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A kidney-specific genetic control module in mice governs endocrine regulation of the cytochrome P450 gene Cyp27b1 essential for vitamin D3 activation

      The vitamin D endocrine system regulates mineral homeostasis through its activities in the intestine, kidney, and bone. Terminal activation of vitamin D3 to its hormonal form, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), occurs in the kidney via the cytochrome P450 enzyme CYP27B1. Despite its importance in vitamin D metabolism, the molecular mechanisms underlying the regulation of the gene for this enzyme, Cyp27b1, are unknown. Here, we identified a kidney-specific control module governed by a renal cell-specific chromatin structure located distal to Cyp27b1 that mediates unique basal and parathyroid hormone (PTH)-, fibroblast growth factor 23 (FGF23)-, and 1,25(OH)2D3-mediated regulation of Cyp27b1 expression. Selective genomic deletion of key components within this module in mice resulted in loss of either PTH induction or FGF23 and 1,25(OH)2D3 suppression of Cyp27b1 gene expression; the former loss caused a debilitating skeletal phenotype, whereas the latter conferred a quasi-normal bone mineral phenotype through compensatory homeostatic mechanisms involving Cyp24a1. We found that Cyp27b1 is also expressed at low levels in non-renal cells, in which transcription was modulated exclusively by inflammatory factors via a process that was unaffected by deletion of the kidney-specific module. These results reveal that differential regulation of Cyp27b1 expression represents a mechanism whereby 1,25(OH)2D3 can fulfill separate functional roles, first in the kidney to control mineral homeostasis and second in extra-renal cells to regulate target genes linked to specific biological responses. Furthermore, we conclude that these mouse models open new avenues for the study of vitamin D metabolism and its involvement in therapeutic strategies for human health and disease.

      Introduction

      The vitamin D endocrine system serves to regulate mineral homeostasis through its actions in the intestine, kidney, and bone (
      • DeLuca H.F.
      Overview of general physiologic features and functions of vitamin D.
      ). Vitamin D3 itself is sequentially activated via two specific chemical modifications that occur first in the liver by CYP2R1 to 25(OH)D3
      The abbreviations used are: 25(OH)D3
      25-hydroxyvitamin D3
      1,25(OH)2D3
      1α,25-dihydroxyvitamin D3
      PTH
      parathyroid hormone
      qPCR
      quantitative real-time PCR
      NRTC
      non-renal target cell
      TPTG
      thyroparathyroid gland
      PTG
      parathyroid gland
      ANOVA
      analysis of variance
      CREB
      cAMP-response element-binding protein
      pCREB
      phosphorylated CREB
      VDR
      vitamin D receptor
      LOD
      limit of detection
      bw
      body weight
      BMD
      bone mineral density
      μCT
      micro-computed tomography
      ChIP-seq
      ChIP-sequencing
      DHS
      DNase I hypersensitivity sequencing
      KS-ERM
      kidney-specific endocrine regulatory module
      NRTC-RM
      non-renal target cell regulatory module
      pol
      polymerase
      MSC
      mesenchymal stem cell
      CTCF
      CCCTC-binding factor.
      and then in the kidney by CYP27B1 to 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active hormonal form of the vitamin (
      • Jones G.
      • Prosser D.E.
      • Kaufmann M.
      Cytochrome P450-mediated metabolism of vitamin D.
      ). Blood levels of 1,25(OH)2D3 are also determined by rates of catabolism that represent the primary function of CYP24A1 in the kidney (
      • Jones G.
      • Prosser D.E.
      • Kaufmann M.
      25-Hydroxyvitamin D-24-hydroxylase (CYP24A1): its important role in the degradation of vitamin D.
      ). CYP27B1 is also expressed at low levels in many non-renal target cells (NRTCs), particularly those of skin and the immune system, where local production of 1,25(OH)2D3 has been suggested to preferentially influence the many pleiotropic, non-calcemic functions of 1,25(OH)2D3 (
      • Adams J.S.
      • Hewison M.
      Extrarenal expression of the 25-hydroxyvitamin D-1-hydroxylase.
      ,
      • Bikle D.D.
      Vitamin D metabolism and function in the skin.
      ). Neither the mechanisms nor the overall biological impact of these non-renal cellular sources of 1,25(OH)2D3 are clear, particularly in the context of circulating 1,25(OH)2D3, which is believed to be derived exclusively from the kidney. Despite these uncertainties, the regulated expression of Cyp27b1 and Cyp24a1 is well-recognized as central to the overall biological activity of the vitamin D endocrine system.
      Cyp27b1 expression is known to be controlled in the kidney by many different factors. PTH represents the primary inducer (
      • DeLuca H.F.
      Parathyroid hormone as a trophic hormone for 1,25-dihydroxyvitamin D3, the metabolically active form of vitamin D.
      ,
      • Garabedian M.
      • Holick M.F.
      • Deluca H.F.
      • Boyle I.T.
      Control of 25-hydroxycholecalciferol metabolism by parathyroid glands.
      ), whereas both 1,25(OH)2D3 (
      • Tanaka Y.
      • DeLuca H.
      Rat renal 25-hydroxyvitamin D3 1- and 24-hydroxylases: their in vivo regulation.
      ) and FGF23 (
      • Shimada T.
      • Hasegawa H.
      • Yamazaki Y.
      • Muto T.
      • Hino R.
      • Takeuchi Y.
      • Fujita T.
      • Nakahara K.
      • Fukumoto S.
      • Yamashita T.
      FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis.
      ,
      • Shimada T.
      • Kakitani M.
      • Yamazaki Y.
      • Hasegawa H.
      • Takeuchi Y.
      • Fujita T.
      • Fukumoto S.
      • Tomizuka K.
      • Yamashita T.
      Targeted ablation of Fgf23 demonstrates an essential physiological role of FGF23 in phosphate and vitamin D metabolism.
      ) represent the major negative regulators of Cyp27b1 expression. Indeed, the ability of PTH, FGF23, and 1,25(OH)2D3 to modulate the expression of Cyp27b1 in the kidney comprises a key element of regulatory signaling that links adaptive vitamin D metabolism to the maintenance of mineral homeostasis. Interestingly, renal expression of Cyp24a1 is reciprocally regulated by these same hormones; PTH suppresses and both 1,25(OH)2D3 and FGF23 induce Cyp24a1 (
      • Chandler J.S.
      • Chandler S.K.
      • Pike J.W.
      • Haussler M.R.
      1,25-Dihydroxyvitamin D3 induces 25-hydroxyvitamin D3–24-hydroxylase in a cultured monkey kidney cell line (LLC-MK2) apparently deficient in the high affinity receptor for the hormone.
      • Zierold C.
      • Darwish H.M.
      • DeLuca H.F.
      Two vitamin D response elements function in the rat 1,25-dihydroxyvitamin D24-hydroxylase promoter.
      ,
      • Quarles L.D.
      Skeletal secretion of FGF-23 regulates phosphate and vitamin D metabolism.
      • Kaufmann M.
      • Lee S.M.
      • Pike J.W.
      • Jones G.
      A high-calcium and phosphate rescue diet and VDR-expressing transgenes normalize serum vitamin D metabolite profiles and renal Cyp27b1 and Cyp24a1 expression in VDR null mice.
      ). This regulatory paradigm in the kidney highlights the critical importance of the coordinated control of both Cyp27b1 and Cyp24a1 expression by these endocrine hormones not only in the maintenance of circulating 1,25(OH)2D3, but in the orchestration of normal mineral homeostasis as well.
      Despite the central importance of these two genes in vitamin D metabolism, little is known of the molecular mechanisms that underpin Cyp27b1 regulation in the kidney by PTH, FGF23, or 1,25(OH)2D3 at the genomic level or of the adaptive relationship that occurs between this modulation and that of Cyp24a1 expression. It has been shown that PTH action at Cyp27b1 involves the PKA signaling pathway and likely the CREB transcription factor (
      • Armbrecht H.J.
      • Hodam T.L.
      • Boltz M.A.
      Hormonal regulation of 25-hydroxyvitamin D3–1α-hydroxylase and 24-hydroxylase gene transcription in opossum kidney cells.
      ) and that 1,25(OH)2D3 action involves the nuclear vitamin D receptor (VDR) (
      • Pike J.W.
      • Meyer M.B.
      • Benkusky N.A.
      • Lee S.M.
      • St John H.
      • Carlson A.
      • Onal M.
      • Shamsuzzaman S.
      Genomic determinants of vitamin D-regulated gene expression.
      ). This contrasts with the actions of the more recently discovered phosphaturic hormone FGF23, where limited insight has been gained with regard to the identities of both the FGF receptor isoform(s) and the transcription factor(s) that are involved in Cyp27b1 expression (
      • Farrow E.G.
      • Davis S.I.
      • Summers L.J.
      • White K.E.
      Initial FGF23-mediated signaling occurs in the distal convoluted tubule.
      ,
      • Portale A.A.
      • Zhang M.Y.
      • David V.
      • Martin A.
      • Jiao Y.
      • Gu W.
      • Perwad F.
      Characterization of FGF23-dependent Egr-1 cistrome in the mouse renal proximal tubule.
      ). Importantly, research over the past decade using unbiased genomic techniques now points to the likelihood that genes such as Cyp27b1 may be regulated via genomic control regions located distal to their transcriptional start sites, a principle that we have found relevant to the vitamin D system (
      • Meyer M.B.
      • Benkusky N.A.
      • Sen B.
      • Rubin J.
      • Pike J.W.
      Epigenetic plasticity drives adipogenic and osteogenic differentiation of marrow-derived mesenchymal stem cells.
      ,
      • Pike J.W.
      • Meyer M.B.
      Regulation of mouse Cyp24a1 expression via promoter-proximal and downstream-distal enhancers highlights new concepts of 1,25-dihydroxyvitamin D(3) action.
      ). The absence of fundamental mechanistic insight relative to Cyp27b1 regulation has impeded progress in fully understanding the vitamin D metabolic system, its role in orchestrating mineral homeostasis, and the pathological impact of alterations in this system that can occur in a wide variety of human maladies, including autoimmune diseases and cancer.
      Herein, we focused our efforts on understanding the expression and regulation of Cyp27b1 in the mouse. Our results identify a complex, multicomponent endocrine regulatory module specific to the kidney that is governed by a chromatin structure that is absent in NRTCs. This module regulates both the basal expression of Cyp27b1 in the kidney as well as its differential modulation by PTH, FGF23, and 1,25(OH)2D3, but it does not control the expression of Cyp27b1 in NRTCs by inflammatory agents such as LPS. These results highlight the differential regulation of Cyp27b1 in vivo and provide a starting point for further delineation of the precise molecular and genomic mechanisms through which both renal and non-renal tissues control the expression of Cyp27b1, production of 1,25(OH)2D3, and therefore its local and endocrine biology.

      Results

       VDR and pCREB are bound upstream of the Cyp27b1 gene in a kidney-specific manner

      Dose- and time-optimized i.p. injections for an acute maximal gene expression response by PTH, FGF23, and 1,25(OH)2D3 into wild-type C57BL/6 (WT) mice lead to a dramatic up-regulation of Cyp27b1 transcripts in the kidney in response to PTH and a significant suppression in response to both FGF23 and 1,25(OH)2D3 (Fig. 1A). Cyp24a1 expression in the kidney, in contrast, is reciprocally suppressed by PTH and induced by both FGF23 and 1,25(OH)2D3 (Fig. 1B). Based upon this regulatory paradigm and the absence of insight into transcriptional activities, we utilized ChIP-seq analysis of the mouse kidney to identify the sites of action of both PTH and 1,25(OH)2D3 at the Cyp27b1 gene locus (the FGF23-activated transcription factor(s) has yet to be identified). ChIP-seq analysis of pCREB and VDR in kidneys of WT mice 1 h after a single injection (a time point previously optimized for primary genomic effects (
      • Meyer M.B.
      • Zella L.A.
      • Nerenz R.D.
      • Pike J.W.
      Characterizing early events associated with the activation of target genes by 1,25-dihydroxyvitamin D3 in mouse kidney and intestine in vivo.
      )) of either vehicle or cognate hormone revealed the presence of pCREB and VDR bound to DNA at the Cyp27b1 locus (Fig. 1, C and D). The former was pre-bound to DNA and generally unaffected by PTH, whereas DNA binding of the latter was largely reinforced upon 1,25(OH)2D3 injection at a single overlapping site within an intron of the upstream Mettl1 gene and at three sites spanning the large intron located in the Mettl21b gene (Fig. 1, C and D, highlighted by gray vertical boxes). Both genes produce methyltransferase-like proteins that are poorly characterized (
      • Cartlidge R.A.
      • Knebel A.
      • Peggie M.
      • Alexandrov A.
      • Phizicky E.M.
      • Cohen P.
      The tRNA methylase METTL1 is phosphorylated and inactivated by PKB and RSK in vitro and in cells.
      ,
      • Malecki J.
      • Aileni V.K.
      • Ho A.Y.Y.
      • Schwarz J.
      • Moen A.
      • Sørensen V.
      • Nilges B.S.
      • Jakobsson M.E.
      • Leidel S.A.
      • Falnes P.
      The novel lysine specific methyltransferase METTL21B affects mRNA translation through inducible and dynamic methylation of Lys-165 in human eukaryotic elongation factor 1α (eEF1A).
      ). Neither pCREB nor VDR was observed immediately upstream of the Cyp27b1 gene's transcriptional start site or in the introns of Mettl1 or Mettl21b in marrow-derived mesenchymal stem cells (MSCs) (Fig. 1, C and D, gray track) following identical hormonal treatment. We also performed a ChIP-seq analysis of the transcription-confirming histone mark H3K36me3 1 h after injection (Fig. 2A). Modulation of H3K36me3 showed that each of these hormones, including FGF23, mediated direct regulation of the Cyp27b1 gene body in the kidney. Equally important, these experimental results identified potential sites of regulatory action for at least two of the three primary mineral-regulating hormones at the Cyp27b1 gene.
      Figure thumbnail gr1
      Figure 1Cyp27b1 expression and genomic occupancy. Gene expression of Cyp27b1 (A) and Cyp24a1 (B) after treatment in 8–9-week-old C57BL/6 wild-type mice with ethanol/PBS vehicle (Veh, gray, n = 6), 230 ng/g bw PTH for 1 h (PTH, blue, n = 6), 10 ng/g bw 1,25(OH)2D3 (1,25D3, black, n = 6) for 6 h, or 50 ng/g bw FGF23 for 3 h (FGF23, green, n = 6). Data are displayed as relative quantitation (RQ, mean ± S.E.) compared with Gapdh. *, p < 0.05 paired t test, treatment versus vehicle. Overlaid ChIP-seq data tracks for pCREB (C), VDR (D), H3K4me1 (E), and H3K9ac (F) from mouse kidney displayed as either basal or vehicle (yellow, n = 3) or treated (blue, n = 3) with PTH, 1,25(OH)2D3, or FGF23 as indicated with concentrations used in A and with a treatment time of 1 h. G, H3K9ac data in Cyp27b1KO mice (C27KO, blue) or VDRKO (blue) versus wild-type (WT, yellow). Overlapping track data appear as green. Regions of interest are highlighted in light gray boxes. Genomic location and scale are indicated (top), and maximum height of tag sequence density for each data track is indicated on the y axis (top left each track, normalized to input and 107 tags). Gene transcriptional direction is indicated by an arrow and exons by boxes.
      Figure thumbnail gr2
      Figure 2Transcriptional signatures modulated with treatment and steady-state ENCODE datasets confirm tissue specificity. A, H3K36me3 overlaid ChIP-seq data tracks displayed as vehicle (yellow, n = 3) or treated (blue, n = 3) with PTH (230 ng/g bw), 1,25(OH)2D3 (10 ng/g bw), or FGF23 (50 ng/g bw) as indicated with a treatment time of 1 h. Overlapping track data appear as green. ENCODE data tracks for CTCF (B), DNase hypersensitivity assay sequence (DNase-seq) (C), and RNA pol II ChIP-seq data (D) are displayed for kidney (yellow), fibroblast, intestine, liver, and bone marrow (gray). Regions of interest are highlighted in light gray boxes. Genomic location and scale are indicated (top), and maximum height of tag sequence density for each data track is indicated on the y axis (top left each track, normalized to input and 107 tags). Gene transcriptional direction is indicated by an arrow and exons by boxes.

       VDR and pCREB are localized to active enhancers in the introns of Mettl1 and Mettl21b

      To determine whether the binding sites were localized to active enhancer regions (
      • Ernst J.
      • Kheradpour P.
      • Mikkelsen T.S.
      • Shoresh N.
      • Ward L.D.
      • Epstein C.B.
      • Zhang X.
      • Wang L.
      • Issner R.
      • Coyne M.
      • Ku M.
      • Durham T.
      • Kellis M.
      • Bernstein B.E.
      Mapping and analysis of chromatin state dynamics in nine human cell types.
      ), ChIP-seq analyses of the histone enhancer signature marks H3K4me1 and H3K9ac were performed in kidneys following a 1-h treatment of either vehicle or each of the three hormones (Fig. 1, E and F). Although broad sites of H3K4me1 enrichment were present across the entire Cyp27b1 locus and at the Cyp27b1 promoter region in the absence of added hormone, enrichment sites unique to the introns of Mettl1 and Mettl21b were also apparent (Fig. 1E). These potential enhancer regions in the kidney aligned precisely with the more punctate pCREB and VDR sites of occupancy, yet were completely absent in similar analyses of MSCs. Results obtained through ChIP-seq analysis of the histone activity mark H3K9ac revealed similar profiles with the exception that PTH up-regulated while FGF23 strongly suppressed H3K9ac enrichment; 1,25(OH)2D3 was without effect (Fig. 1F). These results suggest that both pCREB and VDR localize to unique and potentially active enhancers within specific introns of Mettl1 and Mettl21b and that the H3K9ac mark appears to be directionally regulated by PTH and FGF23 as might be expected, revealing significant activity for both of these hormones. Notably, ChIP-seq analysis of H3K9ac enrichment in kidney extracts from two mouse models of secondary hyperparathyroidism confirmed the profound effect of sustained high PTH at both the putative enhancers and at Cyp27b1 itself (Fig. 1G) (
      • 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.
      ,
      • Dardenne O.
      • Prud'homme J.
      • Arabian A.
      • Glorieux F.H.
      • St-Arnaud R.
      Targeted inactivation of the 25-hydroxyvitamin D(3)-1(α)-hydroxylase gene (CYP27B1) creates an animal model of pseudovitamin D-deficiency rickets.
      ). These results support the novel finding that the regulatory regions that mediate PTH, 1,25(OH)2D3, and perhaps FGF23 action are located in selected introns of the Mettl1 and Mettl21b genes.
      DNase I hypersensitivity sequencing (DHS) analysis of kidneys from untreated WT mice performed by the ENCODE Consortium revealed the presence of distinct chromatin-occupied sites across a broad region containing the Cyp27b1 locus from Tsfm to Tspan31 (Fig. 2C) (
      • Mouse ENCODE Consortium
      • Stamatoyannopoulos J.A.
      • Snyder M.
      • Hardison R.
      • Ren B.
      • Gingeras T.
      • Gilbert D.M.
      • Groudine M.
      • Bender M.
      • Kaul R.
      • Canfield T.
      • Giste E.
      • Johnson A.
      • Zhang M.
      • Balasundaram G.
      • et al.
      An encyclopedia of mouse DNA elements (Mouse ENCODE).
      ). Although common DHS sites are seen in kidney and in many other tissues, only the kidney contained a unique DHS site within the Mettl1 intron and three unique sites in the Mettl21b intron that aligned not only with exclusive kidney enhancer marks but also with specific sites of pCREB and VDR occupancy. A similar profile of RNA pol II occupancy was also apparent consistent with an additional typical feature of enhancers (Fig. 2D) (
      • Liu F.
      Enhancer-derived RNA: a primer.
      ). These findings support the hypothesis that the regions within the Mettl1 and Mettl21b introns likely represent a tissue-specific regulatory module that mediates transcriptional control of Cyp27b1 in the kidney by the three primary mineral-regulating endocrine hormones. Interestingly, this gene-dense region in which Cyp27b1 resides is defined by two major CCCTC-binding factor (CTCF)-occupied sites (Fig. 2B) (
      • Ong C.T.
      • Corces V.G.
      CTCF: an architectural protein bridging genome topology and function.
      ,
      • Nichols M.H.
      • Corces V.G.
      A CTCF Code for 3D genome architecture.
      ), prompting an examination of each gene between these sites. Both Cyp27b1 and Mettl21b but not Mettl1 or any additional genes in the region were similarly regulated by PTH, FGF23, and 1,25(OH)2D3 (Fig. 3, A and B). Although mechanistically unproven, the presence of the two CTCF sites together with similarities in the regulation of both Mettl21b and Cyp27b1 advance speculation that this region may represent or reside in a small topologically associated domain (
      • Nora E.P.
      • Goloborodko A.
      • Valton A.L.
      • Gibcus J.H.
      • Uebersohn A.
      • Abdennur N.
      • Dekker J.
      • Mirny L.A.
      • Bruneau B.G.
      Targeted degradation of CTCF decouples local insulation of chromosome domains from genomic compartmentalization.
      ).
      Figure thumbnail gr3
      Figure 3Co-regulation of neighboring genes within the CTCF boundaries. A, schematic of CTCF-binding locations (yellow boxes) in the Cyp27b1 locus. B, gene expression in 8–9-week-old C57BL/6 wild-type mice of Tsfm, Mettl21b, Mettl1, Cyp27b1, March9, Cdk4, and Tspan31 after treatment with ethanol/PBS vehicle (Veh, gray, n = 6), 230 ng/g bw PTH for 1 h (PTH, blue, n = 6), 10 ng/g bw 1,25(OH)2D3 (1,25D3, black, n = 6) for 6 h, or 50 ng/g bw FGF23 for 3 h (FGF23, green, n = 6). Data displayed as relative quantitation (RQ, mean ± S.E.) compared with Gapdh. *, p < 0.05 paired t test, treatment versus vehicle.

       Deletion of the pCREB/VDR enhancers results in phenotypically unique strains of mice

      To explore the functional capabilities of the putative regulatory regions, we utilized a CRISPR/Cas9 approach (
      • 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.
      ) to create mouse strains with a deletion in the single putative enhancer located in Mettl1 (324 bp) (termed Mettl1-IKO or M1-IKO) and a more complex deletion that removed the three putative enhancers located in Mettl21b (5.5 kb) (termed Mettl21b-IKO or M21-IKO) (Fig. 4A). We also generated a floxed Cyp27b1 mouse through traditional homologous recombination methods and created a global Cyp27b1-deficient mouse (C27KO) analogous to that reported by St-Arnaud and co-workers (
      • Dardenne O.
      • Prud'homme J.
      • Arabian A.
      • Glorieux F.H.
      • St-Arnaud R.
      Targeted inactivation of the 25-hydroxyvitamin D(3)-1(α)-hydroxylase gene (CYP27B1) creates an animal model of pseudovitamin D-deficiency rickets.
      ) (Fig. 4B). Visual inspection of each strain revealed that M21-IKO mice were similar to their WT counterparts, whereas M1-IKO mice were smaller and phenotypically similar to C27KO mice (Fig. 4C). Both of the latter two strains were extremely frail with mortality rates considerably higher than WT mice. Blood calcium and phosphate levels were similar in both M21-IKO and WT mice, whereas PTH levels were slightly elevated, and FGF23 levels were approximately half (Fig. 4D). In contrast, M1-IKO and C27KO mice displayed debilitating hypocalcemia and hypophosphatemia, highly elevated PTH levels, and a near absence of FGF23. Systemic parameters in the three mouse strains correlated directly with BMD measurements (Fig. 5A). μCT analysis, parathyroid gland hypertrophy, growth plate abnormalities, and mineralization (Fig. 5, B–E) indicated that M1-IKO and C27KO mice were similar, although the profile seen in the C27KO mouse was more extreme. Interestingly, differences in the skeletal phenotype of the M21-IKO mice from WT mice were limited to the trabecular compartment (Fig. 5B), potentially due to a bone remodeling issue as mineralization appeared intact (Fig. 5E).
      Figure thumbnail gr4
      Figure 4Genomic knock-out of unique enhancers. A, schematic of CRISPR targeting in the Cyp27b1 locus. VDR ChIP-seq data are shown as a reference. Highlighted regions were excised using two CRISPR-guide RNAs located at the indicated chromosome position. B, schematic of the Cyp27b1KO (C27KO) mouse targeting strategy. C, photograph of gross mouse size and length. D, table of serum calcium (mg/dl), phosphate (mm), PTH (pg/ml), and FGF23 (pg/ml) with n values indicated in parentheses. One-way ANOVA with multiple comparison Tukey post test: *, p < 0.05 KO versus WT.
      Figure thumbnail gr5
      Figure 5Skeletal phenotyping of the M1-IKO and M21-IKO mice. A, BMD measurements in total body, spine, and femur as well as weight measurements for male (blue) and female (red) in wild-type (WT, solid bars, n = 12) and C27KO (n = 6), M1-IKO (n = 14), and M21-IKO (KO, striped, n = 11) mice. *, p < 0.05 paired t test, KO versus WT. B, μCT images (left) and quantification (right) for bone volume/trabecular volume (BV/TV), trabecular number (Tb.Num), trabecular thickness (Tb.Th), trabecular separation (Tb.Sep), cortical thickness (Ct.Th), and cortical porosity (Ct.P). All mice were male (blue) in wild-type (WT, solid bars) and C27KO, M1-IKO, and M21-IKO (KO, striped) mice. n = 6 for all genotypes. *, p < 0.05 paired t test, KO versus WT. C, parathyroid gland hypertrophy is shown in thyroid sections. Parathyroid stains are dark red, location denoted by black arrows. Quantification by ImageJ software for each image (n = 3, M21 n = 1). D, trichrome staining of the femoral head and growth plate from WT, C27KO, M1-IKO, and M21-IKO mice (×4 magnification). Growth plate (red) and collagen (blue) is present and normal in WT and M21-IKO and is absent in C27KO and M1-IKO. E, von Kossa staining of the femoral head and growth plate (×2 magnification). Calcium (black) staining is present and normal in WT and M21-IKO and is decreased in C27KO and M1-IKO.

       Endocrine control of Cyp27b1 expression is altered in M1-IKO and M21-IKO kidneys

      The different phenotypes of the M1-IKO and M21-IKO mice suggested that the regions in Mettl1 and Mettl21b genes were responsible for distinct regulatory functions at the Cyp27b1 gene. To examine this hypothesis, we assessed the steady-state expression of Cyp27b1 and also the ability of each of the three hormones to regulate Cyp27b1 expression in C27KO, M1-IKO, and M21-IKO mice (Fig. 1A). This latter test was executed to determine whether the gene remained sensitive to each individual hormone (Fig. 1A) but not to assess any potential physiological impact. Basal levels of Cyp27b1 were strikingly elevated in C27KO mice compared with controls (Fig. 6, A and B) because, although the gene transcribes a mutant, truncated Cyp27b1 RNA and non-functional enzyme (
      • Dardenne O.
      • Prud'homme J.
      • Arabian A.
      • Glorieux F.H.
      • St-Arnaud R.
      Targeted inactivation of the 25-hydroxyvitamin D(3)-1(α)-hydroxylase gene (CYP27B1) creates an animal model of pseudovitamin D-deficiency rickets.
      ), RNA production, and regulation remained fully intact. Interestingly, although high residual levels of PTH in C27KO mice limited response of Cyp27b1 to exogenous PTH administration, these high levels were unable to prevent FGF23 or 1,25(OH)2D3 from modestly suppressing Cyp27b1 (Fig. 6, A and B). In contrast, although WT mice retained normal sensitivity to each of the three hormones, basal Cyp27b1 expression in M1-IKO mice was strikingly reduced (95%) (Fig. 6A). Because both C27KO and M1-IKO mouse strains exhibit high PTH levels, the only explanation for the dramatic up-regulation of Cyp27b1 expression in C27KO mice coincident with the dramatic down-regulation of the gene in M1-IKO mice is that in the latter strain sensitivity to PTH has been lost. Loss of circulating PTH via thyroparathyroidectomy reduces basal production of 1,25(OH)2D3, an effect that has been previously documented (
      • Garabedian M.
      • Holick M.F.
      • Deluca H.F.
      • Boyle I.T.
      Control of 25-hydroxycholecalciferol metabolism by parathyroid glands.
      ). Cyp27b1 expression in both C27KO and M1-IKO mice exhibits a similar pattern of sensitivity to FGF23 and 1,25(OH)2D3 suppression and loss of sensitivity to PTH induction. Therefore, it is clear that the loss of PTH sensitivity in the M1-IKO mouse is due to deletion of the Mettl1 intronic region. M21-IKO mice exhibit a similar but more limited decrease in basal Cyp27b1 expression (75%), retain complete sensitivity to PTH, yet are insensitive to both FGF23 and 1,25(OH)2D3 treatment (Fig. 6, A and B). Thus, the region that is deleted in the M21-IKO mice appears to contain an independent regulatory feature that influences basal Cyp27b1 expression, perhaps due to the actions of an additional unknown positive factor analogous to that of PTH, as well as regions that mediate suppression by FGF23 and 1,25(OH)2D3. The co-regulation of Mettl21b (Fig. 3) is also lost as a result of these individual deletions, demonstrating that the activities of these enhancers are potentially shared (data not shown). Importantly, CYP27B1 protein levels in M1-IKO and M21-IKO kidneys were also strongly suppressed (Fig. 6E). As expected, high levels of non-functional, mutated CYP27B1 protein were seen in C27KO kidneys, migrating more rapidly than the WT protein due to loss of exons 7–9 (Fig. 6E).
      Figure thumbnail gr6
      Figure 6Gene expression and vitamin D metabolite levels. Gene expression of Cyp27b1 (A), (B, Cyp27b1 treatment fold change compared with vehicle), Cyp24a1 (C), Vdr (D) after treatment in 8–9-week-old C27KO, M1-IKO, and M21-IKO mice compared with WT littermates with ethanol/PBS vehicle (Veh, gray, n = 6), 230 ng/g bw PTH for 1 h (PTH, blue, n = 6), 10 ng/g bw 1,25(OH)2D3 (1,25D3, black, n = 6) for 6 h, or 50 ng/g bw FGF23 for 3 h (FGF23, green, n = 6). Data displayed as relative quantitation (RQ, mean ± S.E.) compared with Gapdh. *, p < 0.05 paired t test, treatment versus vehicle. #, p < 0.05 paired t test, KO Veh versus WT Veh. E, Western blot analysis of CYP27B1 protein expression from mitochondrial extracts compared with loading control VDAC1/PORIN protein expression (top). VDR protein expression compared with β-TUBULIN protein from whole-cell extract (bottom). F, vitamin D serum metabolite concentrations for WT (n = 60), C27KO (C27, n = 6), M1-IKO (M1, n = 28), M21-IKO (M21, n = 19) for 25(OH)D3 in individual animals (ng/ml), 1,25(OH)2D3 (pg/ml), 24,25(OH)2D3 (ng/ml), ratio of 25(OH)D3/24,25(OH)2D3; 25(OH)D3-26,23-Lactone (ng/ml) and 1,24,25(OH)3D3 (pg/ml) displayed as scatter plots (mean ± S.E.). One-way ANOVA with multiple comparison Tukey post test: *, p < 0.05 KO versus WT. 1,24,25(OH)3D3 was below the limit of detection (<LOD) for C27KO and M1-IKO.

       Basal suppression of kidney Cyp27b1 causes secondary suppression of Cyp24a1 and Vdr

      Loss of circulating 1,25(OH)2D3 and up-regulation of PTH in C27KO mice results in suppression of Cyp24a1 and Vdr expression (
      • Nguyen-Yamamoto L.
      • Karaplis A.C.
      • St-Arnaud R.
      • Goltzman D.
      Fibroblast growth factor 23 regulation by systemic and local osteoblast-synthesized 1,25-dihydroxyvitamin D.
      ). Cyp24a1 expression was dramatically down-regulated in both C27KO and M1-IKO mice (Fig. 6C), likely due to the high levels of PTH and low levels of FGF23 (Fig. 4D). Although Cyp24a1 remained sensitive to up-regulation by 1,25(OH)2D3 in the kidneys of each strain, both FGF23 induction and PTH suppression appeared to be fully quenched. Surprisingly, M21-IKO mice also exhibit Cyp24a1 suppression, although this level was less dramatic. Nevertheless, Cyp24a1 retained sensitivity to PTH down-regulation and FGF23 and 1,25(OH)2D3 up-regulation. As basal CYP24A1 protein levels were undetectable in WT, C27KO, M1-IKO, and M21-IKO mice, direct assessment of protein down-regulation was not possible (data not shown). The more modest secondary reduction in Cyp24a1 expression in M21-IKO mice is likely due to the limited increase in PTH but also to a reduction in FGF23, which would limit Cyp24a1 expression (considered below) (
      • Shimada T.
      • Kakitani M.
      • Yamazaki Y.
      • Hasegawa H.
      • Takeuchi Y.
      • Fujita T.
      • Fukumoto S.
      • Tomizuka K.
      • Yamashita T.
      Targeted ablation of Fgf23 demonstrates an essential physiological role of FGF23 in phosphate and vitamin D metabolism.
      ). Vdr was also suppressed in C27KO and M1-IKO kidneys (Fig. 6D), a reduction that was confirmed directly at the protein level (Fig. 6E). The modest up-regulation of Vdr by 1,25(OH)2D3 and FGF23 seen in WT mice appears to be lost in C27KO and M1-IKO mice (Fig. 6D), perhaps due to the low calcemic state of both mouse strains (
      • Healy K.D.
      • Zella J.B.
      • Prahl J.M.
      • DeLuca H.F.
      Regulation of the murine renal vitamin D receptor by 1,25-dihydroxyvitamin D3 and calcium.
      ). Collectively, these studies of Cyp27b1, Cyp24a1, and Vdr expression in C27KO, M1-IKO, and M21-IKO mice suggest that their phenotypes are due to aberrant basal Cyp27b1 expression, selective loss of hormonal regulation in the kidney, and complex reciprocal homeostatic regulation of Cyp24a1. These activities appear to be intrinsic to the regulatory regions themselves, as ChIP-seq analysis of the Cyp27b1 locus in both M1-IKO and M21-IKO mice revealed that the deletion of either the Mettl1 or the Mettl21b intronic enhancer (underlined in red in Fig. 7, A and B) had no effect on either VDR or pCREB binding in the opposite regulatory region. In this analysis, VDR binding in the C27KO mouse was used as a control, as VDR levels in the kidneys of both C27KO and M1-IKO mice show similar down-regulation and thus impact the level of genomic VDR binding in ChIP-seq analyses comparably. A similar analysis of epigenetic signature marks in these mice also revealed a lack of cross-structural alteration (data not shown). Importantly, as highlighted in Fig. 6, A and B, Cyp27b1 expression in M1-IKO mice is insensitive to the high circulating levels of PTH, and this is demonstrated amply through the lack of H3K9ac enrichment that is seen in both C27KO and VDRKO mice (Fig. 7C).
      Figure thumbnail gr7
      Figure 7M1-IKO and M21-IKO enhancer genomic occupancy. Overlaid ChIP-seq data tracks for VDR (A) and pCREB (B) from mouse kidney in wild-type (WT), C27KO, M1-IKO, or M21-IKO mice displayed as either basal or vehicle (Veh) (yellow, n = 3) or treated (blue, n = 3) with 10 ng/g bw 1,25(OH)2D3, for 1 h. C, H3K9ac data in VDRKO (blue), Cyp27b1KO mice (C27KO, blue), or M1-IKO mice (M1-IKO, blue) versus wild-type (WT, yellow). Overlapping track data appear as green. Overlapping track data appear as green. Regions of interest are highlighted in light gray boxes. Deleted genomic regions denoted by red line and red arrowhead. Genomic location and scale are indicated (top) and maximum height of tag sequence density for each data track is indicated on the y axis (top left each track, normalized to input and 107 tags). Gene transcriptional direction is indicated by an arrow and exons by boxes.

       Basal suppression of Cyp27b1 expression in M1-IKO but not M21-IKO kidneys results in altered vitamin D metabolite levels

      We utilized LC-MS/MS techniques to determine 25(OH)D3, 24,25(OH)2D3, and 25(OH)D3-26,23-lactone levels, as well as a novel antibody-based LC-MS/MS method to measure 1,25(OH)2D3 and 1,24,25(OH)3D3 levels in the blood of WT, C27KO, M1-IKO, and M21-IKO mice (Fig. 6F). 25(OH)D3 levels in C27KO, M1-IKO mice were well above those of WT littermate controls with the highest levels observed in the C27KO mice. 24,25(OH)2D3 and 25(OH)D3-26,23-lactone levels, however, were extremely low in the C27KO and M1-IKO mice giving rise to elevated 25(OH)D3/24,25(OH)2D3 ratios consistent with reduced Cyp24a1 expression and in vivo enzyme function. Low circulating levels of 1,25(OH)2D3 and 1,24,25(OH)3D3 (<LOD) in these animals suggest that reduced Cyp24a1 expression arises from either a lack of CYP27B1 itself (C27KO) or its PTH-responsive enhancer (M1-IKO). Although M21-IKO mice exhibited higher levels of expression of Cyp24a1 relative to C27KO and M1-IKO mice, expression levels remained lower than WT (Fig. 6C). While 25(OH)D3 was also elevated in M21-IKO animals, 24,25(OH)2D3 was 2-fold greater than in its WT counterparts. The 25(OH)D3/24,25(OH)2D3 ratio suggests that the rise in 24,25(OH)2D3 results from elevated 25(OH)D3 substrate, but we note that the mean ratio is slightly elevated indicating a somewhat blunted CYP24A1 function. Further analysis of downstream 25(OH)D3-26,23-lactone levels revealed a 4-fold lower concentration than WT and a 20-fold increase in the 25(OH)D3/25(OH)D3-26,23-lactone ratio (data not shown). 1,24,25(OH)3D3 levels were also 3-fold lower in the M21-IKO animals, despite near-normal 1,25(OH)2D3. Taken together, the intermediate level of Cyp24a1 expression observed in M21-IKO animals appears to manifest as altered progression of metabolism through to downstream catabolites, rather than the extreme reduction in serum 24,25(OH)2D3, 25(OH)D3-26,23-lactone, and 1,24,25-(OH)3D3 levels observed in C27KO and M1-IKO mice. 1,25(OH)2D3 concentrations are consistent with the overall systemic and skeletal phenotypes of each of the mouse strains. These results suggest that whereas the general phenotype of M21-IKO mice appears normal, minor changes in the skeleton (Fig. 5) together with alterations in both systemic (Fig. 4D) and vitamin D metabolic (Fig. 6F) profiles appear instrumental in sustaining the near normal levels of 1,25(OH)2D3, thereby preserving mineral homeostasis in these particular mice. We emphasize that C27KO and M1-IKO mice were unable to produce 1,24,25(OH)3D3, a metabolite that is measured in the blood of WT mice and also appears to be produced at low but significant levels in M21-IKO mice. This represents the first robust measurement of a metabolite that, while comprising the first catabolic product of CYP24A1 action on 1,25(OH)2D3, is also the second most biologically active vitamin D metabolite known (
      • Chandler J.S.
      • Pike J.W.
      • Haussler M.R.
      Biosynthesis, purification and receptor binding properties of high specific radioactivity 1α, 24(R),25-trihydroxy-[26,27-methyl-3H]-vitamin D3.
      ,
      • Thomasset M.
      • Cuisinier-Gleizes P.
      • Mathieu H.
      • DeLuca H.F.
      Intestinal calcium-binding protein (CaBP) and bone calcium mobilization in response to 1,24(R),25-(OH)3D3. Comparative effects of 1,25-(OH)2D3 and 24(R),25-(OH)2D3 in rats.
      ). Finally, the central importance of low 1,25(OH)2D3 in the aberrant phenotype in the M1-IKO mouse was confirmed by the ability of a diet containing exogenously added 1,25(OH)2D3 to rescue the abnormal skeletal phenotype of the M1-IKO mouse (Fig. 8A). An additional experiment in which vitamin D was removed from the diet, thereby reducing circulating 25(OH)D3 levels, revealed that although the phenotype of the C27KO mouse was unaffected by this treatment regimen, as expected of a mouse that cannot make 1,25(OH)2D3, the phenotype of the M1-IKO mouse worsened and after 5 weeks was similar to that of the C27KO mouse (Fig. 8, B and C).
      Figure thumbnail gr8
      Figure 8Dietary treatment for M1-IKO mice. A, bone mineral density (BMD) measurements in total body, spine, and femur as well as weight measurements for male (blue) and female (red) in wild-type (WT, solid bars) and C27KO or M1-IKO (KO, striped) mice fed chow diet (−) or chow supplemented with 1 ng/g 1,25(OH)2D3 (+). Two-way ANOVA with multiple comparison Tukey post-test is shown. n = 4 for both sex and treatment. *, p < 0.05 KO versus WT. #, p < 0.05 1,25D3 diet versus chow. B, table of serum 25(OH)D3 (ng/ml), 24,25(OH)2D3 (ng/ml), calcium (mg/dl), phosphate (mm), PTH (pg/ml), and FGF23 (pg/ml) in mice fed a purified diet containing vitamin D3 (+) versus purified diet without vitamin D3 (−) with n values indicated in parentheses. One-way ANOVA with multiple comparison Tukey post test: *, p < 0.05 KO versus WT. C, BMD measurements in total body, spine, and femur as well as weight measurements for male (blue) and female (red) in wild-type (WT, solid bars) and C27KO or M1-IKO (KO, striped) mice fed a purified diet containing vitamin D3 (+) versus purified diet without vitamin D3 (−). n = 4 for both sex and treatment. Two-way ANOVA with multiple comparison Tukey post-test is shown. *, p < 0.05 KO versus WT. #, p < 0.05 diet versus diet without vitamin D3.

       Repressive abilities of the intronic Mettl21b enhancer

      Similarities between the ability of 1,25(OH)2D3 and FGF23 to suppress Cyp27b1 expression in the kidney and their union in the intron of Mettl21b prompted the idea that 1,25(OH)2D3 might suppress Cyp27b1, at least in part, through FGF23 by inducing transcription of the Fgf23 gene and raising its blood levels, an established function of 1,25(OH)2D3 (
      • Liu S.
      • Tang W.
      • Zhou J.
      • Stubbs J.R.
      • Luo Q.
      • Pi M.
      • Quarles L.D.
      Fibroblast growth factor 23 is a counter-regulatory phosphaturic hormone for vitamin D.
      ,
      • Haussler M.R.
      • Whitfield G.K.
      • Haussler C.A.
      • Sabir M.S.
      • Khan Z.
      • Sandoval R.
      • Jurutka P.W.
      1,25-Dihydroxyvitamin D and Klotho: a tale of two renal hormones coming of age.
      ). Additional evidence includes the following. 1) ChIP-seq studies across the Cyp27b1 locus suggested that although both PTH induced and FGF23 suppressed H3K9ac enrichment within the intronic regions of Mettl1 and Mettl21b and at the Cyp27b1 promoter, 1,25(OH)2D3 was without effect at this time point (Fig. 1F). 2) a reduction of VDR binding within the Mettl21b intron in the M1-IKO mouse due to Vdr down-regulation did not appear to limit the ability of either 1,25(OH)2D3 or FGF23 to suppress Cyp27b1 expression (Fig. 7). To test this hypothesis, we correlated the ability of 1,25(OH)2D3 to suppress Cyp27b1 expression over time in WT kidneys with its ability to induce Fgf23 in bone. The results revealed that following an acute but modest up-regulation of Cyp27b1 expression, 1,25(OH)2D3 provoked a rapid suppression of Cyp27b1 that reached near baseline within 4–6 h (Fig. 9A). This suppression was fully sustained for at least 24 h, an unusual feature of 1,25(OH)2D3 activity alone, and likely much longer given previous studies (
      • Chow E.C.
      • Quach H.P.
      • Vieth R.
      • Pang K.S.
      Temporal changes in tissue 1α,25-dihydroxyvitamin D3, vitamin D receptor target genes, and calcium and PTH levels after 1,25(OH)2D3 treatment in mice.
      ). Importantly, Fgf23 transcripts were also induced by 1,25(OH)2D3 in the skeleton within this same time frame (Fig. 9B). Based upon this finding, we then correlated these temporal responses with serum levels of PTH and FGF23 as well as calcium and phosphate. Although PTH was mildly suppressed by 1,25(OH)2D3, FGF23 was increased from a residual baseline of 200 to ∼1700 pg/ml at 12 h (Fig. 9A). Both calcium and phosphate were also induced by 1,25(OH)2D3 within this time period, an in vivo response to the hormone that has been well-established. These results support the idea that both 1,25(OH)2D3 and FGF23 may act either sequentially or together to suppress Cyp27b1 expression through a sustained effect mediated by the up-regulation of FGF23. It is worth noting that the mobilization of phosphate may also play an important role because both 1,25(OH)2D3 and phosphate are the most striking regulators of FGF23 that are currently known (
      • Quarles L.D.
      Role of FGF23 in vitamin D and phosphate metabolism: implications in chronic kidney disease.
      ). A similar time course of Cyp27b1 response to 1,25(OH)2D3 was also seen in M1-IKO kidneys (Fig. 9E), although this response was less robust because the basal expression of Cyp27b1 and serum levels of FGF23 are already highly suppressed (Figs. 4D and 6A). In WT mice, the temporary reduction of FGF23 and elevation in PTH allows PTH to transiently increase Cyp27b1 expression. However, Cyp27b1 expression is not acutely increased in the M1-IKO mice due to its insensitivity to PTH. Importantly, although this study suggests that the suppression of Cyp27b1 by 1,25(OH)2D3 may involve the induction of FGF23 either directly by the hormone or via its mobilization of phosphate, proof of this hypothesis will require delineation of the exact sites of FGF23 action in the Mettl21b intron following identification of the transcription factor(s) involved and/or elimination of the ability of 1,25(OH)2D3 to up-regulate Fgf23 expression.
      Figure thumbnail gr9
      Figure 9Time course of 1,25(OH)2D3 repression of Cyp27b1. A, overlaid data for Cyp27b1 gene expression in the kidney (black), serum FGF23 (green), serum PTH (blue), serum phosphate (gray), and serum calcium (orange) in 8–9-week-old wild-type C57BL/6 mice after 10 ng/g dose of 1,25(OH)2D3 for 0, 1–4, 6, 12, and 24 h. Fgf23 (B) expression from the L5 vertebrae and Cyp24a1 (C) and Vdr (D) gene expression from the kidney using same time points. Gene expression data displayed as relative quantitation (RQ, mean ± S.E.) compared with Gapdh. n = 6 for all time points. One-way ANOVA with multiple comparison Tukey post-test: *, p < 0.05 time point versus 0 h. E, overlaid data for Cyp27b1 gene expression as in A for the M1-IKO mice, as well as Cyp24a1 and Vdr gene expression. PTH was excluded due to extremely high and unchanged serum levels.

       Regulation of Cyp27b1 expression in NRTCs is functionally unique from the kidney

      Previous studies have suggested that Cyp27b1 is expressed in a number of NRTCs and may be responsible for local conversion of 25(OH)D3 to 1,25(OH)2D3 (
      • Adams J.S.
      • Hewison M.
      Extrarenal expression of the 25-hydroxyvitamin D-1-hydroxylase.
      ). However, its expression does not appear to be regulated by either PTH or FGF23 and perhaps only modestly by 1,25(OH)2D3. Cyp27b1 expression is extremely low in NRTCs, and these hormones do not regulate Cyp27b1 in skin, bone, liver, and other specific cell types, although 1,25(OH)2D3 modestly suppressed Cyp27b1 in the skin and in thyroparathyroid gland tissue (TPTG) (Fig. 10A and 11A). Based upon ChIP-seq analysis of MSCs (Figure 1, Figure 2), we reasoned that the absence of Cyp27b1 regulation by PTH and FGF23 and, in specific NRTC cases, by 1,25(OH)2D3 could be explained by the absence of the endocrine enhancers. To test this idea, we compared the basal expression levels of Cyp27b1 in several NRTCs from C27KO, M1-IKO, and M21-IKO mice with those of individual littermate controls. Basal levels of Cyp27b1 expression in skin, calvaria, and L5 vertebrae were unaffected by either of the two genomic deletions (Fig. 10B). Interestingly, both basal and ex vivo T cell receptor-inducible levels of Cyp27b1 expression in T cells isolated from either WT or M1-IKO spleens were also unaffected (Fig. 10C) (
      • Hewison M.
      Vitamin D and innate and adaptive immunity.
      ). These general findings provide key support for the idea that the regulatory module that mediates the actions of PTH, FGF23, and 1,25(OH)2D3 at Cyp27b1 is absent in NRTCs and thus prevents similar regulation by the three endocrine hormones. Based upon tissue selectivity, we have termed this the kidney-specific endocrine regulatory module (KS-ERM). Surprisingly, although the calcium and phosphate hormones are unable to control Cyp27b1 expression in NRTCs, the ability of the inflammatory modulator LPS to induce Cyp27b1 was fully apparent (Fig. 10B). LPS induction of Cyp27b1 expression has been previously described, particularly in immune cells such as macrophages, where it is suggested to be involved in the ability of vitamin D to modulate both innate and acquired immunity (
      • Adams J.S.
      • Rafison B.
      • Witzel S.
      • Reyes R.E.
      • Shieh A.
      • Chun R.
      • Zavala K.
      • Hewison M.
      • Liu P.T.
      Regulation of the extrarenal CYP27B1-hydroxylase.
      ). Importantly, the ability of LPS to induce the Cyp27b1 gene in these cell types was unaffected by the enhancer deletions introduced into the M1-IKO and M21-IKO mice, although LPS activity appeared to be potentially unmasked in the kidneys of these mutant mice as a consequence of the reduced basal Cyp27b1 expression in both strains (Fig. 10C). We conclude that LPS and T cell receptor activation of Cyp27b1 in distinct NRTCs is mediated by a regulatory region(s) we have termed the NRTC-regulatory module (NRTC-RM) whose location lies outside that defined by the two separate components of the KS-ERM (Fig. 10D).
      Figure thumbnail gr10
      Figure 10Non-renal target cell expression of Cyp27b1. A, gene expression of Cyp27b1 after treatment in 8–9-week-old C57BL/6 wild-type mice with ethanol/PBS vehicle (Veh, gray, n = 6), 230 ng/g bw PTH for 1 h (PTH, blue, n = 6), 10 ng/g bw 1,25(OH)2D3 (1,25D3, black, n = 6) for 6 h, or 50 ng/g bw FGF23 for 3 h (FGF23, green, n = 6) examined in kidney, skin, calvaria, L5 vertebrae, and liver. B, Cyp27b1 gene expression in kidney, skin, calvaria, and L5 vertebrae after treatment with 10 mg/kg lipopolysaccharides (LPS, red, n = 6) for 6 h versus vehicle (Veh, gray, n = 6). C, Cyp27b1 gene expression in CD4+ and CD8+ T cells in M1-IKO or WT littermate mice activated with α-CD3/CD28 antibody for 3 h (left, orange, n = 6) or 100 nm 1,25(OH)2D3 for 3 h (right, black, n = 6) versus vehicle (Veh, gray, n = 6). Data displayed as relative quantitation (RQ, mean ± S.E.) compared with Gapdh. *, p < 0.05 paired t test, treatment versus vehicle. D, schematic diagram of the KS-ERM) incorporating the M1 and M21 enhancer control regions compared with the NRTC-RM. Repressive elements are shown in red; activating elements are shown in green, and unknown elements are shown in blue.
      Figure thumbnail gr11
      Figure 11Parathyroid gland expression and genomic occupancy of Cyp27b1. A, gene expression of Cyp27b1 after treatment in 8–9-week- old C57BL/6 wild-type mice with ethanol/PBS vehicle (Veh, gray, n = 6), 230 ng/g bw PTH for 1 h (PTH, blue, n = 6), 10 ng/g bw 1,25(OH)2D3 (1,25D3, black, n = 6) for 6 h, 50 ng/g bw FGF23 for 3 h (FGF23, green, n = 6), or 10 mg/kg lipopolysaccharides (LPS, red, n = 6) examined in the TPTG. *, p < 0.05 paired t test, treatment versus vehicle. B, gene expression of Cyp27b1 in C27KO, M1-IKO, and M21-IKO mice (KO, striped, n = 6) basally (vehicle) compared with wild-type (WT, solid, n = 6) littermates in the TPTG. *, p < 0.05 paired t test, KO versus WT. Data displayed as relative quantitation (RQ, mean ± S.E.) compared with Gapdh. Overlaid data tracks for genomic occupancy by ChIP-seq of H3K4me1 and H3K9ac basally (yellow) in kidney, TPTG, and MSC (C) or C27KO and M1-IKO (blue) versus WT (yellow) (D). Overlapping track data appear as green. Regions of interest are highlighted in light gray boxes. Genomic location and scale are indicated (top), and maximum height of tag sequence density for each data track is indicated on the y axis (top left each track, normalized to input and 107 tags). Gene transcriptional direction is indicated by an arrow and exons by boxes.

       Cyp27b1 locus in TPTG tissue contains signatures of both NRTCs and renal cells

      Cyp27b1 is expressed at reduced levels in isolated cells of the parathyroid gland (PTG) and in ex vivo cultures of both PTG cells and organs of several species (
      • Ritter C.S.
      • Haughey B.H.
      • Armbrecht H.J.
      • Brown A.J.
      Distribution and regulation of the 25-hydroxyvitamin D3 1α-hydroxylase in human parathyroid glands.
      ,
      • Segersten U.
      • Correa P.
      • Hewison M.
      • Hellman P.
      • Dralle H.
      • Carling T.
      • Akerström G.
      • Westin G.
      25-hydroxyvitamin D(3)-1α-hydroxylase expression in normal and pathological parathyroid glands.
      ). However, the regulatory properties of the Cyp27b1 gene itself have not been explored in this tissue in vivo. We therefore examined in a concluding set of studies the potential for Cyp27b1 regulation in the parathyroid gland, based upon the strong linkage between the function of this organ and its central involvement in the regulation of both 1,25(OH)2D3 metabolism and mineral homeostasis. This was enabled by excising total TPTG tissue after hormone injection into WT, C27KO, M1-IKO, or M21-IKO mice and conducting both RNA expression and ChIP-seq analysis in this set of glands. Although the tissue is more complex, this approach allowed us to avoid time-consuming isolation methods and ex vivo culturing and to assess analyses directly from the mice. Although much has been made of vitamin D action to suppress expression of the PTH gene, loss of the VDR by selective gene knock-out in the parathyroid glands of mice has revealed only a minor impact on PTH expression, suggesting that the primary actions of vitamin D in this tissue remain to be understood (
      • Meir T.
      • Levi R.
      • Lieben L.
      • Libutti S.
      • Carmeliet G.
      • Bouillon R.
      • Silver J.
      • Naveh-Many T.
      Deletion of the vitamin D receptor specifically in the parathyroid demonstrates a limited role for the receptor in parathyroid physiology.
      ). Of the three hormones and LPS administered to WT mice, only 1,25(OH)2D3 was statistically active and modestly suppressed Cyp27b1 expression (Fig. 11A). Loss of the enhancer in the M1-IKO mouse resulted in a striking suppression of Cyp27b1 much like that seen in the kidney; this suppression was not seen in the TPTG of M21-IKO mice (Fig. 11, A and B). We therefore treated WT mice with a single dose of 1,25(OH)2D3 and examined the Cyp27b1 locus using ChIP-seq for the presence of the VDR at sites observed in the kidney (Fig. 1D). Despite the presence of the VDR at accepted target genes for 1,25(OH)2D3 in the TPTG, including Cyp24a1, VDR occupancy was not detected anywhere within the Cyp27b1 locus (data not shown). To explore further, we examined the epigenetic landscape surrounding the Cyp27b1 gene in the TPTG, and we assessed the basal levels of the enhancer signature marks H3K4me1 and H3K9ac and compared them with those in the kidney and in MSCs. Although several minor similarities were apparent, neither of these epigenetic marks displayed a pattern consistent with that seen in the kidney (Fig. 11C). Similar to the kidney, H3K4me1 shows a minor enrichment in the M21-IKO region (furthest left highlighted region, Fig. 11C) as well as the M1-IKO region; however, H3K9ac is more comparable to the MSC control and devoid of activity in both regions. We then examined basal epigenetic H3K4me1 and H3K9ac landscapes across the Cyp27b1 gene in TPTG tissue derived from either C27KO or M1-IKO mice and compared them with those obtained from their WT littermates (Fig. 11D). Although modest H3K4me1 enrichment was seen in a single region in the Mettl21b intron and perhaps in the Mettl1 intron in these contrasting ChIP-seq analyses, there was no evidence of residual enrichment of either the H3K4me1 or H3K9ac activity marks in either intron. These results suggest that despite minor functional similarities, the Cyp27b1 gene locus in the TPTG reflects an enhancer landscape that is more similar to NRTCs than to the kidney. We conclude that the KS-ERM is a control module unique to the kidney, providing regulatory capabilities at the Cyp27b1 gene that mediate unique sensitivity to PTH, FGF23, and 1,25(OH)2D3.

      Discussion

      The phenotypes of the mice carrying KS-ERM deletions contrast with those of WT mice and suggest that reduced expression of Cyp27b1 stimulates striking adaptive homeostatic responses that involve the differential production of PTH and FGF23 as well as 1,25(OH)2D3. These responses impact not only Cyp27b1 and Cyp24a1 expression but vitamin D metabolism as well and highlight both the genomic as well as homeostatic circuits that are essential for the maintenance of normal mineral balance. A summary of the key players involved in this homeostatic response and our working hypothesis is illustrated in Fig. 12A, where the physiological actions of PTH, FGF23, and 1,25(OH)2D3 are likewise shown reflecting the specific mouse models that have been used to describe the interrelationships. Sites of activity controlled by the enhancers deleted in the M1-IKO and M21-IKO strains are also depicted as well. As documented, most of these models are functionally linked to the aberrant regulation of Cyp27b1 expression or activity in the kidney, which leads to abnormal 1,25(OH)2D3 production. Decreased circulating levels of 1,25(OH)2D3 result in hypocalcemia and hypophosphatemia, prompting a rise in PTH and a suppression of FGF23. In the case of normal mice, homeostatic changes in PTH and FGF23 correct the underexpression of Cyp27b1 and suppress Cyp24a1 expression, raising blood 1,25(OH)2D3 levels that together with PTH and FGF23 normalize extracellular calcium and phosphate metabolism.
      Figure thumbnail gr12
      Figure 12Homeostatic regulation of calcium and phosphate and conservation to human. A, schematic diagram for the homeostatic regulation of calcium and phosphate. B, in silico VISTA analysis of conservation between mouse and human CYP27B1 genomic loci. Conservation score reaches significance at 75% (indicated by black middle line) for exons (blue) and M1 and M21 enhancer regions (coral, highlighted by gray boxes). Other genomic features are as indicated in the figure.
      In M1-IKO mice, however, and prompted by the down-regulation of Cyp27b1, the gene's up-regulation is disrupted due to the lack of Cyp27b1 sensitivity to PTH. Thus, calcium and phosphate lowering persists, causing a sustained rise in PTH and a further decrease in FGF23. These two hormonal changes also suppress Cyp24a1 expression that both decreases 1,25(OH)2D3 turnover and compromises 24,25(OH)2D3 and 1,24,25(OH)3D3 production, thereby eliciting an elevation in 25(OH)D3 substrate levels. This rise may contribute to a homeostatic effort to raise circulating blood levels of 1,25(OH)2D3, which is largely futile in the case of the M1-IKO mice. In contrast, the reduced expression of Cyp27b1 in M21-IKO mice with retained gene sensitivity to PTH is sufficient to mediate appropriate up-regulation of 1,25(OH)2D3 to maintain a normal calcium and phosphate balance. This largely prevents uncontrolled up-regulation of PTH while stimulating FGF23 synthesis. The homeostatic response also prevents the striking suppression of Cyp24a1 observed in the M1-IKO mice, thereby contributing to the successful preservation of 1,25(OH)2D3. It is possible that the full brunt of the loss of Cyp27b1 sensitivity to FGF23 and 1,25(OH)2D3 suppression in the M21-IKO mice may be observed only under conditions in which 1,25(OH)2D3 and FGF23 are elevated, and both hypercalcemia and hyperphosphatemia are evident (
      • Haussler M.R.
      • Whitfield G.K.
      • Haussler C.A.
      • Sabir M.S.
      • Khan Z.
      • Sandoval R.
      • Jurutka P.W.
      1,25-Dihydroxyvitamin D and Klotho: a tale of two renal hormones coming of age.
      ). Nevertheless, the current results in M21-IKO mice highlight not only the nature of the exquisite homeostatic circuits that prevail in vivo to maintain mineral balance but demonstrate how the levels of key players are also modulated to achieve a normalized calcium and phosphate outcome. The inability of PTH to induce Cyp27b1 expression in M1-IKO mice almost fully compromises the capacity of Cyp27b1 and Cyp24a1 to maintain 1,25(OH)2D3 levels, highlighting the essential role of PTH in the control of skeletal growth and development as well as the maintenance of mineral homeostasis.
      The presence of a unique module in the kidney that mediates the regulation of Cyp27b1 by key hormones involved in mineral homeostasis together with its absence in NRTCs suggests that the primary role of Cyp27b1 in the kidney is to produce circulating blood levels of 1,25(OH)2D3 that can orchestrate the expression of genes in the intestine, kidney, and bone, which are responsible for global mineral homeostasis. The absence of this endocrine module in NRTCs and the simultaneous presence of an independent module outside the KS-ERM that controls Cyp27b1 expression in NRTCs (NRTC-RM) suggests that the expression of Cyp27b1 in these cell types serves an alternative function to regulate the production of autocrine 1,25(OH)2D3 that controls cell-specific gene activation and local pleiotropic biological activities (
      • Hewison M.
      • Burke F.
      • Evans K.N.
      • Lammas D.A.
      • Sansom D.M.
      • Liu P.
      • Modlin R.L.
      • Adams J.S.
      Extra-renal 25-hydroxyvitamin D3–1α-hydroxylase in human health and disease.
      ). If these separate roles of Cyp27b1 in the kidney and NRTCs are correct, it implies the existence of two relatively distinct spheres of vitamin D influence, the first comprising the control of mineral metabolism and the second consisting of the regulation of numerous biological activities that are cell type-specific and independent of mineral metabolism. Most importantly, this thesis indicates that the mechanistic basis for these two biologically active domains of vitamin D action are largely determined by the differential regulation of Cyp27b1 in these two tissue classes. These two domains are unlikely to be fully independent, however, considering that extracellular calcium and phosphate levels impact all cell types and that endocrine regulation by vitamin D also influences circulating 25(OH)D3, the common substrate for Cyp27b1 activity in both tissue types. Conversely, certain biological actions of vitamin D in NRTCs are also likely to impact the orchestration of mineral homeostasis by the intestine, kidney, and bone as well. For example, the potential impact of vitamin D on insulin secretion, utilization, and energy metabolism is likely to impact the maintenance of mineral homeostasis (
      • Bouillon R.
      • Carmeliet G.
      • Lieben L.
      • Watanabe M.
      • Perino A.
      • Auwerx J.
      • Schoonjans K.
      • Verstuyf A.
      Vitamin D and energy homeostasis–of mice and men.
      ). We argue that the current mouse models we have created, as well as additional models in preparation, will allow us to isolate the activities of each sphere of influence independently, thereby permitting a precise determination of not only the impact of locally produced 1,25(OH)2D3 on the biological activities of NRTCs but of the dependence of these activities on circulating 1,25(OH)2D3 produced by the kidney. This is important because a central clinical issue currently is the impact of vitamin D supplementation on 25(OH)D3 levels that are hypothesized to represent a primary determinant of the local production of 1,25(OH)2D3 (
      • Bouillon R.
      Comparative analysis of nutritional guidelines for vitamin D.
      ,
      • Bouillon R.
      Optimal vitamin D supplementation strategies.
      ). Of greater importance, however, is the fact that both spheres of vitamin D influence may also be altered in disease, as illustrated in chronic kidney disease (
      • Quarles L.D.
      Role of FGF23 in vitamin D and phosphate metabolism: implications in chronic kidney disease.
      ), autoimmune disease (
      • Cantorna M.T.
      • Hayes C.E.
      • DeLuca H.F.
      1,25-Dihydroxyvitamin D3 reversibly blocks the progression of relapsing encephalomyelitis, a model of multiple sclerosis.
      ), and cancer (
      • Feldman D.
      • Krishnan A.V.
      • Swami S.
      • Giovannucci E.
      • Feldman B.J.
      The role of vitamin D in reducing cancer risk and progression.
      ), where both genetic and epigenetic changes could affect both the production and utilization of 1,25(OH)2D3. These possibilities are likely to influence the utility of vitamin D analogues in disease and open the question as to whether tissue-selective modulation of Cyp27b1 expression may represent an alternative approach to vitamin D therapeutics.
      The implications for the locations of regulatory enhancers for the expression of both Cyp27b1 and Mettl21b are significant relative to genome-wide association studies attempting to correlate single nucleotide polymorphisms to the prevalence of autoimmune disease, particularly multiple sclerosis (
      • International Multiple Sclerosis Genetics Consortium, Wellcome Trust Case Control Consortium 2
      • Sawcer S.
      • Hellenthal G.
      • Pirinen M.
      • Spencer C.C.
      • Patsopoulos N.A.
      • Moutsianas L.
      • Dilthey A.
      • Su Z.
      • Freeman C.
      • Hunt S.E.
      • Edkins S.
      • Gray E.
      • Booth D.R.
      • et al.
      Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis.
      • Raychaudhuri S.
      • Remmers E.F.
      • Lee A.T.
      • Hackett R.
      • Guiducci C.
      • Burtt N.P.
      • Gianniny L.
      • Korman B.D.
      • Padyukov L.
      • Kurreeman F.A.
      • Chang M.
      • Catanese J.J.
      • Ding B.
      • Wong S.
      • van der Helm-van Mil A.H.
      • et al.
      Common variants at CD40 and other loci confer risk of rheumatoid arthritis.
      ,
      • Ramagopalan S.V.
      • Dyment D.A.
      • Cader M.Z.
      • Morrison K.M.
      • Disanto G.
      • Morahan J.M.
      • Berlanga-Taylor A.J.
      • Handel A.
      • De Luca G.C.
      • Sadovnick A.D.
      • Lepage P.
      • Montpetit A.
      • Ebers G.C.
      Rare variants in the CYP27B1 gene are associated with multiple sclerosis.
      • Sundqvist E.
      • Bäärnhielm M.
      • Alfredsson L.
      • Hillert J.
      • Olsson T.
      • Kockum I.
      Confirmation of association between multiple sclerosis and CYP27B1.
      ). In these studies, disease-associated SNPs have been identified in an extended region that contains the CYP27B1 gene as well as neighboring genes that include METTL1 and METTL21B (FAM119B) (
      • Alcina A.
      • Fedetz M.
      • Fernández O.
      • Saiz A.
      • Izquierdo G.
      • Lucas M.
      • Leyva L.
      • García-León J.A.
      • Abad-Grau Mdel M.
      • Alloza I.
      • Antigüedad A.
      • Garcia-Barcina M.J.
      • Vandenbroeck K.
      • Varadé J.
      • de la Hera B.
      • et al.
      Identification of a functional variant in the KIF5A-CYP27B1-METTL1-FAM119B locus associated with multiple sclerosis.
      ,
      • Karaky M.
      • Alcina A.
      • Fedetz M.
      • Barrionuevo C.
      • Potenciano V.
      • Delgado C.
      • Izquierdo G.
      • Matesanz F.
      The multiple sclerosis-associated regulatory variant rs10877013 affects expression of CYP27B1 and VDR under inflammatory or vitamin D stimuli.
      ). Indeed, some genotypes appear to be preferential for the altered expression of CYP27B1, although observed co-regulation of the METTL21B gene has introduced some complexity. Importantly, the human CYP27B1 locus and adjacent genes on chromosome 12 are syntenic on the reverse strand with the mouse haplotype. In fact, a VISTA analysis of genomic conservation indicates not only that this is the case but that the only non-exonic regions that reach significance are those in which we have identified enhancers (Fig. 12B). Our discovery that the regulatory regions that control the mouse Cyp27b1 gene in the kidney are embedded within the introns of Mettl1 and Mettl21b and that Mettl21b is also co-regulated by PTH, FGF23, and 1,25(OH)2D3 are therefore potentially relevant. We hypothesize that the SNPs that have been identified in this region and particularly in the METTL1 gene in humans may hint at the locations of important regulatory enhancers for both CYP27B1 and METTL21B. It is also of significance that if non-coding SNPs in these genomic regions are linked to the expression of CYP27B1, our data would suggest that multiple sclerosis is associated with endocrine production of 1,25(OH)2D3 and not local production by CNS-invasive immune cells. Thus, it will be of interest to determine whether the regulation of the human CYP27B1 gene in the kidney is modulated through a distal organization similar to that in the mouse and to determine the functional nature of the SNPs that have been identified. In that regard, our studies may have broader implications for vitamin D in other human autoimmune diseases as well.
      In summary, a kidney-specific regulatory module has been identified upstream of the Cyp27b1 gene that controls both the basal and hormone-regulated expression of the gene in the kidney in mice in vivo. This module is absent in the Cyp27b1 region in NRTCs, which accounts for the absence of regulation by PTH, FGF23, and 1,25(OH)2D3 in these cell types. Dissection of this region in mice via CRISPR/Cas9 genome-editing methods has revealed distinct elements responsible for basal expression in the kidney as well as for PTH induction and 1,25(OH)2D3 and FGF23 suppression. The phenotypes of these mouse strains highlight both the genomic and homeostatic circuits that control Cyp27b1 expression. We conclude that these studies open new avenues to study vitamin D metabolism and its involvement in both mineral metabolism and in other pleiotropic biology in vivo in health and disease.

      Experimental procedures

       Reagents

      The following reagents were used for in vivo injections: 1α,25(OH)2D3 was obtained from SAFC Global (Madison, WI); PTH(1–84) human, was obtained from Bachem (H-1370.0100 Torrance, CA); mouse FGF23 (2629-FG-025) was from R&D Systems, Minneapolis, MN; and LPS (L6529) was from Sigma.
      Antibodies used for ChIP-seq analysis of VDR (C-20, sc-1008, lot no. H1216) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). H3K4me1 (ab8895, lot no. GR283603-1) and H3K36me3 (ab9050, lot no. GR273247-1) were purchased from Abcam (Cambridge, MA). Phosphorylated CREB (Ser-133) (pCREB, 06-519, lot no. 2762242) and H3K9ac (06-942, lot no. 2664263) were purchased from Millipore Corp. (Billerica, MA).
      Traditional genotyping PCR was completed with GoTaq (Promega, Madison, WI), and all real-time qPCR was completed with the StepOnePlus using TaqMan for gene expression assays (Applied Biosystems, Foster City, CA). Primers were obtained from IDT (Coralville, IA).

       Gene expression

      Dissected tissues were frozen immediately in liquid nitrogen and stored at −80 °C. Frozen tissues were homogenized in TRIzol reagent (Life Technologies, Inc.), and RNA was isolated per the manufacturer's instructions. 1 μg of isolated total RNA was DNase-treated, reverse-transcribed using the High Capacity cDNA kit (Applied Biosystems), and then diluted to 100 μl with RNase/DNase free water. qPCR was performed using primers specific to a select set of differentially expressed genes by TaqMan analyses. TaqMan gene expression probes (Applied Biosystems) were used for RT-PCR and are found in Table 1.
      Table 1Primers used for analyses
      Sequence-based reagentsSequence/sourceRef.
      TaqMan primers
          GapdhApplied Biosystems4352339E
          Cyp27b1Applied BiosystemsMm01165918
          Cyp24a1Applied BiosystemsMm00487244
          VdrApplied BiosystemsMm00437297
          Mettl1Applied BiosystemsMm00487686
          Mettl21bApplied BiosystemsMm01165909
          Tspan31Applied BiosystemsMm00482543
          TsfmApplied BiosystemsMm00508436
          March9Applied BiosystemsMm01165913
          Cdk4Applied BiosystemsMm00726334
      Primers for CRISPR
          M1-IKO Guide 1GATTAGTTGACCTTTCCTCCTGGThis paper
          M1-IKO Guide 2CAGGAACTCCAGACCATGAGAGGThis paper
          M21-IKO Guide 1CCTACCTTCCCGCTACTGTTGGGThis paper
          M21-IKO Guide 2CCCTTCCTTAGGGACTTCATGGGThis paper
      Primers for genotyping
          C27KO KO-FACTTTTCTGATTCAGGGATGAAGGTTTAGCThis paper
          C27KO KO-RGAAGGAGGGCAGATTAGATATTCTAGGATGCThis paper
          C27KO WT-FCTCCTGCCAGAGTCTATCCCTGThis paper
          C27KO WT-RGAAGGAGGGCAGATTAGATATTCTAGGATGCThis paper
          M1-IKO spanFAGTGGAGTTTGCAGACATAGGCTThis paper
          M1-IKO spanRTTACCTGTCTATAGGGAAGATGThis paper
          M1-IKO internalFTCTACTCTGGGTCTGTGGCCTTThis paper
          M1-IKO internalRAGCTAGACAGAACAACCGGGGThis paper
          M21-IKO spanFTTCCTCCACTGAGACAAGAGTTAThis paper
          M21-IKO spanRCCTTGCTACTTTCCAACAGCCTGCCTThis paper
          M21-IKO internalFATGTATCCTCTCCCTCCTGAACAThis paper
          M21-IKO internalRCCCCATACAATAGGGTTTCTCTCTGThis paper

       ChIP followed by sequencing (ChIP-seq)

      ChIP was performed using antibodies listed under “Reagents.” ChIP was performed as described previously with several modifications (
      • Meyer M.B.
      • Zella L.A.
      • Nerenz R.D.
      • Pike J.W.
      Characterizing early events associated with the activation of target genes by 1,25-dihydroxyvitamin D3 in mouse kidney and intestine in vivo.
      ). In brief, kidney and TPTG samples were obtained from mice treated with 1,25(OH)2D3, PTH, FGF23, or the corresponding vehicle for 1 h. Two kidneys or 10 TPTGs were combined per replicate, rinsed with ice-cold 1× PBS, and then subjected to immunoprecipitation using either a control IgG or the indicated antibodies. Statistical analysis and data processing for ChIP-seq assays 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.
      ). The isolated DNA (or input DNA acquired prior to precipitation) was then validated by qPCR and further prepared for ChIP-seq analysis. ChIP-seq libraries were prepared as described 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.
      ). Conservation analysis was performed with VISTA Point comparing the mouse (mm9) with human (hg19) genomic regions surrounding the CYP27B1 gene (
      • Frazer K.A.
      • Pachter L.
      • Poliakov A.
      • Rubin E.M.
      • Dubchak I.
      VISTA: computational tools for comparative genomics.
      ).

       CRISPR-generated and transgenic mice

      The guides used for CRISPR–Cas9-mediated genome editing were optimized for the least number of potential off-target sites and fewest sites within coding exons using the Zhang Laboratory CRISPR Design tool (crispr.mit.edu)3 and cross-referenced with Liu lab CRISPR-DO (cistrome.org/crispr).
      Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.
      The guides (see Table 1) were annealed and cloned into plasmids pX330 or pX458 obtained from Zhang Laboratory via Addgene (Cambridge, MA) as described recently (
      • Meyer M.B.
      • Benkusky N.A.
      • Onal M.
      • Pike J.W.
      Selective regulation of Mmp13 by 1,25(OH)2D3, PTH, and Osterix through distal enhancers.
      ). Resulting PCR products were then transcribed in vitro utilizing the T7 MEGAshortscript kit (Life Technologies, Inc.) (
      • Wang H.
      • Yang H.
      • Shivalila C.S.
      • Dawlaty M.M.
      • Cheng A.W.
      • Zhang F.
      • Jaenisch R.
      One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.
      ). The mixture of 50 ng/μl of the produced RNA guides and 40 ng/μl Cas9 protein in injection buffer (5 mm Trizma base, 5 mm Tris-HCl, 0.1 mm EDTA, pH 7.4) was injected into the pronucleus of 1 day-fertilized embryos isolated from hyper-ovulating female C57BL/6 mice, as described previously (
      • Meyer M.
      • de Angelis M.H.
      • Wurst W.
      • Kühn R.
      Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases.
      ), and implanted into recipient females at the University of Wisconsin-Madison Biotechnology Center Transgenic Animal Facility. The resultant pups were genotyped with spanning primers (Table 1), cloned, and sequenced. The top 10 predicted potential off-target sites were examined by PCR and sequencing analysis.
      The VDR-null (VDRKO) strain was produced by targeted ablation of the second zinc finger of the VDR DNA-binding domain (
      • 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.
      ). VDRKO mice were fed a standard rodent chow diet (5008; Lab Diet, St. Louis, MO). Cyp27b1 null (C27KO) mice were produced by GenOway (Strasbourg, France) with a strategy similar to that previously described (
      • Onal M.
      • St John H.C.
      • Danielson A.L.
      • Pike J.W.
      Deletion of the distal Tnfsf11 RL-D2 enhancer that contributes to PTH-mediated RANKL expression in osteoblast lineage cells results in a high bone mass phenotype in mice.
      ). In brief, the mouse Cyp27b1 gene was amplified by PCR using genomic DNA from C57BL/6 ES cells as a template and cloned into the pCR4-TPOP vector (Invitrogen) to create a targeting vector. Two loxP sites were inserted in intron 6 and downstream of exon 9 and a positive selection marker, neomycin resistance gene, which is flanked by FRT sites, was introduced between exon 9 and downstream loxP site. Diphtheria toxin A was also introduced upstream of the Cyp27b1 gene as a negative selection marker to reduce the isolation of non-homologous recombined ES cell clones and to enhance the chance to isolate ES cell clones containing the distal loxP site. The targeting vector was linearized and electroporated into ES cells. The recombined ES cell clones identified by the selection, PCR confirmation, and Southern blot analysis were then injected into C57BL/6 blastocysts to create chimeric mice, and the injected blastocysts were reimplanted into OF1 pseudopregnant females and allowed to develop. To remove the selection marker flanked by FRT sites and exons 7–9 of the Cyp27b1 gene flanked by loxP sites, the recombinant mice were sequentially bred with Flp-recombinase expressing deleter mice and then Cre-recombinase expressing deleter mice. For genotyping, a primer set amplifying a DNA region between the last exon and downstream of the last exon (880 bp) and a primer set amplifying a DNA region between intron 5 and downstream of the last exon (560 bp) were used for WT and the deleted alleles, respectively (Table 1).

       Animal studies

      Genetically modified mice were outbred with C57BL/6 mice (Envigo, Indianapolis, IN, and The Jackson Laboratory, Bar Harbor, ME) as heterozygotes. Mice were housed in high-density ventilated caging in the Animal Research Facility of the University of Wisconsin-Madison under 12-h light/dark cycles, 72 °F temperature, and 45% humidity. All mice used in this study were maintained on a standard rodent chow diet (5008, Lab Diet, St. Louis, MO), aged 8–9 weeks, and backcrossed five or more generations with WT mice unless otherwise indicated. For the 1,25(OH)2D3 rescue diet study, 3-week-old mice were fed either a 1,25(OH)2D3 rescue diet (5008 chow diet + 1 ng/g 1,25(OH)2D3) or standard chow (5008) for 5 weeks, at which time the animals were sacrificed, and tissues were collected. For the vitamin D3-deficiency study, 3-week-old mice were fed either a vitamin D3-deficient diet containing 0.6% calcium and 0.4% phosphorus (TD.01102, Envigo, Madison, WI) or a control diet containing 0.6% calcium, 0.4% phosphorus, and 2200 IU vitamin D/kg (TD.97191, Envigo) for 5 weeks, at which time animals were sacrificed, and tissues were collected. All experiments and tissue collections were performed in the procedure rooms in the Research Animal Facility of the University of Wisconsin-Madison. All animal studies were reviewed and approved by the Research Animal Care and Use Committee of the University of Wisconsin-Madison (A005478).
      Animals were injected intraperitoneally with 10 ng/g body weight (bw) 1,25(OH)2D3 (in propylene glycol), 230 ng/g bw PTH(1–84) (in PBS), 50 ng/g bw FGF23 (in PBS + 0.1% BSA), 10 mg/kg bw LPS (in PBS), or vehicle (EtOH or PBS). Animals were sacrificed, and tissues were collected 1 h after PTH injection, 3 h after FGF23 injection, and 6 h after 1,25(OH)2D3 and LPS injections. Unless otherwise indicated, all experiments were done with equal numbers of males and females (n ≥ 6). There were no differences in our data between males and females; therefore, the data presented combine both males and females in all studies unless otherwise indicated.

       Western blot analysis

      Mitochondrial extracts from whole kidneys were prepared with a Potter-Elvehjem homogenization tube using 5 ml of homogenization buffer (250 mm sucrose, 19 mm HEPES, 10 mm KCl, pH 7.4, plus protease inhibitor mixture (Roche Applied Science)). The resulting homogenate was centrifuged (4000 × g) for 1 min (4 °C). The supernatant was centrifuged (9000 × g) for 20 min (4 °C), and the resulting pellet was resuspended in 1 ml of homogenization buffer and centrifuged (9000 × g) for 10 min (4 °C). The pellet was solubilized in 500 μl of SDS loading buffer (100 mm Tris-HCl, pH 6.8, 4% SDS, 0.2% bromphenol blue, 20% glycerol, 200 mm DTT). 30 μl of sample was heated to 95 °C for 5 min and subjected to 12% SDS-PAGE. The kidney mitochondrial extracts were subjected to Western blot analysis as described 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.
      ) using primary antibodies to CYP27B1 (G-20, sc-49644, Santa Cruz Biotechnology, Inc., lot no. B1114, 1:500) and VDAC/Porin (ab15895, Abcam, lot no. GR264581-2, 1:5000) followed by secondary antibodies donkey α-goat IgG-HRP (sc-2020, Santa Cruz Biotechnology, Inc., 1:2000) and goat α-rabbit IgG-HRP (sc-2004, Santa Cruz Biotechnology, Inc., 1:5000), respectively.
      Whole-kidney tissue lysates were prepared using Tissue Lysis Buffer (10 mm Tris-Cl, pH 8, 300 mm KCl, 1 mm EDTA, 2 mm DTT, and protease inhibitor mixture), and protein concentrations were measured using protein assay (Bio-Rad). 80 μg of lysates were denatured and subjected to 12% SDS-PAGE. Subsequently, whole-kidney lysates were subjected to Western blot analysis, as described 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.
      ), using primary antibodies to VDR (9A7 (
      • Pike J.W.
      • Donaldson C.A.
      • Marion S.L.
      • Haussler M.R.
      Development of hybridomas secreting monoclonal antibodies to the chicken intestinal 1 α,25-dihydroxyvitamin D3 receptor.
      ), 1:2000) and β-tubulin (H-235, sc-9104, Santa Cruz Biotechnology, Inc.; lot no. E0913, 1:5000) followed by secondary antibodies goat α-rat IgG-HRP (sc-2032, Santa Cruz Biotechnology, Inc., 1:2000) and goat α-rabbit IgG-HRP (1:5000), respectively. Because of the overlapping molecular weights of VDR and β-tubulin, 1 aliquot of sample was run over two gels and immunoblotted as described above.

       Blood chemistry

      Cardiac blood was collected at the time of sacrifice. Collected blood was split into serum- or EDTA-treated plasma, incubated at room temperature for 30 min, followed by centrifugation at 6000 rpm for 12 min (twice) to obtain serum or EDTA plasma. Serum calcium and phosphate levels were measured using QuantiChromTM calcium assay kit (DICA-500, BioAssay Systems, Hayward, CA) and QuantiChromTM phosphate assay kit (DIPI-500, BioAssay Systems). Circulating intact FGF23 and PTH were measured in EDTA plasma via mouse/rat FGF-23 (intact) ELISA kit (60-6800, Immutopics, San Clemente, CA) and mouse PTH(1–84) ELISA kit (60-2305, Immutopics), respectively.

       Quantification of serum vitamin D metabolites

      Serum 25(OH)D3, 25(OH)D3-26,23-lactone, and 24,25(OH)2D3were quantified by LC-MS/MS, using previously published methods (
      • Kaufmann M.
      • Lee S.M.
      • Pike J.W.
      • Jones G.
      A high-calcium and phosphate rescue diet and VDR-expressing transgenes normalize serum vitamin D metabolite profiles and renal Cyp27b1 and Cyp24a1 expression in VDR null mice.
      ,
      • Kaufmann M.
      • Gallagher J.C.
      • Peacock M.
      • Schlingmann K.P.
      • Konrad M.
      • DeLuca H.F.
      • Sigueiro R.
      • Lopez B.
      • Mourino A.
      • Maestro M.
      • St-Arnaud R.
      • Finkelstein J.S.
      • Cooper D.P.
      • Jones G.
      Clinical utility of simultaneous quantitation of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D by LC-MS/MS involving derivatization with DMEQ-TAD.
      ), except that the starting volume of serum was reduced to 25 μl and diluted with 275 μl of water after addition of internal standard where an equivalent of 19 μl of serum was analyzed per injection. Vitamin D metabolite levels were determined in individual animals. Concentration of 25(OH)D3-26,23-lactone was estimated using the calibration line for 24,25(OH)2D3 and recovery of d6-24,25(OH)2D3. We observed that simultaneous assay of 1,25(OH)2D3 and 1,24,25(OH)3D3 was possible based on cross-reactivity of an anti-1,25(OH)2D3 antibody slurry (Immundiagnostik) with 1,24,25(OH)3D3. A six-point calibrator for 1,25-(OH)2D3 and 1,24,25(OH)3D3 was created in vitamin D-stripped serum (MSG2000; Golden West Biologicals), containing 5–300 pg/ml 1,25(OH)2D3 (Cerilliant) and 1–25 pg/ml 1,24,25(OH)3D3. 150-μl aliquots of serum were equilibrated with 200 pg/ml d6-1,25(OH)2D3 and 12.5 pg/ml d6-1,24,25(OH)3D3, which were generated in-house by HPLC purification from a CYP24A1 expression system incubated with d6-1,25(OH)2D3 based on previously described methodology (
      • Kaufmann M.
      • Prosser D.E.
      • Jones G.
      Bioengineering anabolic vitamin D-25-hydroxylase activity into the human vitamin D catabolic enzyme, cytochrome P450 CYP24A1, by a V391L mutation.
      ). The serum was incubated with 100 μl of anti-1,25(OH)2D3 antibody slurry (
      • Laha T.J.
      • Strathmann F.G.
      • Wang Z.
      • de Boer I.H.
      • Thummel K.E.
      • Hoofnagle A.N.
      Characterizing antibody cross-reactivity for immunoaffinity purification of analytes prior to multiplexed liquid chromatography-tandem mass spectrometry.
      ) for 2 h at room temperature with orbital shaking at 1200 rpm. The slurry was isolated by vacuum filtration and rinsed four times with 400-μl aliquots of water, and vitamin D metabolites were eluted two times with 400 μl of ethanol. The ethanol eluate was evaporated to dryness and derivatized with 4-[2-(3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxo-2-quinoxalinyl)ethyl]-3H-1,2,4-triazole-3.5-(4H)-dione (DMEQ-TAD) as described previously. The sample was re-dissolved in 50 μl of mobile phase consisting of 50:50 (% by volume) methanol/water, and 35 μl (110-μl eq of serum) was injected into the LC-MS/MS system as described previously (
      • Kaufmann M.
      • Morse N.
      • Molloy B.J.
      • Cooper D.P.
      • Schlingmann K.P.
      • Molin A.
      • Kottler M.L.
      • Gallagher J.C.
      • Armas L.
      • Jones G.
      Improved screening test for idiopathic infantile hypercalcemia confirms residual levels of serum 24,25-(OH)2D3 in affected patients.
      ). Multiple reaction monitoring transitions used for analysis of 1,25-(OH)2D3 and 1,24,25(OH)3D3 were as follows: m/z 762 → 468 + 762 → 484 and 778 → 468 + 778 → 484, respectively.

       BMD and μCT analysis

      At 8–9 weeks of age, BMDs of the CRISPR-generated mice and their WT littermates were measured and analyzed by dual X-ray absorptiometry with a PIXImus densitometer (GE-Lunar Corp., Madison, WI), as described previously (
      • Onal M.
      • St John H.C.
      • Danielson A.L.
      • Pike J.W.
      Deletion of the distal Tnfsf11 RL-D2 enhancer that contributes to PTH-mediated RANKL expression in osteoblast lineage cells results in a high bone mass phenotype in mice.
      ). μCT analyses were performed using a high-resolution benchtop Scanco uCT50 (Scanco Medical, Bassersdorf, Switzerland) at the Vanderbilt Center for Bone Biology. Tomographic images were acquired at 70 peak kilovoltages and 200 μA with an isotropic voxel size of 12.0 mm and at an integration time of 400 ms with 1000 projections per 360° rotation. Reconstructed μCT images were binarized, using a Gaussian noise filter with σ = 0.3 and support = 1 and a threshold of 470 mg of hydroxyapatite/ccm for trabecular bone or 0.3, 1, and 733 mg of HA/cm3 for midshaft cortical bone. The volume of interest for trabecular bone in the distal femoral metaphysis was the entire medullary area located 0.360–1.560 mm proximal to the distal growth plate. The midshaft volume of interest was a 1.2-mm-long segment of cortical bone centered between the femoral head and surface of the distal condyles. All volumetric structural parameters were calculated from the 3D renderings using Scanco Evaluation software following standard methods and nomenclature (
      • Bouxsein M.L.
      • Boyd S.K.
      • Christiansen B.A.
      • Guldberg R.E.
      • Jepsen K.J.
      • Müller R.
      Guidelines for assessment of bone microstructure in rodents using micro-computed tomography.
      ).

       Immunohistochemistry

      Mice were perfused with PBS followed by 4% paraformaldehyde. The collected tissues were immersed overnight in 4% paraformaldehyde and then overnight again in 30% sucrose. Thyroparathyroid tissues were processed to prepare frozen tissue blocks using Neg-50 (Richard-Allan Scientific) and cut into 10-μm sections with an HM5050E microtome (Microm). The tissue sections were stained with hematoxylin stain solution (3530-32; Ricca Chemical Co.) and eosin-Y (Fisher). The stained tissue sections were mounted with Permount (Fisher). Images for histological assay were taken using an ECLIPSE Ti-S microscope (Nikon) and QICAM 12-bit Mono Fast 1394 cooled camera (QImaging). PTG sizes were determined using ImageJ software. After μCT analysis, undecalcified tibiae were embedded in methyl methacrylate, sectioned on an automatic retractable Microtom 355 with a D-profile, tungsten carbide steel knife at 4 μm. Adjacent sections were stained with Masson trichrome (
      • Perrien D.S.
      • Akel N.S.
      • Edwards P.K.
      • Carver A.A.
      • Bendre M.S.
      • Swain F.L.
      • Skinner R.A.
      • Hogue W.R.
      • Nicks K.M.
      • Pierson T.M.
      • Suva L.J.
      • Gaddy D.
      Inhibin A is an endocrine stimulator of bone mass and strength.
      ) or with von Kossa to visualize mineralized bone.

       T cell activation

      Spleen CD4+ or CD8+ T cells were isolated by negative selection using EasySep mouse CD4+ or CD8+ T cell isolation kit (Stemcell Technologies, Vancouver, British Columbia, Canada) per the manufacturer's instructions. Isolated CD4+ or CD8+ T cells were plated in 24-well plates with a density of 1 × 106 cells/well and treated 3 h with vehicle (PBS) or 25 μl/well of CD3/CD28 mouse T-activator Dynabeads (Gibco, Life Technologies, Inc.). In addition, isolated CD4+ or CD8+ T cells were plated in 24-well plates with a density of 1 × 106 cells/well and treated 3 h with vehicle (EtOH) or 100 nm 1,25(OH)2D3.

       Statistical evaluation

      Data were analyzed using GraphPad Prism 7 software (GraphPad Software, Inc., La Jolla, CA) and in consultation with the Statistics Department, University of Wisconsin. All values are reported as the means ± S.E., and differences between group means were evaluated using one-way ANOVA, two-way ANOVA, or Student's t test as indicated in the figure legends.

      Author contributions

      M. B. M. and J. W. P. conceived the paper; M. B. M., N. A. B., M. O., S. M. L., and M. K. performed the investigation; M. B. M. and J. W. P. wrote the original draft; N. A. B., M. K., and G. J. reviewed and edited the paper, M. B. M. performed data curation.

      Acknowledgments

      We thank Dr. Dan Perrien at the Vanderbilt Center for Bone Biology for μCT analysis and growth plate slide preparation. We thank the University of Wisconsin Biotechnology Center Transgenic Animal Facility for generating the CRISPR/Cas9 enhancer-deleted mice. We also thank Professor Mark R. Haussler for historical perspective and careful reading of the manuscript. Through a Queen's University/Waters Corp. agreement, Waters generously provided the LC-MS/MS instrumentation used in this study.

      Author Profile

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