological alteration or progression of a disease state in vivo can also influence VDR expression in specific tissues; the administration of a factor or induction of differentiation in cells in culture can also provoke VDR gene expression as well. Indeed, numerous attempts to correlate VDR expression levels with human disease states have been reported (
      • Ding N.
      • Yu R.T.
      • Subramaniam N.
      • Sherman M.H.
      • Wilson C.
      • Rao R.
      • Leblanc M.
      • Coulter S.
      • He M.
      • Scott C.
      • Lau S.L.
      • Atkins A.R.
      • Barish G.D.
      • Gunton J.E.
      • Liddle C.
      • et al.
      A vitamin D receptor/SMAD genomic circuit gates hepatic fibrotic response.
      ), although most with little direct success. With the exception of bone cells, however, little is known of the molecular mechanisms through which this regulation occurs, primarily because most studies have focused on delineating these mechanisms via transient transfection approaches that involve VDR gene promoter plasmid constructs (
      • Wietzke J.A.
      • Welsh J.
      Phytoestrogen regulation of a vitamin D3 receptor promoter and 1,25-dihydroxyvitamin D3 actions in human breast cancer cells.
      ); the results of studies of this type have been largely disappointing and frequently incorrect. Initial studies in bone cells using unbiased ChIP-chip analysis, however, provided some resolution to this issue by revealing that the mouse gene was not regulated by 1,25(OH)2D3, all-trans-retinoic acid (atRA), or parathyroid hormone (PTH) via cis elements located proximal to the promoter, but rather through distal elements situated either within intronic regions downstream of the Vdr gene promoter or within the upstream intergenic region (
      • Zella L.A.
      • Meyer M.B.
      • Nerenz R.D.
      • Lee S.M.
      • Martowicz M.L.
      • Pike J.W.
      Multifunctional enhancers regulate mouse and human vitamin D receptor gene transcription.
      ,
      • Zella L.A.
      • Kim S.
      • Shevde N.K.
      • Pike J.W.
      Enhancers located within two introns of the vitamin D receptor gene mediate transcriptional autoregulation by 1,25-dihydroxyvitamin D3.
      ). Indeed, these studies suggest that autoregulation by 1,25(OH)2D3 in bone cells is likely mediated via two separate intronic sites as well as through an upstream element; the activities of atRA and PTH, in contrast, have not been fully defined. A vitamin D-response element (VDRE) was identified in one of these intronic enhancers that mediated 1,25(OH)2D3 activity, however (
      • Zella L.A.
      • Kim S.
      • Shevde N.K.
      • Pike J.W.
      Enhancers located within two introns of the vitamin D receptor gene mediate transcriptional autoregulation by 1,25-dihydroxyvitamin D3.
      ). These early studies support the idea that like many other genes examined through unbiased methodologies, the Vdr gene is likely to be regulated through multiple distal regulatory regions in not only bone cells but perhaps other tissues as well.
      In this study, we explore further the mechanisms that underlie the regulation of Vdr gene expression in several cell types. We confirm through ChIP-sequencing (ChIP-seq) analysis the locations of the three regulatory patches that mediate the autoregulatory activity of 1,25(OH)2D3 as well as the up-regulation of the gene by atRA and PTH in bone cells, and we show that these elements are conserved within the mesenchymal lineage. The epigenetic landscape that highlights key features across the Vdr gene locus and supports the enhancer function of these three regions is also profiled. We then demonstrate that these enhancers are indeed responsible for mediating VDR, retinoic acid receptor (RAR), and cAMP-regulated enhancer-binding protein (CREB) action using stably selected wild type and enhancer mutant BAC clone bone cell lines and by creating enhancer mutant daughter cell lines via CRISPR/Cas9 genome editing methods. We also identify the binding sites for atRA-induced RAR and PTH-induced CREB that are located within these enhancers. The roles of the enhancers determined in cell culture were then validated in vivo using transgenic strains that express the VDR from wild type or enhancer(s)-deleted VDR BAC transgenes. Finally, we show using ChIP-seq analysis that binding sites for the VDR across the Vdr gene locus differ in cells derived from the kidney cortex and from the intestinal epithelium; many of these new sites are highlighted by unique histone marks and characterized by DNase I hypersensitivity sites (DHSs) as well. We conclude that although the VDR binds to three separate regulatory regions across the Vdr gene locus to control this gene's expression in mesenchyme-derived cells, the capacity of 1,25(OH)2D3 to regulate Vdr expression in other tissues, such as intestine and kidney, may be mediated through enhancers that are different from those identified in mesenchyme-derived skeletal cells.

      Experimental Procedures

      Reagents

      Minimum Eagle's medium α modification and FBS were purchased from Mediatech, Inc., and Hyclone Laboratories, respectively. Penicillin/streptomycin, G418, and Lipofectamine PLUS were obtained from Invitrogen. 1,25(OH)2D3 was obtained from SAFC, and forskolin (Fsk), dibutyryl-cAMP (Bt2cAMP), and atRA were from Sigma. FuGENE HD, Glo Lysis Buffer, and Bright-Glo Luciferase Assay System were purchased from Promega. Protein Assay was obtained from Bio-Rad. High Capacity cDNA reverse transcription kit and all TaqMan primers were obtained from Life Technologies, Inc. (Applied Biosystems), and information on TaqMan primers is available upon request. All ChIP antibodies were purchased from Santa Cruz Biotechnology, Inc., Millipore, or Abcam as reported previously (
      • Meyer M.B.
      • Benkusky N.A.
      • Lee C.H.
      • Pike J.W.
      Genomic determinants of gene regulation by 1,25-dihydroxyvitamin D3 during osteoblast-lineage cell differentiation.
      ). Primers were obtained from IDT, and the sequences are available upon request. QuantiChrom calcium assay kit was obtained from BioAssay Systems.

      Cell Culture

      Mouse MC3T3-E1 osteoblastic cells were an early passage line from Sudo et al. (
      • Sudo H.
      • Kodama H.A.
      • Amagai Y.
      • Yamamoto S.
      • Kasai S.
      In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria.
      ). UAMS-32PB (UAMS-PB) cells, which are mouse UAMS-32 stromal/osteoblastic cells expressing blasticidin-resistant PTH receptor (
      • Fu Q.
      • Manolagas S.C.
      • O'Brien C.A.
      Parathyroid hormone controls receptor activator of NF-κB ligand gene expression via a distant transcriptional enhancer.
      ), were kindly provided by Charles O'Brien (University of Arkansas Medical School). Mesenchymal stem cells (MSCs) were derived from C57BL/6 mice (Harlan Teklad) as reported previously (
      • Case N.
      • Xie Z.
      • Sen B.
      • Styner M.
      • Zou M.
      • O'Conor C.
      • Horowitz M.
      • Rubin J.
      Mechanical activation of β-catenin regulates phenotype in adult murine marrow-derived mesenchymal stem cells.
      ). The three cell lines mentioned above and UAMS-PB cells in which genomes were edited by the CRISPR/Cas9 system as described below were cultured in minimum Eagle's medium α modification supplemented with 10% FBS and 1% penicillin/streptomycin. MC3T3-E1 stable cell lines that we generated as described below were cultured in minimum Eagle's medium α modification supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.2 mg/ml G418.

      Construction of VDR BAC Clones and Generation of MC3T3-E1 BAC Stable Cell Lines

      The wild type mouse VDR BAC clone was previously constructed from BAC clone RP23-136G8 containing the mouse Vdr gene locus and contiguous upstream and downstream intergenic sequences (
      • Zella L.A.
      • Meyer M.B.
      • Nerenz R.D.
      • Lee S.M.
      • Martowicz M.L.
      • Pike J.W.
      Multifunctional enhancers regulate mouse and human vitamin D receptor gene transcription.
      ). In brief, the BAC clone RP23-136G8 was genetically engineered by BAC recombineering techniques to contain an HA tag (HA) at the translation start site of the mouse Vdr gene and a reporter cassette containing an internal ribosome entry site-driven luciferase reporter and a TK promoter (TK)-driven neomycin resistance gene in the 3′ UTR of mouse Vdr gene (
      • Zella L.A.
      • Meyer M.B.
      • Nerenz R.D.
      • Lee S.M.
      • Martowicz M.L.
      • Pike J.W.
      Multifunctional enhancers regulate mouse and human vitamin D receptor gene transcription.
      ). To delete the enhancer regions from the wild type mouse VDR BAC clone, we used the galactokinase (galK) system as described previously (
      • Zella L.A.
      • Meyer M.B.
      • Nerenz R.D.
      • Lee S.M.
      • Martowicz M.L.
      • Pike J.W.
      Multifunctional enhancers regulate mouse and human vitamin D receptor gene transcription.
      ,
      • Warming S.
      • Costantino N.
      • Court D.L.
      • Jenkins N.A.
      • Copeland N.G.
      Simple and highly efficient BAC recombineering using galK selection.
      ), and the deletions were confirmed by PCR and sequencing. To generate MC3T3-E1 BAC stable cell lines, either wild type or one of the enhancer(s)-deleted mouse VDR BAC clones were stably transfected into the cells using a Nucleofector (Lonza) and collections of stable cells were selected with 0.2 mg/ml G418 as described previously (
      • Meyer M.B.
      • Goetsch P.D.
      • Pike J.W.
      A downstream intergenic cluster of regulatory enhancers contributes to the induction of CYP24A1 expression by 1α,25-dihydroxyvitamin D3.
      ).

      CRISPR/Cas9 Genome Editing in UAMS-PB Cells

      CRISPR/Cas9 reagents, pSpCas9(BB)-2A-GFP (pX458, Addgene 48138) and pSpCas9(BB)-2A-Puro (pX459, Addgene 62988) were obtained from the Zhang laboratory via Addgene (
      • Cong L.
      • Ran F.A.
      • Cox D.
      • Lin S.
      • Barretto R.
      • Habib N.
      • Hsu P.D.
      • Wu X.
      • Jiang W.
      • Marraffini L.A.
      • Zhang F.
      Multiplex genome engineering using CRISPR/Cas systems.
      ). All guides were designed as described previously (
      • Meyer M.B.
      • Benkusky N.A.
      • Pike J.W.
      Selective distal enhancer control of the Mmp13 gene identified through clustered regularly interspaced short palindromic repeat (CRISPR) genomic deletions.
      ), and each pair of guides (guide1 and guide2) without protospacer adjacent motif was then cloned into pX458 and pX459, respectively. Guide sequences (sequence-protospacer adjacent motif) were as follows (a “g” was added preceding the sequence as necessary for the U6 promoter): for U1 deletion, guide1-gCTACCCACAATAACTCCAGCTGG, and guide2-CTCAAGGATAAGACATGAGCTGG; for S1 deletion, guide1-gTCACACACTCTCCCGTATCT, and guide2-GTGTGTCTTCTTTAATACGGAGG; for S3 deletion, guide1-gAGAAGATGGGACGCACATCCAGG, and guide2-GAATCTTGTAGGAGTCTCCCTGG. As reported previously (
      • Meyer M.B.
      • Benkusky N.A.
      • Pike J.W.
      Selective distal enhancer control of the Mmp13 gene identified through clustered regularly interspaced short palindromic repeat (CRISPR) genomic deletions.
      ), each pair of the cloned plasmids was co-transfected into UAMS-PB cells using FuGENE HD, and single clones in which each enhancer region was deleted were identified using FACS and further PCR. The deletions in the clones were confirmed by PCR and sequencing. The genome-edited UAMS-PB cells and wild type UAMS-PB cells (control) were treated with 10−7 m 1,25(OH)2D3, 10−6 m Fsk, or 10−6 m atRA for 24 h and harvested for gene expression analysis.

      Plasmids

      The reporter plasmids used in this study were constructed by cloning the DNA fragments obtained by DNA amplification of the regions described in each figure from mouse VDR BAC clone (RP23-136G8) into the pTK-luc vector (empty). The sequences of the reporter plasmids were confirmed by sequencing. A dominant negative CREB (A-CREB) expression plasmid (pCMV500-A-CREB) and its control plasmid (pCMV500) (
      • Ahn S.
      • Olive M.
      • Aggarwal S.
      • Krylov D.
      • Ginty D.D.
      • Vinson C.
      A dominant-negative inhibitor of CREB reveals that it is a general mediator of stimulus-dependent transcription of c-fos.
      ) were kindly provided by Dr. Charles Vinson (National Institutes of Health, Bethesda). pCH110-β-gal and pRSV-β-gal were used as transfection controls to normalize luciferase activity.

      Luciferase Assay

      To measure luciferase activities in MC3T3-E1 BAC stable cells, the cells were seeded into 24-well plates at a density of 7.5 × 104 cells per well and treated with the indicated concentrations of 1,25(OH)2D3, Fsk, and atRA for 24 h. For transient reporter assay, MC3T3-E1 cells were co-transfected with the plasmids indicated below using Lipofectamine PLUS, as described previously (
      • Zella L.A.
      • Meyer M.B.
      • Nerenz R.D.
      • Lee S.M.
      • Martowicz M.L.
      • Pike J.W.
      Multifunctional enhancers regulate mouse and human vitamin D receptor gene transcription.
      ). For the analysis of the U1 enhancer, cells were transfected with 150 ng each of pRSV-β-galactosidase, the reporter plasmid, and either pCMV500-A-CREB or pCMV500. For analysis of the S3 enhancer, 50 ng of pCH110-β-galactosidase and 250 ng of the reporter plasmids were co-transfected. The transfected cells were then treated with 10−6 m Fsk or atRA for 24 h. Cell lysates from either BAC stable cells or transiently transfected cells were obtained using Glo Lysis Buffer and cleared via centrifugation. Luciferase activities were measured using the Bright-Glo Luciferase Assay System and normalized to total protein quantified via Protein Assay or to β-galactosidase activity for either the stable cell lines or the transiently transfected cells, respectively, as described previously (
      • Zella L.A.
      • Meyer M.B.
      • Nerenz R.D.
      • Lee S.M.
      • Martowicz M.L.
      • Pike J.W.
      Multifunctional enhancers regulate mouse and human vitamin D receptor gene transcription.
      ,
      • Yamamoto H.
      • Shevde N.K.
      • Warrier A.
      • Plum L.A.
      • DeLuca H.F.
      • Pike J.W.
      2-Methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 potently stimulates gene-specific DNA binding of the vitamin D receptor in osteoblasts.
      ).

      Generation of BAC Transgenic Mice and VDR Null Mice Containing BAC Transgenes

      We have previously reported the wild type mouse VDR BAC transgenic mouse strain (
      • Lee S.M.
      • Bishop K.A.
      • Goellner J.J.
      • O'Brien C.A.
      • Pike J.W.
      Mouse and human BAC transgenes recapitulate tissue-specific expression of the vitamin D receptor in mice and rescue the VDR-null phenotype.
      ). To generate transgenic mice containing U1- or U1/S1-deleted VDR BAC clone, each BAC clone was introduced into mice as a transgene as described previously (
      • Lee S.M.
      • Bishop K.A.
      • Goellner J.J.
      • O'Brien C.A.
      • Pike J.W.
      Mouse and human BAC transgenes recapitulate tissue-specific expression of the vitamin D receptor in mice and rescue the VDR-null phenotype.
      ). Genotypes of the transgenic mice were identified by luciferase assay using lysates obtained from tail clips, as described previously (
      • Lee S.M.
      • Bishop K.A.
      • Goellner J.J.
      • O'Brien C.A.
      • Pike J.W.
      Mouse and human BAC transgenes recapitulate tissue-specific expression of the vitamin D receptor in mice and rescue the VDR-null phenotype.
      ). VDR null mice carrying the wild type or U1/S1-deleted mouse VDR BAC clone (WT-BAC/VDR−/− or ΔU1/S1-BAC/VDR−/−, respectively) were generated through a breeding strategy previously outlined (
      • Lee S.M.
      • Bishop K.A.
      • Goellner J.J.
      • O'Brien C.A.
      • Pike J.W.
      Mouse and human BAC transgenes recapitulate tissue-specific expression of the vitamin D receptor in mice and rescue the VDR-null phenotype.
      ) using the VDR null mice created by Li et al. (
      • Li Y.C.
      • Pirro A.E.
      • Amling M.
      • Delling G.
      • Baron R.
      • Bronson R.
      • Demay M.B.
      Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia.
      ). Genotyping methods for WT-BAC/VDR−/− mice were reported previously (
      • Lee S.M.
      • Bishop K.A.
      • Goellner J.J.
      • O'Brien C.A.
      • Pike J.W.
      Mouse and human BAC transgenes recapitulate tissue-specific expression of the vitamin D receptor in mice and rescue the VDR-null phenotype.
      ). The genotypes of ΔU1/S1-BAC/VDR−/− mice were determined by PCR with primers that detect deletion of U1 and S1 enhancers to determine the presence of the transgene and primers for the endogenous Vdr allele using tail genomic DNAs as templates. The presence of the transgene was confirmed by luciferase assay of tail lysate as described previously (
      • Lee S.M.
      • Bishop K.A.
      • Goellner J.J.
      • O'Brien C.A.
      • Pike J.W.
      Mouse and human BAC transgenes recapitulate tissue-specific expression of the vitamin D receptor in mice and rescue the VDR-null phenotype.
      ).

      Animal Study

      Transgenic mice were outbred with C57BL/6 mice (Harlan) as heterozygotes. All mice used in this study were maintained on a standard rodent chow diet (5008; Harlan Teklad) except VDR null mice and ΔU1/S1-BAC/VDR−/− mice to examine alopecic phenotype at 6 months of age. In this case, the mice were fed a rescue diet containing 20% lactose, 2% calcium, and 1.25% phosphate diet (TD.96348; Harlan Teklad). For gene expression analysis, mice were injected i.p. with 1,25(OH)2D3 (10 ng/g body weight), Bt2cAMP (0.1 ng/g body weight), or atRA (1 ng/g body weight), and tissues for RNA preparation were collected 6, 1, or 4 h after the treatments, respectively. Vehicle controls for 1,25(OH)2D3 and atRA or Bt2cAMP were a mixture of ethanol and propylene glycol or PBS, respectively. The injected mice were 8–10-week-old mice of both genders. Mice were exposed to a 12-h light-dark cycle. All animal studies were reviewed and approved by the Research Animal Care and Use Committee of the University of Wisconsin-Madison.

      Characterization of Phenotypes of ΔU1/S1-BAC/VDR−/− Mice

      Serum calcium concentration was measured using QuantiChrom calcium assay kit as described previously (
      • Lee S.M.
      • Bishop K.A.
      • Goellner J.J.
      • O'Brien C.A.
      • Pike J.W.
      Mouse and human BAC transgenes recapitulate tissue-specific expression of the vitamin D receptor in mice and rescue the VDR-null phenotype.
      ). Bone mineral densities (BMDs) of 8-week-old mice of both sexes were separately quantified, as described previously (
      • Lee S.M.
      • Bishop K.A.
      • Goellner J.J.
      • O'Brien C.A.
      • Pike J.W.
      Mouse and human BAC transgenes recapitulate tissue-specific expression of the vitamin D receptor in mice and rescue the VDR-null phenotype.
      ). The alopecic phenotype was visually assessed at 6 months of age.

      Gene Expression Analysis

      Total RNAs were prepared from cells or mouse tissues using TRI Reagent (Molecular Research Center) and then subjected to reverse transcription using the High Capacity cDNA reverse transcription kit following the manufacturer's protocols as described previously (
      • Lee S.M.
      • Bishop K.A.
      • Goellner J.J.
      • O'Brien C.A.
      • Pike J.W.
      Mouse and human BAC transgenes recapitulate tissue-specific expression of the vitamin D receptor in mice and rescue the VDR-null phenotype.
      ,
      • Meyer M.B.
      • Benkusky N.A.
      • Pike J.W.
      Selective distal enhancer control of the Mmp13 gene identified through clustered regularly interspaced short palindromic repeat (CRISPR) genomic deletions.
      ). Gene expression was assessed by TaqMan-mediated quantitative PCR (qPCR) on a StepOnePlus (Applied Biosystems).

      Chromatin Immunoprecipitation Coupled to DNA Sequencing Analysis (ChIP-seq)

      Chromatin immunoprecipitation followed by sequencing (ChIP-seq) analyses was previously performed in MC3T3-E1 cells (
      • Meyer M.B.
      • Benkusky N.A.
      • Lee C.H.
      • Pike J.W.
      Genomic determinants of gene regulation by 1,25-dihydroxyvitamin D3 during osteoblast-lineage cell differentiation.
      ,
      • Meyer M.B.
      • Benkusky N.A.
      • Pike J.W.
      The RUNX2 cistrome in osteoblasts: characterization, down-regulation following differentiation, and relationship to gene expression.
      ), MSCs (
      • Meyer M.B.
      • Benkusky N.A.
      • Pike J.W.
      1,25-Dihydroxyvitamin D3 induced histone profiles guide discovery of VDR action sites.
      ), and UAMS-PB cells (
      • Meyer M.B.
      • Benkusky N.A.
      • Pike J.W.
      Selective distal enhancer control of the Mmp13 gene identified through clustered regularly interspaced short palindromic repeat (CRISPR) genomic deletions.
      ). Briefly, the cells were treated with vehicle or 10−7 m 1,25(OH)2D3 for 3 h and subjected to immunoprecipitation using either a control IgG or the indicated antibodies as described previously (
      • Meyer M.B.
      • Benkusky N.A.
      • Lee C.H.
      • Pike J.W.
      Genomic determinants of gene regulation by 1,25-dihydroxyvitamin D3 during osteoblast-lineage cell differentiation.
      ,
      • Meyer M.B.
      • Benkusky N.A.
      • Pike J.W.
      The RUNX2 cistrome in osteoblasts: characterization, down-regulation following differentiation, and relationship to gene expression.
      ). ChIP-seq analyses in mouse kidney (
      • Meyer M.B.
      • Benkusky N.A.
      • Pike J.W.
      Selective distal enhancer control of the Mmp13 gene identified through clustered regularly interspaced short palindromic repeat (CRISPR) genomic deletions.
      ) and intestine (
      • Lee S.M.
      • Riley E.M.
      • Meyer M.B.
      • Benkusky N.A.
      • Plum L.A.
      • DeLuca H.F.
      • Pike J.W.
      1,25-Dihydroxyvitamin D3 controls a cohort of vitamin D receptor target genes in the proximal intestine that is enriched for calcium-regulating components.
      ) were previously reported. In brief, kidney and intestine samples were obtained from mice treated with 1,25(OH)2D3 (10 ng/g body weight) for 1 h and subjected to immunoprecipitation using either a control IgG or the indicated antibodies as described previously (
      • Meyer M.B.
      • Benkusky N.A.
      • Lee C.H.
      • Pike J.W.
      Genomic determinants of gene regulation by 1,25-dihydroxyvitamin D3 during osteoblast-lineage cell differentiation.
      ,
      • Meyer M.B.
      • Benkusky N.A.
      • Pike J.W.
      The RUNX2 cistrome in osteoblasts: characterization, down-regulation following differentiation, and relationship to gene expression.
      ). Statistical analysis and data processing for ChIP-seq assay were performed as reported previously (
      • Meyer M.B.
      • Benkusky N.A.
      • Pike J.W.
      The RUNX2 cistrome in osteoblasts: characterization, down-regulation following differentiation, and relationship to gene expression.
      ). A genome-wide analysis of the mouse kidney ChIP-seq data sets will be published elsewhere.

      Statistical Analysis

      All data are presented as the mean ± S.E. Student's unpaired t test was used to identify significant differences (p < 0.05).

      Results

      ChIP-seq Analysis of VDR/RXR-binding Sites at the Vdr Gene Locus in Mesenchymal and Differentiated Osteoblastic Cell Lines

      Previous ChIP-chip analyses in MC3T3-E1 osteoblastic cells using antibodies to both VDR and RXR revealed the presence of at least three dominant enhancers in the Vdr gene locus that bound both proteins in response to 1,25(OH)2D3, two located within separate introns at +19 and + 29 kb termed S3 and S1, respectively, and one located 6 kb upstream of the TSS termed U1 (
      • Zella L.A.
      • Meyer M.B.
      • Nerenz R.D.
      • Lee S.M.
      • Martowicz M.L.
      • Pike J.W.
      Multifunctional enhancers regulate mouse and human vitamin D receptor gene transcription.
      ). Further analysis of these sites upon activation by atRA suggested the presence of RAR at the S3 region and the presence of CREB at the U1 region following treatment with the protein kinase A (PKA) activator and PTH surrogate, Fsk. A VDRE for the VDR/RXR heterodimer was identified at S1, the most robust vitamin D-responsive site within the gene (
      • Zella L.A.
      • Kim S.
      • Shevde N.K.
      • Pike J.W.
      Enhancers located within two introns of the vitamin D receptor gene mediate transcriptional autoregulation by 1,25-dihydroxyvitamin D3.
      ). To characterize these sites in more detail, we conducted an analysis of VDR and RXR binding across this gene using ChIP-seq analysis and contrasted the results obtained in the established MC3T3-E1 cell line with those obtained in osteoblastic UAMS-PB cells. As documented in Fig. 1, the ChIP-seq data overlay (HUB) tracks across the Vdr gene locus conducted in the absence and presence of 1,25(OH)2D3 in MC3T3-E1 cells fully support the original conclusion that VDR and RXR are inducible at this locus and that three dominant enhancers S1, S3, and U1 serve to autoregulate Vdr gene expression. Importantly, a VDR enhancer profile similar to that obtained in MC3T3-E1 cells was also identified for the VDR in the UAMS-PB cell line, a finding that strongly supports the validity of each of these three enhancers in this cell type despite their cell line nature. As regulatory enhancers are frequently established early on within the development of a cell lineage, we also examined in a separate analysis whether these three individual Vdr gene enhancers were similarly located within an MSC precursor with the proven potential to give rise to not only osteoblastic cells but to additional mesenchymal lineage-derived cells such as chondrocytes and adipocytes as well (
      • Case N.
      • Xie Z.
      • Sen B.
      • Styner M.
      • Zou M.
      • O'Conor C.
      • Horowitz M.
      • Rubin J.
      Mechanical activation of β-catenin regulates phenotype in adult murine marrow-derived mesenchymal stem cells.
      ). As seen in Fig. 1, the ChIP-seq tracks obtained from this precursor cell type reveal that VDR and RXR binding is indeed present in this undifferentiated cell type and is dominant at S1, S3, and U1. These results confirm that the autoregulation of the Vdr gene by 1,25(OH)2D3 is mediated by three primary regulatory enhancers that appear early on within the mesenchymal stem cell lineage and that are retained following osteoblast differentiation. Importantly, ChIP-seq analysis of the VDR across the Vdr gene from not only osteoblasts but adipocytes differentiated from this MSC line also revealed these three enhancers, suggesting that the VDR is autoregulated in a similar manner in adipocytes as well (data not shown).
      Figure thumbnail gr1
      FIGURE 1.Cistrome of VDR and RXR at the mouse Vdr gene in MC3T3-E1, MSC, and UMAS-PB cells. Representative ChIP-seq tracks of triplicate samples of MC3T3-E1 cells (MC3T3), MSCs (MSC), and UMAS-PB cells (UAMS) at the mouse Vdr gene locus for VDR and RXR are presented as tag density tracks normalized to input and 107 tags (y axis). Exons and introns of Vdr and Tmem106c genes are shown in boxes and lines, respectively, above the tracks. Transcriptional direction of a gene is indicated by an arrow at the TSS, and genomic location and scale are provided. Maximum height of tag density for the data track is indicated on each track. Bindings of the transcription factors in vehicle- and 1,25(OH)2D3-treated samples are shown in yellow and blue, respectively. Overlapped binding activity is shown in green. The bone enhancer regions (S1, S3 and U1) are indicated below the tracks.

      Assessment of Epigenetic Histone Signatures across the Vdr Gene Locus in Osteoblast Lineage Cells

      Although a general histone H4 acetylation profile was developed across the Vdr gene locus in earlier studies (
      • Zella L.A.
      • Meyer M.B.
      • Nerenz R.D.
      • Pike J.W.
      The enhanced hypercalcemic response to 20-epi-1,25-dihydroxyvitamin D3 results from a selective and prolonged induction of intestinal calcium-regulating genes.
      ), a more complete understanding of the structural and functional significance of novel histone marks that has emerged in the ensuing years prompted us to examine the profiles of several specific histone modifications across the Vdr gene locus and to determine whether 1,25(OH)2D3 might exert an impact on their levels. Accordingly, we conducted ChIP-seq analysis using qualified antibodies to the histone modifications, H3K4 mono-methylation (H3K4me1; enriched across enhancers), H3K36 tri-methylation (H3K36me3; enriched across gene transcription units), and H3K9 acetylation and H4K5 acetylation (H3K9ac and H4K5ac; enriched at sites that undergo dynamic chromatin decondensation) as documented in Fig. 2, where the HUB tracks contrast the results obtained in the absence and presence of hormone. As can be seen, landscape profiles for all ofthe histone modifications examined were generally similar between the MC3T3-E1 cell line and that of its undifferentiated MSC precursor. Importantly, all three Vdr enhancers in both cell types were clearly enriched for the H3K4me1 enhancer signature mark. Interestingly, this mark was also present at additional sites within the first intron, near the Vdr gene TSS and at several upstream sites as well (highlighted in Fig. 2), hinting at the potential for additional enhancers for this gene. H4K5ac enrichment, however, was apparent at sites that were also enriched for H3K4me1, suggesting the possibility of chromatin decondensation at each of these regions of activity. It is also worth noting that specific VDR-binding sites vertically align precisely within valleys in the levels of H4K5ac and H3K4me1, particularly in the MC3T3-E1 cell analyses. These reductions are believed to highlight both nucleosome deficiency as well as the presence of additional chromatin regulatory factors that could serve to facilitate VDR/RXR binding at these sites. These results suggest not only that the three identified enhancers mediate the autoregulatory activity of the VDR/RXR heterodimer at the Vdr gene locus in bone cells but that additional regulatory patches within the Vdr gene locus may be present as well. An effect of 1,25(OH)2D3 to enhance the levels of histone modification can be observed across the Vdr gene, although this effect appears to be the most robust at sites of H4K5ac.
      Figure thumbnail gr2
      FIGURE 2.Binding profiles of histone marks at mouse Vdr gene in MC3T3-E1 cells and MSCs. Representative ChIP-seq tracks of triplicate samples of MC3T3-E1 cells (MC3T3) and MSCs (MSC) at mouse Vdr gene locus for the indicated histone marks are presented as tag density tracks normalized to input and 107 tags (y axis). Exons and introns of Vdr and Tmem106c genes are shown in boxes and lines, respectively, above the tracks. Transcriptional direction of a gene is indicated by an arrow at the TSS, and genomic location and scale are provided. Maximum height of tag density for the data track is indicated on each track. Bindings of the modified histones in vehicle- and 1,25(OH)2D3-treated samples are shown in yellow and blue, respectively. Overlapped binding activity is shown in green. The bone enhancer regions (S1, S3, and U1) are indicated below the tracks. Additional potential enhancer regions are boxed and highlighted in pale yellow.

      Defining the Functional Activities of the Three Vdr Gene Enhancers in Gene Context

      Recent studies suggest that many if not most genes are regulated by multiple enhancers frequently located distal to the promoters of the genes they regulate (
      • Meyer M.B.
      • Benkusky N.A.
      • Pike J.W.
      Selective distal enhancer control of the Mmp13 gene identified through clustered regularly interspaced short palindromic repeat (CRISPR) genomic deletions.
      ,
      • Bishop K.A.
      • Wang X.
      • Coy H.M.
      • Meyer M.B.
      • Gumperz J.E.
      • Pike J.W.
      Transcriptional regulation of the human TNFSF11 gene in T cells via a cell type-selective set of distal enhancers.
      ,
      • Kim S.
      • Yamazaki M.
      • Zella L.A.
      • Shevde N.K.
      • Pike J.W.
      Activation of receptor activator of NF-κB ligand gene expression by 1,25-dihydroxyvitamin D3 is mediated through multiple long-range enhancers.
      ). Given this scenario, it is no longer realistic to examine the activities of regulatory regions in the absence of either chromatin or gene context. As a result, we exploited a wild type mouse VDR BAC clone as seen in Fig. 3A that we had previously constructed and had found capable of not only recapitulating the expression of the endogenous Vdr gene when introduced as a transgene into mice but also of rescuing the aberrant phenotype of the VDR null mouse (
      • Lee S.M.
      • Bishop K.A.
      • Goellner J.J.
      • O'Brien C.A.
      • Pike J.W.
      Mouse and human BAC transgenes recapitulate tissue-specific expression of the vitamin D receptor in mice and rescue the VDR-null phenotype.
      ). Accordingly, we utilized recombinant engineering to delete DNA segments within this minigene corresponding to S1, S3, or U1 and created double deletions of U1 and S1, U1 and S3, and S1 and S3, as summarized in Fig. 3B. We then prepared individual collections of MC3T3-E1 cells containing these stably integrated BAC clone constructs and explored their ability to mediate luciferase response to 1,25(OH)2D3, atRA and Fsk. As can be seen in Fig. 3C, although 1,25(OH)2D3 strongly induced cells containing the wild type Vdr minigene following a 24-h treatment, cells with individual deletions of S1, S3, and U1 resulted in a differential reduction in responsivity to 1,25(OH)2D3, but in each case retained a residual response to the hormone. Deletion of combinations of U1 and S1, U1 and S3, and S1 and S3 also resulted in residual response to 1,25(OH)2D3, but it was most strikingly reduced when both S1 and S3 were deleted. In contrast, although Fsk strongly induced cells containing the wild type minigenes, cells with a deletion of U1 or combinations containing U1 exhibited the most striking reduction in response to Fsk. Finally, although atRA induced cells containing the wild type minigene, only cells that contained a deletion of S3 or combinations containing deletion of S3 were no longer inducible by atRA. We conclude from these experiments that VDR binding at S1, S3, and U1 contributes to the functional up-regulation of Vdr gene expression and that the activities of Fsk and atRA are mediated largely by U1 and S3, respectively.
      Figure thumbnail gr3
      FIGURE 3.Structure of wild type and enhancer-deleted mouse VDR BAC clones and reporter assay of MC3T3-E1 VDR BAC stable cell lines. A, wild type mouse VDR BAC clone includes the entire mouse Vdr gene locus and its surrounding intergenic segments. Exons and introns are represented by black and blue boxes, respectively. Direction of transcription is indicated by an arrow at the TSS. Insertion sites of a reporter cassette and HA tag are shown in the 3′-UTR and in the translation start site (the 3rd exon), respectively. Location of each enhancer is indicated by an arrow. The sizes of upstream and downstream intergenic sequence included in the BAC clone are indicated. B, locations of the enhancer regions deleted in the BAC clone are shown as distances from TSS, and the size of each deletion is provided. C, luciferase activities were measured from stable MC3T3-E1 cells carrying wild type (WT) and enhancer(s)-deleted (U1 KO, S1 KO, S3 KO, U1/S1 KO, U1/S3 KO, and S1/S3 KO) mouse VDR BAC clones after 24 h of incubation with the indicated concentrations of either vehicle (V) or VDR-inducing hormones, 1,25(OH)2D3, Fsk, and atRA and normalized to total protein. The normalized luciferase activities are presented as average fold induction ± S.E. versus vehicle-treated sample for each stable cell lines. Neor, neomycin resistance gene; TK, human thymidine kinase promoter; LUC, luciferase; IRES, internal ribosome entry site; HA, hemagglutinin tag; bp, base pair; V, vehicle-treated sample. *, p < 0.05 versus vehicle-treated sample.

      Individual Deletion of the Vdr Gene Enhancers S1, S3, and U1 by CRISPR/Cas9 Genome Editing in UAMS-PB Cells Confirms Their Functional Roles in Vdr Gene Transcriptional Output

      As integration sites can influence the activities of BAC clones, we used the CRISPR/Cas9 genome editing method to create a series of daughter cell lines derived from the parental UAMS-PB cell, which contained genomic deletions that removed S1, S3, or U1, as summarized in Fig. 4A. Individual cell lines were then treated with either vehicle, 1,25(OH)2D3, Fsk, or atRA for 24 h, and the level of VDR transcripts was measured following RNA isolation. As can be seen in Fig. 4B, although deletion of the U1 region had little effect on the basal expression of VDR transcripts compared with expression in the unmodified wild type parental cells, complete loss of response appeared to be limited to that induced by Fsk. Accordingly, only a modest reduction in response to 1,25(OH)2D3 and atRA was observed. Similarly, although deletion of S1 also had only a minor effect on basal activity, it strongly reduced but did not eliminate response to 1,25(OH)2D3. In contrast, the response to both atRA and Fsk remained intact. Finally, although deletion of S3 had limited effect on basal expression of VDR transcripts, a complete loss of response was limited to that induced by atRA; no reduction in response to 1,25(OH)2D3 or Fsk was observed. These studies generally confirm individual functions of each of the three Vdr gene enhancers, while reinforcing the idea that S1 remains the dominant autoregulatory enhancer for 1,25(OH)2D3 in the Vdr gene locus. The fact that changes in the basal expression of the VDR transcript are limited in these studies suggests that these three enhancers do not play key roles in bone-specific basal expression of the Vdr gene.
      Figure thumbnail gr4
      FIGURE 4.Assessment of individual roles of the enhancers in hormone responses using CRISPR/Cas9 genome editing. A, locations of the enhancer regions deleted in genome-edited UAMP-PB clones by CRISPR/Cas9 system are shown as distances from the TSS, and the size of each deletion is provided. B, enhancer-deleted clones (U1 KO, S1 KO, and S3 KO) and their parental UAMS-PB cells (WT) were incubated for 24 h with vehicle (Veh), 1,25(OH)2D3 (1,25, 10−7 m), Fsk (10−6 m), or atRA (10−6 m), and expression of Vdr was measured following RNA isolation by qPCR. The expression levels were normalized to Gapdh (relative quantity, RQ) in a triplicate set of assays and presented as the mean ± S.E. *, p < 0.05 compared with vehicle-treated sample of each cell line. #, p < 0.05 compared with wild type parental UAMS-PB cell treated with the same hormone.

      Defining the DNA Sequence Elements for atRA and Fsk

      Earlier studies using MC3T3-E1-transfected enhancer/reporterconstructs and mutagenesis revealed the presence of a VDRE in S1 (
      • Zella L.A.
      • Kim S.
      • Shevde N.K.
      • Pike J.W.
      Enhancers located within two introns of the vitamin D receptor gene mediate transcriptional autoregulation by 1,25-dihydroxyvitamin D3.
      ). The absence of 1,25(OH)2D3-inducible activity measurable in the S3 and U1 DNA segments using transfected constructs prevented further definition of the sites of action of 1,25(OH)2D3 in these two enhancers, although several potential VDREs appeared to be present near the VDR peak maxima within S3 and U1 (
      • Zella L.A.
      • Meyer M.B.
      • Nerenz R.D.
      • Lee S.M.
      • Martowicz M.L.
      • Pike J.W.
      Multifunctional enhancers regulate mouse and human vitamin D receptor gene transcription.
      ). Binding sites for CREB and RAR in the U1 and S3 enhancers, respectively, were not determined. To address this issue, we utilized a motif-finding tool (MatInspector, Genomatix) to identify two potential CREB-response elements (CREs) at the peak maximal for CREB in the U1 region, as seen in Fig. 5A, and we created two constructs in a TK promoter-luciferase expression vector, one (U1-2) containing both potential CREs and the second (U1-1) containing only a single CRE. The sequences of the CREs are shown. As seen in Fig. 5B, examination of U1-1 together with an additional version in which the single CRE had been mutated employing transient transfection analysis in MC3T3-E1 cells revealed that CRE1 was indeed active. As documented in Fig. 5C, examination of U1-2 as well as additional versions in which CRE1 and CRE2 were mutated either individually or in combination indicated that both CREs contributed to the activation of the constructs by Fsk. Importantly, cotransfection of a vector expressing a dominant negative version of CREB was capable of strongly reducing Fsk inducible activity of CREB confirming the role of CREB in this activation. Following conceptual identification of a single potential retinoic acid-response element (RARE) in the S3 enhancer, whose location and sequence is depicted in Fig. 5D, we prepared a series of three constructs (S3-1, S3-2, and S3-3) in the same expression vector as illustrated that allowed us to confirm the functionality of this regulatory element as well. As is documented in Fig. 5E, both enhancer constructs that contained the potential RARE remained capable of mediating response to atRA, although the construct in which the potential RARE was removed was unresponsive. Importantly, mutation of the single RARE in construct S3-1, as seen in Fig. 5F, resulted in complete abrogation of response to atRA as well. These experiments document in unequivocal terms the binding sites within S3 and U1 for RAR and CREB, respectively, and demonstrate that they are indeed functional.
      Figure thumbnail gr5
      FIGURE 5.Identification of the DNA sequence elements for Fsk and atRA responses in U1 and S3 enhancers. A and D, schematic structures show the location of each DNA fragment inserted into control reporter, pTK-luc (Empty), to generate the reporters (A, U1-1 and U1-2; D, S3-1, S3-2, and S3-3) as a relative distance (base pair) from TSS of Vdr gene and putative CREs (CRE1 and CRE2; A) and RARE (D) as white boxes. The locations of the putative sites are presented as a relative distance (base pair) from TSS of Vdr gene in parentheses. Wild type (WT) and mutant (MUT1 for CRE1, MUT2 for CRE2, and MUT for RARE) nucleotide sequences (underlined) of the putative sites in U1 (A) and S3 (D) regions are provided. B, C, E, and F, indicated reporter plasmids were cotransfected with β-gal expression vector (B, E, and F) or β-gal expression vector and either A-CREB expression vector (gray or dark green bars, +) or its control vector (white or green bars; −) (C) into MC3T3-E1 cells. Luciferase activity was measured after 24 h of incubation with Fsk (B and C, green or dark green bars, 10−6 m), atRA (E and F, purple bars, 10−6 m), or vehicle controls (Veh; white bars), normalized to β-gal activity in a triplicate set of assays and presented as the mean ± S.E. *, p < 0.05 compared with vehicle control. TK, human thymidine kinase promoter; LUC, luciferase.

      Confirmation of the Role of the Vdr Gene Enhancers S1, S3, and U1 in Mice in Vivo

      The above experiments suggest that Vdr gene enhancers S1, S3, and U1 play both unique as well as overlapping functional roles in the regulation of Vdr gene expression by several hormones in osteoblastic cells in culture. The previous demonstration that a wild type mouse VDR BAC transgene was capable of recapitulating the expression and regulation of the endogenous Vdr gene in mice in vivo (
      • Lee S.M.
      • Bishop K.A.
      • Goellner J.J.
      • O'Brien C.A.
      • Pike J.W.
      Mouse and human BAC transgenes recapitulate tissue-specific expression of the vitamin D receptor in mice and rescue the VDR-null phenotype.
      ) suggested that the roles of S1, S3, and U1 might also be examined in vivo as well. To this end, two separate mouse strains were prepared via injection of mouse VDR BAC clone transgenes documented in Fig. 3A containing deletion of either the U1 enhancer (U1 KO BAC) or both the U1 and the S1 enhancers (U1/S1 KO BAC). Both transgenes were expressed, although the levels of the VDR protein produced were at least 10-fold lower than those expressed endogenously in normal mice or as detected as a result of expression from the wild type mouse VDR BAC transgene (data not shown). As much lower levels of VDR expression than that seen endogenously in normal mice have been found to rescue the phenotype of the VDR null mouse, we crossed the U1/S1 KO BAC mouse into the VDR null mouse background via the breeding scheme used to transfer the wild type VDR BAC transgene into this strain, and we examined the ability of the more complex double mutant transgene to rescue aberrant systemic and skeletal features of the VDR null mouse. In contrast to that of the wild type VDR BAC transgene, neither the systemic features of hypocalcemia nor the consequence of low blood calcium at the level of BMD was fully rescued, as documented in Fig. 6, A and B, respectively, as the mice remained hypocalcemic and exhibited a reduced BMD, although to a lesser degree than that seen in VDR null mice. Interestingly, this level of VDR expression was fully able to prevent the blockade in hair follicle cycling, as evidenced by the absence of alopecia seen in VDR null mice in Fig. 6C. Previous studies have suggested that this feature of VDR action can be mediated by extremely low levels of VDR expression as well (
      • Lee S.M.
      • Goellner J.J.
      • O'Brien C.A.
      • Pike J.W.
      A humanized mouse model of hereditary 1,25-dihydroxyvitamin D-resistant rickets without alopecia.
      ), although the mechanism for this action is unknown. As the regulation of VDR gene expression is likely to be independent of the level of VDR transcript expression from appropriately integrated transgenes, however, we examined the ability of a single injection of 1,25(OH)2D3, atRA, or Bt2cAMP (an additional PKA activator) to up-regulate the expression of the VDR transcript from this mutant transgene. As is documented in Fig. 6, D–F, although VDR transcript expression from both the endogenous mouse gene and its corresponding wild type transgene (in the VDR null mouse background) were fully activated by each of the three regulatory agents, only atRA was capable of up-regulating VDR transcripts from the U1/S1 KO BAC transgene (Fig. 6F). Indeed, response of the transgene in the absence of the U1 and S1 enhancers was either blunted or prevented in response to 1,25(OH)2D3 (Fig. 6D) or to Bt2cAMP (Fig. 6E), respectively. As seen, the use of appropriate positive control genes supported the unique activity of each of the activating agents.
      Figure thumbnail gr6
      FIGURE 6.Phenotypes of ΔU1/S1-BAC/VDR−/− mice and regulation of Vdr expression by hormones in the mice. A and B, serum calcium levels (A) and female and male total body BMDs (B) in wild type littermates (WT, white bars), VDR null (VDR KO, black bars), WT-BAC/VDR−/− (WT-BAC, gray bars), and ΔU1/S1-BAC/VDR−/− (U1/S1-BAC, hatched gray bars) mice were measured at 8 weeks of age. Each value is the average of 5–7 mice per strain ± S.E. *, p < 0.05 compared with wild type mice, #, p < 0.05 compared with VDR null mice. C, images show gross appearance of 6-month-old mice representative of the strains indicated. D–F, WT-BAC/VDR−/− (WT-BAC) and ΔU1/S1-BAC/VDR−/− (U1/S1-BAC) mice and their wild type littermates (WT) were intraperitoneally injected with vehicle (white bars), 1,25(OH)2D3 (D, pale brown bars, 10 ng/g body weight), Bt2cAMP (E, green bars, 0.1 ng/g body weight), or atRA (F, purple bars, 1 ng/g body weight), and expression of Vdr and the positive response genes to each treatment in the indicated tissues were measured following RNA isolation by qPCR. Expression levels of the target gene transcripts were normalized to Gapdh and expressed as the mean for each strain ± S.E. (5–7 mice per group) normalized to the levels measured in the vehicle-treated wild type mouse strain. *, p < 0.05 compared with vehicle-treated sample of each strain.
      Because VDR expression from both the U1 KO BAC and the U1/S1 KO BAC transgenes were similar and the latter was unable to fully rescue the VDR null mouse phenotype, we examined the in vivo regulation of the U1 KO BAC transgene exclusively in the background of the wild type mouse by employing a transgene-selective TaqMan primer. The results of this analysis, as seen in Fig. 7, demonstrate that although response to Bt2cAMP remained absent, almost full recovery of response to 1,25(OH)2D3 and retention of response to atRA was apparent. Although these results do not prove that the S3 enhancer is directly responsible for the activation of the Vdr gene by atRA, they provide strong evidence that the S1 enhancer is a dominant mediator of 1,25(OH)2D3 activity at the Vdr gene and that the U1 enhancer is the exclusive mediator of the PKA pathway.
      Figure thumbnail gr7
      FIGURE 7.Regulation of Vdr expression by hormones in U1 KO BAC transgenic mice. U1 KO BAC transgenic mice (U1 KO-BAC) and wild type littermates (WT) were intraperitoneally injected with vehicle (white bars), 1,25(OH)2D3 (A, 1,25, pale brown bars, 10 ng/g body weight), Bt2cAMP (B, cAMP, green bars, 0.1 ng/g body weight), or atRA (C, purple bars, 1 ng/g body weight). Expression of either endogenous Vdr gene (Vdr) in WT mice or Vdr transcribed from the U1-deleted transgene (HA-Vdr) in the transgenic mice and the positive response genes to each treatment in the indicated tissues were measured following RNA isolation by qPCR. Expression levels of the target gene transcripts were normalized to Gapdh and expressed as the mean for each strain ± S.E. (5–7 mice per group) normalized to the levels measured in the vehicle-treated wild type mouse strain. *, p < 0.05 compared with vehicle-treated sample of each strain.

      Selective Regulation of the Vdr Gene by 1,25(OH)2D3, atRA, and a PKA Activator in the Bone

      The ability of 1,25(OH)2D3, atRA, and PTH to regulate Vdr expression in tissues other than bone in vivo is controversial (
      • Zella L.A.
      • Kim S.
      • Shevde N.K.
      • Pike J.W.
      Enhancers located within two introns of the vitamin D receptor gene mediate transcriptional autoregulation by 1,25-dihydroxyvitamin D3.
      ). To address this issue for primary tissue targets of vitamin D action, we treated normal mice with a single injection of 1,25(OH)2D3, atRA, or Bt2cAMP, isolated RNA from the calvaria, upper small intestine, and the kidney cortex, and examined the ability of these treatments to up-regulate Vdr gene expression. As can be seen in Fig. 8, whereas the Vdr expression was up-regulated by each of the three agents in bone tissue, only 1,25(OH)2D3 was active in the kidney, and neither 1,25(OH)2D3 nor atRA up-regulated Vdr expression in the intestine. As the intestine is not known to contain PTH receptors, we did not examine this tissue for response to Bt2cAMP. Our results suggest that in normal mice at 8 weeks of age, the panel of three agents was active on the Vdr gene primarily in bone with only modest response to 1,25(OH)2D3 in the kidney. This result, however, does not rule out the potential for Vdr regulation in these tissues by these agents under separate physiological or pathophysiological conditions.
      Figure thumbnail gr8
      FIGURE 8.Regulation of endogenous Vdr expression by hormones in wild type mice. Wild type mice (WT) were intraperitoneally injected with vehicle (white bars), 1,25(OH)2D3 (1,25, pale brown bars, 10 ng/g body weight), atRA (purple bars, 1 ng/g body weight), or Bt2cAMP (cAMP, green bars, 0.1 ng/g body weight) as indicated, and the expression of the endogenous Vdr gene in the indicated tissues was measured following RNA isolation by quantitative polymerase chain reaction. Expression levels of the target gene transcripts were normalized to Gapdh and expressed as the mean for each strain ± S.E. (5–7 mice per group) normalized to the levels measured in the vehicle-treated wild type mouse strain. *, p < 0.05 compared with vehicle-treated sample.

      ChIP-seq Analysis and the Presence of DHSs Reveal Tissue-selective Sets of Regulatory Enhancers in the Vdr Gene Locus

      In a final set of experiments, we explored the underlying basis for this selective regulation of Vdr gene expression by examining VDR binding activity across the Vdr gene in the kidney cortex using ChIP-seq analysis, and we contrasted the binding sites identified with those observed in bone cells. The distribution of histone marks across the Vdr gene locus in the kidney that signify the presence of regulatory enhancers (H3K4me1) or reflect changes in chromatin architecture and function in response to 1,25(OH)2D3 (H3K9ac and H3K27 acetylation (H3K27ac)) was similarly evaluated in parallel. Accordingly, normal (vitamin D-sufficient) mice were treated with a single dose of 1,25(OH)2D3, and the kidney cortex was isolated 1 h later and subjected to ChIP-seq analysis using antibodies to the VDR, H3K4me1, H3K9ac, and H3K27ac. As can be seen in Fig. 9A, strong 1,25(OH)2D3-inducible VDR binding was clearly observed at S1 in the kidney as well as at several additional sites nearby, which included a site at +27 kb. Importantly, most of these sites exhibited some residual VDR binding in the absence of exogenously added 1,25(OH)2D3, presumably due to the presence of endogenous ligand in vitamin D-sufficient animals. Interestingly, VDR was not seen bound to the S3 or U1 regions but was strongly bound to several regions located further upstream at −30, −35, and −37 kb (Fig. 9A, highlighted in pale yellow). Virtually all sites of VDR binding, including those located upstream at −30, −35, and −37 kb, were not only strongly enriched for the H3K4me1 enhancer signature mark but also for the chromatin activity marks H3K9ac and H3K27ac as well. Surprisingly, these upstream sites (Fig. 9A, highlighted) did not align with those seen in mesenchymal cells. Whether these unique upstream sites represent enhancers that can regulate Vdr gene expression is unknown, although it is clear that the presence of 1,25(OH)2D3 promotes an elevation in the H3K27ac mark. These sites are also upstream of neighboring Tmem106c; however, this gene is not regulated by 1,25(OH)2D3 (data not shown). Interestingly, the locations of all of these histone marks are also uniquely characterized as hotspots or sites of DNase I hypersensitivity in kidney obtained from similarly aged C57BL/6 mice using data made available by Professor John Stammatoyannopoulos and co-workers at the University of Washington as part of the ENCODE Consortium and documented on the UCSC Genome Browser website (ENCODE Data Coordination Center) (
      • Dunham I.
      • Kundaje A.
      • Aldred S.F.
      • Collins P.J.
      • Davis C.A.
      • Doyle F.
      • Epstein C.B.
      • Frietze S.
      • Harrow J.
      • Kaul R.
      • Khatun J.
      • Lajoie B.R.
      • Landt S.G.
      • Lee B.K.
      • Pauli F.
      • et al.
      An integrated encyclopedia of DNA elements in the human genome.
      ). Importantly, as seen in Fig. 9A, all the VDR-binding sites, including those at S1 as well as those located upstream at −30, −35, and −37 kb, exhibited DHS. Although it is possible that the VDR itself is responsible for this hypersensitivity because the mice were vitamin D-sufficient and therefore contain VDR bound at these sites, it is more likely that DHSs are influenced by prebound protein complexes that serve to displace nucleosomes, alter chromatin architecture, and enable VDR accessibility. The presence of VDR at the dominant S1 enhancer in the kidney and its absence at S3 and U1 may explain the modest response of the Vdr gene to 1,25(OH)2D3 seen in the kidney in vivo, although it is possible that the more distal binding activity of the VDR could contribute as well. Whether functional S3 and U1 enhancers (irrespective of VDR binding) are present is unclear, although neither enhancers appear to be strongly marked by appropriate histone modifications nor are they marked by DHS. It is possible that the loss of chromatin access at these sites underlies that loss of response to atRA and PTH and reduces response to 1,25(OH)2D3.
      Figure thumbnail gr9
      FIGURE 9.Cistrome of Vdr gene in mouse tissues in vivo. A, representative ChIP-seq tracks of triplicate samples for the VDR and the indicated histone marks and DHS profiles (ENCODE Consortium) at mouse Vdr gene locus in kidney are presented. B, representative ChIP-seq tracks of triplicate samples for the VDR and DHS profiles at mouse Vdr gene locus in intestine are presented. C, DHS profiles of the indicated tissues are provided. Bindings of the VDR and the modified histones in vehicle- and 1,25(OH)2D3-treated samples are shown in yellow and blue, respectively. Overlapped binding activity is shown in green. DHSs (DHS-seq) are shown in brown. The y axes represent tag densities normalized to input and 107 tags for ChIP-seq or raw taq densities for DHS-seq. Exons and introns of Vdr and Tmem106c genes are shown in boxes and lines, respectively, above the tracks. Transcriptional direction of a gene is indicated by an arrow at TSS, and genomic location and scale are provided. Maximum height of tag density for the data track is indicated on each track. The bone enhancer regions (S1, S3, and U1) are indicated below the tracks. Additional potential enhancer regions are boxed and highlighted in pale yellow.
      A similar ChIP-seq analysis limited exclusively to the VDR was conducted using intestinal epithelial cells. As can be seen in Fig. 9B, VDR binding was not apparent at the bone cell enhancers S1, S3, or U1, perhaps corresponding to the inability of 1,25(OH)2D3 treatment to up-regulate the Vdr gene in the intestine in vivo. Novel sites of significant VDR binding were observed within the first intron at +4.6 kb, however, and upstream at not only −10 and −20 kb but also at −37 kb, which was also identified in kidney cells as well. It is unclear why this distal binding activity is unable to mediate up-regulation of the Vdr gene in the intestine, although perhaps inducibility requires simultaneous coactivation through additional pathways or under alternative physiological conditions. Although the levels of histone modification have yet to be established in the intestine, the results in Fig. 9B show that these regions of VDR binding are also sites of DHS as well. Overall, these data reveal that sites of VDR binding at specific gene loci can differ substantially in a highly tissue/cell type-dependent manner.

      Lack of DHS at the Vdr Gene in Certain Tissues Highlights the Potential for VDR Expression in a Limited Cell Type(s) within Tissues

      Differences in the apparent locations of VDR-binding sites in key Vdr gene-expressing tissues such as bone, intestine, and kidney, the presence of histone signatures that highlight these and additional enhancer sites in a tissue-selective manner, and the strong correlation between these regions and DHS suggest that the distribution of the latter features at the Vdr gene locus may be useful in predicting the overall level of Vdr gene expression in tissues and that the level of histone modifications may also be useful in that prediction as well. We therefore examined the DHS profiles that characterize the Vdr gene in liver, spleen, muscle, and brain as documented in Fig. 9C. As can be seen, sites present in the regulatory regions and the Vdr promoter in bone, kidney, and intestine (FIGURE 2., FIGURE 9., A and B) are almost completely absent in each of these tissues. The absence of these sites correlates directly with the very low levels of VDR known to be expressed in liver, spleen, muscle, and brain and suggest that chromatin access may represent a primary determinant of the overall expression of the VDR in these tissues.

      Discussion

      In this study, we describe experiments aimed at understanding the enhancer determinants of Vdr gene expression in bone cells and other primary target tissues that orchestrate the regulation of mineral homeostasis in response to 1,25(OH)2D3. Early studies identified three distal intronic/intergenic enhancers within the Vdr gene locus in osteoblasts that mediate the autoregulatory actions of 1,25(OH)2D3 and the independent regulatory actions of PTH and atRA as well (
      • Zella L.A.
      • Meyer M.B.
      • Nerenz R.D.
      • Lee S.M.
      • Martowicz M.L.
      • Pike J.W.
      Multifunctional enhancers regulate mouse and human vitamin D receptor gene transcription.
      ,
      • Zella L.A.
      • Kim S.
      • Shevde N.K.
      • Pike J.W.
      Enhancers located within two introns of the vitamin D receptor gene mediate transcriptional autoregulation by 1,25-dihydroxyvitamin D3.
      ). The current studies using ChIP-seq analysis confirm the presence of these regulatory sites not only in osteoblastic cells but in their mesenchymal stem cell precursors that also give rise to chondrocytes and adipocytes. Importantly, each of these regulatory sites exhibited a significant enrichment in H3K4me1 that represents the signature of an enhancer structure together with changes in the level of H3K9ac in response to 1,25(OH)2D3 that are indicative of chromatin decondensation and increased gene activity. Based upon the above findings, we confirmed the functional roles and the identity of the transcription factor binding sites within these enhancers using BAC clone stable cell lines, CRISPR/Cas9-mediated enhancer-deleted daughter cell lines, and transient transfection analysis, as well as through the generation of mice transgenic for either wild type or mutant VDR transgenes that contained specific enhancer deletions. In a final set of experiments, we used ChIP-seq analysis to contrast genetic and epigenetic features of the Vdr gene locus described in bone cells with those found in the kidney and intestinal epithelial cells of mice in vivo. These studies revealed striking differences in the enhancer landscape across the Vdr gene locus between bone, kidney, and intestine, differences that were evident at the level of VDR binding, histone enhancer signature marks, and DHS. Differences in VDR binding at the Vdr gene locus in response to 1,25(OH)2D3 activation in vivo correlated with the limited effect of the hormone to induce Vdr gene expression in the kidney and the absence of response observed in the intestine. In both tissues, however, a series of additional VDR-binding sites was observed that were unique to either the intestine, the kidney, or both and were also marked by enhancer histone signatures and DHS. Although the roles of these sites are currently unknown, the likelihood that these regions participate in Vdr gene regulation in these tissues is high, although perhaps under different developmental or physiological settings. We conclude that although expression of the Vdr gene is certain to be regulated in a cell type-specific fashion by many different transcription factors that act at unique binding sites within the Vdr gene locus, the gene may also be selectively regulated in different cell types by the VDR through the use of unique sets of cell type-specific enhancers whose access can be determined via chromatin architecture.
      Our results point to the unequivocal role of three enhancers within the Vdr gene locus in mediating both the autoregulatory actions of 1,25(OH)2D3 on Vdr expression and the regulatory actions of atRA and PKA activators in two bone cell lines as well as in a precursor MSC that generically gives rise to osteoblasts as well as other mesenchymal cell types, including adipocytes. Interestingly, a similar enhancer profile was also seen at terminal differentiation in osteoblast-derived osteocytes (
      • St John H.C.
      • Bishop K.A.
      • Meyer M.B.
      • Benkusky N.A.
      • Leng N.
      • Kendziorski C.
      • Bonewald L.F.
      • Pike J.W.
      The osteoblast to osteocyte transition: epigenetic changes and response to the vitamin D3 hormone.
      ). The experiments documented herein using both BAC clones and a series of enhancer-deleted cell lines show definitively that the VDR binds to the enhancers S1, S3, and U1 and that atRA and PKA activators, via RAR and CREB, activate uniquely S3 and U1. Although previous enhancer mutagenesis studies identified a functional VDRE within the S1 region (
      • Zella L.A.
      • Kim S.
      • Shevde N.K.
      • Pike J.W.
      Enhancers located within two introns of the vitamin D receptor gene mediate transcriptional autoregulation by 1,25-dihydroxyvitamin D3.
      ), current studies have now identified both the single RARE located within the S3 region and two CREs located within the U1 enhancer. The regulation of the VDR gene by atRA was first localized in the human VDR gene to a region that is consistent with that identified as S3 (
      • Miyamoto K.
      • Kesterson R.A.
      • Yamamoto H.
      • Taketani Y.
      • Nishiwaki E.
      • Tatsumi S.
      • Inoue Y.
      • Morita K.
      • Takeda E.
      • Pike J.W.
      Structural organization of the human vitamin D receptor chromosomal gene and its promoter.
      ), although the RARE within was not identified at that time and remains currently unresolved. It is certainly possible, however, that additional sites of transcription factor binding within these regions may also be present, given the uncertainties that accompany the analyses of enhancer activity via transient transfection analysis. Indeed, we have identified several potential VDRE motifs in both S3 and U1, although none of these appeared to be active when assessed independently via the enhancer assays utilized in the studies above (
      • Zella L.A.
      • Meyer M.B.
      • Nerenz R.D.
      • Lee S.M.
      • Martowicz M.L.
      • Pike J.W.
      Multifunctional enhancers regulate mouse and human vitamin D receptor gene transcription.
      ). Perhaps most important is the confirmation that S1 and U1 participate in the regulation of the Vdr gene by 1,25(OH)2D3 and PKA activators, respectively, in the mouse in vivo, and by inference that S3 mediates atRA activity in the bone in vivo as well. This confirmation is important in view of the fact that studies are emerging to suggest that the altered environment of many primary cells placed in culture can impact the epigenome and alter sites of regulation (
      • Romanoski C.E.
      • Link V.M.
      • Heinz S.
      • Glass C.K.
      Exploiting genomics and natural genetic variation to decode macrophage enhancers.
      ). Whether these hormone-regulated enhancers contribute to the basal expression of the Vdr gene in bone cells in vivo is unknown given the problems associated with the preparation and analysis of recombinant activity in mice carrying different transgenes with varied copy numbers and with different sites of integration. The lack of effect of enhancer deletion on the basal activity of the Vdr gene in CRISPR/Cas9-derived UAMS-PB daughter cells, however, would suggest that these enhancers likely have limited impact on basal expression in bone and perhaps in the kidney and raise the question as to the location and nature of those that control this function. Regardless, these studies support the original hypothesis that at least three distal enhancers located within introns as well as upstream are key to the hormonal regulation of the Vdr gene in bone.
      The unique distribution of VDR binding activity across the Vdr gene in the kidney and intestine and the generally unique profiles of VDR binding relative to that seen in bone cells were surprising. Accordingly, the VDR was not seen at any of the three bone-specific sites in the intestine but was observed at S1 in the kidney. These profiles appear to correlate with the modest ability of 1,25(OH)2D3 to up-regulate Vdr expression in kidney under normal conditions in mice and the lack of the hormone's ability to up-regulate Vdr expression in the intestine. The ability of 1,25(OH)2D3 to autoregulate the Vdr gene in tissues other than bone in vivo is controversial, although this hormone is known to up-regulate Vdr expression in other cell types in culture, including not only those of mesenchymal but of hematopoietic and epithelial origin as well. The underlying cis mechanisms for the regulation of the VDR in these cell types are unknown. Interestingly, VDR binding at the Vdr gene locus in the kidney and intestine was noted within the first intron and at multiple intergenic sites upstream of U1; in some cases, these overlapped within the two tissues, and in others they did not. As all of these sites in the kidney were characterized by histone modifications indicative of enhancers, it seems likely that VDR binding simply highlights the presence of legitimate enhancers despite the lack of our understanding of the receptor's role at these sites. Equally important, virtually all of these regions in the kidney, whether VDR was bound or not, were also characterized by the annotated presence of DHS. Although the epigenetic histone landscape across the Vdr gene in the intestine has not yet been evaluated, VDR-binding sites in that tissue were also highlighted by DHS, with the caveat that the latter was conducted in tissue from the lower intestine rather than the upper intestine as evaluated herein. As many genes are similarly regulated by 1,25(OH)2D3 in different segments of the intestine (
      • Lee S.M.
      • Riley E.M.
      • Meyer M.B.
      • Benkusky N.A.
      • Plum L.A.
      • DeLuca H.F.
      • Pike J.W.
      1,25-Dihydroxyvitamin D3 controls a cohort of vitamin D receptor target genes in the proximal intestine that is enriched for calcium-regulating components.
      ), we have no reason to believe important differences would exist at the Vdr gene locus. We speculate that all of the genomic activities identified in the more distal upstream intergenic portions of the mouse Vdr gene seen here are reminiscent of significant epigenetic histone activity seen in previous ChIP-chip studies of the highly homologous human VDR gene (
      • Zella L.A.
      • Meyer M.B.
      • Nerenz R.D.
      • Lee S.M.
      • Martowicz M.L.
      • Pike J.W.
      Multifunctional enhancers regulate mouse and human vitamin D receptor gene transcription.
      ). This may be important because very early studies of VDR expression in human tissues suggested the presence of low abundance transcripts that contained apparent exonic sequence originating from this far upstream region of the gene (
      • Crofts L.A.
      • Hancock M.S.
      • Morrison N.A.
      • Eisman J.A.
      Multiple promoters direct the tissue-specific expression of novel N-terminal variant human vitamin D receptor gene transcripts.
      ). Detection of these sequences was specific to normal kidney and from an intestinal carcinoma as well as parathyroid gland adenoma (
      • Crofts L.A.
      • Hancock M.S.
      • Morrison N.A.
      • Eisman J.A.
      Multiple promoters direct the tissue-specific expression of novel N-terminal variant human vitamin D receptor gene transcripts.
      ). Although these data suggest the possibility of a second uniquely active VDR gene promoter, current awareness of enhancer-mediated enhancer RNA production and the structural and functional similarities between enhancers and promoters (
      • Kim T.K.
      • Shiekhattar R.
      Architectural and functional commonalities between enhancers and promoters.
      ) suggests that additional studies will be necessary to resolve the nature of this far upstream activity in both the mouse and human genes.
      It is particularly interesting that DHS profiles across the Vdr gene locus reflect the presence of either regulatory enhancers or open chromatin structure in the kidney as highlighted by the presence of specific histone modifications. In addition, although analyses of these modifications in the intestine are not yet available, DHS in the lower intestine also aligns with virtually all the sites to which the VDR binds in the upper intestine. Finally, although DHSs have not been obtained for bone cells, the similarity between DHSs in cells of the mesenchymal lineage such as fibroblasts and fat cells (see the data available on the UCSC Genome Browser website) (
      • Dunham I.
      • Kundaje A.
      • Aldred S.F.
      • Collins P.J.
      • Davis C.A.
      • Doyle F.
      • Epstein C.B.
      • Frietze S.
      • Harrow J.
      • Kaul R.
      • Khatun J.
      • Lajoie B.R.
      • Landt S.G.
      • Lee B.K.
      • Pauli F.
      • et al.
      An integrated encyclopedia of DNA elements in the human genome.
      ) and the locations of histone modifications in osteoblastic cell lines suggest that the DHSs that are available are also aligned with these modifications as well. As DHS is likely due to the presence of chromatin and/or DNA-binding protein complexes and nucleosome absence, these profiles appear to be unique to specific cell types. Indeed, at the Vdr gene locus they probably reflect the potential for both the gene's basal as well as regulated transcriptional activity in tissues known to express the VDR in a robust manner, such as intestine, kidney, bone, and the parathyroid glands as well as many others. If this hypothesis is correct, the absence of a similar profile across this gene in tissues such as muscle, brain, liver, and certain immune cells would lead to the prediction that VDR expression is either uniquely regulated in those tissues or that its expression levels are exceedingly low. Indeed, direct analysis of VDR expression in tissues such as muscle and brain as well as the difficulty of detecting the VDR via Western blot analysis or by immunohistochemistry in these tissues provide strong support for the latter interpretation. As the functional genomic activity of the VDR is likely highly dependent in biochemical terms upon the overall expression of a threshold level of VDR protein, it seems unlikely that this low level of the VDR could represent equal dispersion across all cell types in complex tissues such as muscle and brain. Thus, we speculate that VDR expression as measured in those tissues and as assessed by unique DHS profiles must be much higher but restricted to a limited subset of cells within the tissue or organ being analyzed. Recent studies support this idea by revealing that the VDR may be localized to stellate cells of the liver (
      • Ding N.
      • Yu R.T.
      • Subramaniam N.
      • Sherman M.H.
      • Wilson C.
      • Rao R.
      • Leblanc M.
      • Coulter S.
      • He M.
      • Scott C.
      • Lau S.L.
      • Atkins A.R.
      • Barish G.D.
      • Gunton J.E.
      • Liddle C.
      • et al.
      A vitamin D receptor/SMAD genomic circuit gates hepatic fibrotic response.
      ), myoblasts of the muscle (
      • Girgis C.M.
      • Mokbel N.
      • Cha K.M.
      • Houweling P.J.
      • Abboud M.
      • Fraser D.R.
      • Mason R.S.
      • Clifton-Bligh R.J.
      • Gunton J.E.
      The vitamin D receptor (VDR) is expressed in skeletal muscle of male mice and modulates 25-hydroxyvitamin D (25OHD) uptake in myofibers.
      ), and specific neurons in the brain (
      • Cui X.
      • Pelekanos M.
      • Liu P.Y.
      • Burne T.H.
      • McGrath J.J.
      • Eyles D.W.
      The vitamin D receptor in dopamine neurons; its presence in human substantia nigra and its ontogenesis in rat midbrain.
      ). In these tissues, detection of the VDR protein by immunocytochemistry suggests that the levels of the receptor may be equivalent to those seen in cells of bona fide 1,25(OH)2D3 tissue targets such as intestine, kidney, and bone. At the very least, however, the correlation between DHS and histone modifications provides the likely identification of sites within the Vdr gene that might serve as regulatory foci, and motifs within these sites could provide clues as to the transcription factors that are involved. Our current studies lead to the realization that individual genes may be differentially regulated by the same transcription factor in different cell types through utilization of unique enhancers made available within the gene locus through selective chromatin access. Accordingly, future studies will be needed to confirm this hypothesis.
      In summary, we have identified key regulatory regions within the mouse Vdr gene locus that permit the up-regulation of Vdr expression by 1,25(OH)2D3, atRA, and PKA activators (PTH) in bone cells. Surprisingly, only one of these VDR binding regions is conserved in the kidney, and none are conserved in the intestine. This finding is consistent with the observation that 1,25(OH)2D3 only modestly up-regulates Vdr expression in the kidney and has no effect on expression in the intestine in vivo. Unexpectedly, VDR binding was noted at many unique sites upstream of those identified and confirmed in bone, sites that show hypersensitivity to DNase I treatment and are decorated by key histone modifications that are consistent with the presence and activity of enhancers. Although the function of these sites is currently unknown, it is clear that DHS data could be used to predict the overall expression of the Vdr gene in potential target tissues.

      Author Contributions

      S. M. L. and J. W. P. conceived the study and wrote the paper. S. M. L. performed the experiments with cell lines and animals and organized all the data. M. B. M. processed and interpreted the ChIP-seq data. N. A. B. provided technical assistance for ChIP and preparation of sequencing library. C. A. O. generated transgenic mouse strains. All authors reviewed the results and approved the final version of the manuscript.

      Acknowledgments

      We acknowledge the ENCODE Consortium and the Stammatoyannopoulos group at the University of Washington for DNase I hypersensitivity data sets. We thank members of the Pike laboratory for their helpful contributions to this work. We also acknowledge David Nehls, Regina Berget and Douglas Jacobson for the animal husbandry associated with this study.

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