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Vitamin D metabolism centers on kidney regulation of Cyp27b1 by mineralotropic hormones, including induction by parathyroid hormone (PTH), suppression by fibroblast growth factor 23 (FGF23) and 1,25-dihydroxyvitamin D

The primary function of vitamin D in higher vertebrates is to regulate mineral homeostasis through direct actions on intestinal calcium and phosphate absorption, bone mineral resorption, and renal mineral reabsorption Vitamin D itself is inactive and must undergo sequential modification via two specific chemical reactions, first in the liver to 25(OH)D3 by the enzyme CYP2R1, and then in the kidney to 1,25-dihydroxyvitamin D3 by the tightly regulated enzyme CYP27B1 Calcitriol, whose level is also modulated via renal CYP24A1-mediated degradation, is then secreted into the blood as an active endocrine hormone and delivered to distant target tissues Here, Pike and Meyer discuss the importance of adequate blood levels of vitamin D and the models for characterizing features of NRTC calcitriol production

Fibroblast growth factor 23 (FGF23) is a bone-derived hormone involved in the control of phosphate (P) homeostasis and vitamin D metabolism. Despite advances, however, molecular details of this gene’s regulation remain uncertain. In this report, we created mouse strains in which four epigenetically marked FGF23 regulatory regions were individually deleted from the mouse genome using CRISPR/Cas9 gene-editing technology, and the consequences of these mutations were then assessed on Fgf23 expression and regulation in vivo. An initial analysis confirmed that bone expression of Fgf23 and circulating intact FGF23 (iFGF23) were strongly influenced by both chronic dietary P treatment and acute injection of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. However, further analysis revealed that bone Fgf23 expression and iFGF23 could be rapidly upregulated by dietary P within 3 and 6 hours, respectively; this acute upregulation was lost in the FGF23-PKO mouse containing an Fgf23 proximal enhancer deletion but not in the additional enhancer-deleted mice. Of note, prolonged dietary P treatment over several days led to normalization of FGF23 levels in the FGF23-PKO mouse, suggesting added complexity associated with P regulation of FGF23. Treatment with 1,25(OH)2D3 also revealed a similar loss of Fgf23 induction and blood iFGF23 levels in this mouse. Finally, normal lipopolysaccharide (LPS) induction of Fgf23 expression was also compromised in the FGF23-PKO mouse, a result that, together with our previous report, indicates that the action of LPS on Fgf23 expression is mediated by both proximal and distal Fgf23 enhancers. These in vivo data provide key functional insight into the genomic enhancers through which Fgf23 expression is mediated.

Vitamin D(3) is terminally bioactivated in the kidney to 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) via cytochrome P450 family 27 subfamily B member 1 (CYP27B1), whose gene is regulated by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and 1,25(OH)(2)D(3). Our recent genomic studies in the mouse have revealed a complex kidney-specific enhancer module within the introns of adjacent methyltransferase-like 1 (Mettl1) and Mettl21b that mediate basal and PTH-induced expression of Cyp27b1 and FGF23- and 1,25(OH)(2)D(3)-mediated repression. Gross deletion of these segments in mice has severe effects on Cyp27b1 regulation and skeletal phenotype but does not affect Cyp27b1 expression in nonrenal target cells (NRTCs). Here, we report a bimodal activity in the Mettl1 intronic enhancer with components responsible for PTH-mediated Cyp27b1 induction and 1,25(OH)(2)D(3)-mediated repression and additional activities, including FGF23 repression, within the Mettl21b enhancers. Deletion of both submodules eliminated basal Cyp27b1 expression and regulation in the kidney, leading to systemic and skeletal phenotypes similar to those of Cyp27b1-null mice. However, basal expression and lipopolysaccharide-induced regulation of Cyp27b1 in NRTCs was unperturbed. Importantly, dietary normalization of calcium, phosphate, PTH, and FGF23 rescued the skeletal phenotype of this mutant mouse, creating an ideal in vivo model to study nonrenal 1,25(OH)(2)D(3) production in health and disease. Finally, we confirmed a conserved chromatin landscape in human kidney that is similar to that in mouse. These findings define a finely balanced homeostatic mechanism involving PTH and FGF23 together with protection from 1,25(OH)(2)D(3) toxicity that is responsible for both adaptive vitamin D metabolism and mineral regulation.

Our recent genomic studies identified a complex kidney‐specific enhancer module located within the introns of adjacent Mettl1 (M1) and Mettl21b (M21) genes that mediate basal and PTH induction of Cyp27b1, as well as suppression by FGF23 and 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3]. The tissue specificity for this regulatory module appears to be localized exclusively to renal proximal tubules. Gross deletion of these segments in mice has severe consequences on skeletal health, and directly affects Cyp27b1 expression in the kidney. Deletion of both the M1 and M21 submodules together almost completely eliminates basal Cyp27b1 expression in the kidney, creating a renal specific pseudo‐null mouse, resulting in a systemic and skeletal phenotype similar to that of the Cyp27b1‐KO mouse caused by high levels of both 25‐hydroxyvitamin D3 [25(OH)D3] and PTH and depletion of 1,25(OH)2D3. Cyp24a1 levels in the double KO mouse also decrease because of compensatory downregulation of the gene by elevated PTH and reduced FGF23 that is mediated by an intergenic module located downstream of the Cyp24a1 gene. Outside of the kidney in nonrenal target cells (NRTCs), expression of Cyp27b1 in these mutant mice was unaffected. Dietary normalization of calcium, phosphate, PTH, and FGF23 rescues the aberrant phenotype of this mouse and normalizes the skeleton. In addition, both the high levels of 25(OH)D3 were reduced and the low levels of 1,25(OH)2D3 were fully eliminated in these mutant mice as a result of the rescue‐induced normalization of renal Cyp24a1. Thus, these hormone‐regulated enhancers for both Cyp27b1 and Cyp24a1 in the kidney are responsible for the circulating levels of 1,25(OH)2D3 in the blood. The retention of Cyp27b1 and Cyp24a1 expression in NRTCs of these endocrine 1,25(OH)2D3‐deficient mice suggests that this Cyp27b1 pseudo‐null mouse will provide a model for the future exploration of the role of NRTC‐produced 1,25(OH)2D3 in the hormone's diverse noncalcemic actions in both health and disease. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

During bone marrow stromal cell (BMSC) differentiation, both Wnt signaling and the development of a rigid cytoskeleton promote commitment to the osteoblastic over adipogenic lineage. β-catenin plays a critical role in the Wnt signaling pathway to facilitate downstream effects on gene expression. We show that β-catenin was additive with cytoskeletal signals to prevent adipogenesis, and β-catenin knockdown promoted adipogenesis even when the actin cytoskeleton was depolymerized. β-catenin also prevented osteoblast commitment in a cytoskeletal-independent manner, with β-catenin knockdown enhancing lineage commitment. Chromatin immunoprecipitation (ChIP)-sequencing demonstrated binding of β-catenin to the promoter of enhancer of zeste homolog 2 (EZH2), a key component of the polycomb repressive complex 2 (PRC2) complex that catalyzes histone methylation. Knockdown of β-catenin reduced EZH2 protein levels and decreased methylated histone 3 (H3K27me3) at osteogenic loci. Further, when EZH2 was inhibited, β-catenin's anti-differentiation effects were lost. These results indicate that regulating EZH2 activity is key to β-catenin's effects on BMSCs to preserve multipotentiality. © 2020 American Society for Bone and Mineral Research.

Objectives First-rib resection is a key component in the treatment of Paget-Schroetter syndrome. We report our experience with robotic first-rib resection. Methods Patients diagnosed with Paget-schroetter syndrome underwent thrombolysis followed by robotic first-rib resection. The diagnosis was made by preoperative venography (VA) and magnetic resonance venography. The robot was used to dissect the first rib, disarticulate the costosternal joint and divide the scalene muscles. Success of the first-rib resection was assessed by physical examination, venogram and magnetic resonance venography. Results Eighty-three robotic first-rib resections were performed (49 men and 34 women). The mean age of the patients was 24 years ± 8.5 years. The operative time was 127.6 min ± 20.8 min. The median hospitalization was 4 days. There were no surgical complications, neurovascular injuries or mortality. Patients with a patent subclavian vein on the postoperative venogram (57 of 83 patients, 69%) were anticoagulated with warfarin for 3 months. In the remaining 27 patients with a persistent postoperative occlusion of the subclavian vein, 21 (21 of 83 patients, 24%) underwent angioplasty and were anticoagulated with warfarin for 3 months, and 6 (6 of 83, 7%) required stent placement to achieve complete vein patency. Patients who underwent stent placement received antiplatelet therapy in addition to warfarin anticoagulation for 3 months. At a median follow-up of 24 months, all patients had an open subclavian vein with a patency rate of 100%. Conclusions The robotic transthoracic first-rib resection is feasible and allows for a minimally invasive resection of the first rib, while minimizing neurovascular complications.

The vitamin D receptor (VDR) mediates the pleiotropic biological actions of 1,25-dihydroxyvitamin D(3)(1,25(OH)(2)D(3)). These actions include orchestration of mineral homeostasis which is coordinated by the kidney, intestine, bone and parathyroid gland wherein the VDR transcriptionally regulates expression of the genes involved in this complex process. Mutations in human VDR (hVDR) cause hereditary vitamin D resistant rickets, a genetic syndrome characterized by hypocalcemia, hyperparathyroidism and rickets resulting from dysregulation of mineral homeostasis. Expression of the VDR is regulated by external stimuli in a tissue-specific manner. However, the mechanisms of this tissue-specificity remain unclear. Studies also suggest that phosphorylation of hVDR at serine 208 impacts the receptor’s transcriptional activity. These experiments were conducted in vitro, however, and therefore limited in their conclusions. In this report, we summarize (1) our most recently updated ChIP-seq data from mouse tissues to identify regulatory regions responsible for the tissues-specific regulation of the VDR and (2) our studies to understand the mechanism of hormonal regulation of Vdr expression in bone and kidney in vivo using transgenic mouse strains generated by mouse mini-genes that contain comprehensive genetic information capable of recapitulating endogenous Vdr gene regulation and expression. We also defined the functional human VDR gene locus in vivo by using a human mini-gene comparable to that in the mouse to generate a humanized VDR mouse strain in which the receptor is expressed at normal levels (normal expressor). The present report also shows that a humanized mouse model in which the VDR is expressed at levels about 10- fold lower than the normal expressor mouse rescued the VDR-null phenotype despite its reduced transcriptional activity relative to wildtype expression. We also generated an additional humanized mouse model expressing hVDR bearing a mutation converting serine 208 to alanine (hVDR-S208A). In spite of the mutation, target gene expression induced by the ligand was unchanged relative to a mouse strain expressing comparable levels of wildtype hVDR. Further characterization also showed that serum calcium and parathyroid hormone levels were normal and alopecia was not observed in this hVDR-S208A mouse strain as well. Taken together, our in vivo studies using ChIP-seq analyses and the mini-gene transgenic mice improve our understanding of the tissue-specific regulatory mechanisms of controlling VDR expression and the mechanisms of action of the VDR.

Recent studies of transcription have revealed an advanced set of overarching principles that govern vitamin D action on a genome-wide scale. These tenets of vitamin D transcription have emerged as a result of the application of now well-established techniques of chromatin immunoprecipitation coupled to next-generation DNA sequencing that have now been linked directly to CRISPR-Cas9 genomic editing in culture cells and in mouse tissues in vivo. Accordingly, these techniques have established that the vitamin D hormone modulates sets of cell-type specific genes via an initial action that involves rapid binding of the VDR–ligand complex to multiple enhancer elements at open chromatin sites that drive the expression of individual genes. Importantly, a sequential set of downstream events follows this initial binding that results in rapid histone acetylation at these sites, the recruitment of additional histone modifiers across the gene locus, and in many cases, the appearance of H3K36me3 and RNA polymerase II across gene bodies. The measured recruitment of these factors and/or activities and their presence at specific regions in the gene locus correlate with the emerging presence of cognate transcripts, thereby highlighting sequential molecular events that occur during activation of most genes both in vitro and in vivo. These features provide a novel approach to the study of vitamin D analogs and their actions in vivo and suggest that they can be used for synthetic compound evaluation and to select for novel tissue- and gene-specific features. This may be particularly useful for ligand activation of nuclear receptors given the targeting of these factors directly to genetic sites in the nucleus.

Fibroblast growth factor 23 (FGF23) production is regulated by both calciotropic hormones and inflammation. Consistent with this, elevated FGF23 levels are associated with inflammatory markers as well as parathyroid hormone (PTH) in various disease states, including chronic kidney disease (CKD). However, the molecular mechanisms underpinning Fgf23 transcription in response to these regulators are largely unknown. We therefore utilized chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) data from an osteocyte cell line to identify potential regulatory regions of the Fgf23 gene. Based on ChIP-seq analysis of enhancer-associated histone modifications, including H3K4 methylation and H3K9 acetylation, we discovered several potential enhancers for Fgf23, one of which was located 16kb upstream of the gene's transcriptional start site. Deletion of this putative enhancer from the mouse genome using CRISPR-Cas9 technology led to lower bone, thymus, and spleen expression of Fgf23 mRNA without altering circulating levels of the intact hormone, although as previously reported, only bone displayed significant basal expression. Nevertheless, lack of the -16kb enhancer blunted FGF23 upregulation in a tissue-specific manner by the acute inflammatory inducers lipopolysaccharide (LPS), interleukin-1-beta (IL-1β), and tumor necrosis factor-alpha (TNFα) in bone, non-osseous tissues, and in circulation. Lack of the -16kb enhancer also inhibited PTH-induced bone Fgf23 mRNA. Moreover, the absence of this Fgf23 enhancer in an oxalate diet-induced murine CKD model prevented the early onset induction of osseous, renal, and thymic Fgf23 mRNA levels and led to a significant blunting of elevated circulating intact FGF23 levels. These results suggest that -16kb enhancer mediates the induction of Fgf23 by inflammation and PTH and facilitates the increase in FGF23 expression in a murine model of CKD. As exemplified herein, these Fgf23 enhancer-deleted mice will provide a unique model in which to study the role of FGF23 expression in inflammatory diseases.

The vitamin D endocrine system is essential for calcium metabolism and skeletal integrity. 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] regulates bone mineral homeostasis and acts directly on osteoblasts. In the present study we characterized the transcriptional regulation of the class 3 semaphorin (Sema3) gene family by 1,25(OH)2D3 in osteoblastic cells. Class 3 semaphorins are secreted proteins that regulate cell growth, morphology and migration, and were recently shown to be involved in bone homeostasis. In ST2, MC3T3-E1 and primary calvarial osteoblast cell cultures we found that all members of the Sema3 gene family were expressed, and that Sema3e and Sema3f were the most strongly induced 1,25(OH)2D3 target genes among the studied cell types. In addition, transcription of Sema3b and Sema3c was upregulated, whereas Sema3d and Sema3g was downregulated by 1,25(OH)2D3 in different osteoblastic cells. Chromatin immunoprecipitation analysis linked to DNA sequencing (ChIP-seq analysis) revealed the presence of the vitamin D receptor at multiple genomic loci in the proximity of Sema3 genes, demonstrating that the genes are primary 1,25(OH)2D3 targets. Furthermore, we showed that recombinant SEMA3E and SEMA3F protein were able to inhibit osteoblast proliferation. However, recombinant SEMA3s did not affect ST2 cell migration. The expression of class 3 semaphorins in osteoblasts together with their regulation by 1,25(OH)2D3 suggests that these genes, involved in the regulation of bone homeostasis, are additional mediators for 1,25(OH)2D3 signaling in osteoblasts.

Epidermal lineages and injury induced regeneration are controlled by transcriptional programs coordinating cellular signaling and epigenetic regulators, but the mechanism remains unclear. Previous studies showed that conditional deletion of the transcriptional coactivator Mediator 1 (Med1) changes epidermal lineages and accelerates wound re-epithelialization. Here, we studied a molecular mechanism by which Med1 facilitates these processes, in particular, by focusing on TGFβ signaling through genome wide transcriptome analysis. The expression of the TGF ligands (Tgfβ1/β2) and their downstream target genes is decreased in both normal and wounded Med1 null skin. Med1 silencing in cultured keratinocytes likewise reduces the expression of the ligands (TGFβ1/β2) and diminishes activity of TGFβ signaling as shown by decreased p-Smad2/3. Silencing Med1 increases keratinocyte proliferation and migration in vitro. Epigenetic studies using chromatin immuno-precipitation and next generation DNA sequencing reveals that Med1 regulates transcription of TGFβ components by forming large clusters of enhancers called super-enhancers at the regulatory regions of the TGFβ ligand and SMAD3 genes. These results demonstrate that Med1 is required for the maintenance of the TGFβ signaling pathway. Finally, we show that pharmacological inhibition of TGFβ signaling enhances epidermal lineages and accelerates wound re-epithelialization in skin similar to that seen in the Med1 null mice, providing new insights into epidermal regeneration.

Cyp27b1 and Cyp24a1 are reciprocally regulated in the kidney by the key hormones PTH, FGF23, and 1,25(OH)(2)D(3). Our recent genomic studies in mice identified a complex kidney-specific enhancer module located within the introns of adjacent Mettl1 (M1) and Mettl21b (M21) genes that mediate basal and PTH induction of Cyp27b1 as well as suppression by FGF23 and 1,25(OH)(2)D(3). Gross deletion of these segments in mice has severe consequences on skeletal health, and directly affects Cyp27b1 expression in the kidney. Deletion of both M1 and M21 submodules together fully eliminates basal Cyp27b1 expression in the kidney, leading to a systemic and skeletal phenotype similar to that of the Cyp27b1-KO mouse due to depletion of 1,25(OH)(2)D(3) and high PTH. Cyp24a1 levels in the double KO mouse were low due to compensatory regulation by elevated PTH and reduced FGF23. However, expression of Cyp27b1 and retention of its regulation by inflammation (LPS) in the NRTCs remained unperturbed. Dietary normalization of calcium, phosphate, PTH, and FGF23 rescues this aberrant phenotype and normalizes the skeletal issues. Cyp24a1 is controlled by its own unique enhancers for 1,25(OH)(2)D(3), FGF23, and PTH. We were also able to eliminate these activities in mice. Collectively, the hormone-mediated enhancer regulation of both Cyp27b1 and Cyp24a1 in the kidney is responsible for the circulating levels of 1,25(OH)(2)D(3) in the blood which in turn primarily affects calcium and phosphate regulation. Importantly, we can now manipulate this system with our enhancer deletion animal models to study 1,25(OH)(2)D(3) production in non-renal target cells and tissues not only in disease, where it is known to affect the immune system, but also in healthy individuals. Here we will review our studies that have defined a finely balanced homeostatic control mechanism employed by PTH and FGF23 with catastrophic toxicity protection from 1,25(OH)(2)D(3) in the genomic regulation of vitamin D metabolism and its accompanied control of mineral maintenance.

Cytochrome P450 family 27 subfamily B member 1 (CYP27B1) and CYP24A1 function to maintain physiological levels of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) in the kidney. Renal Cyp27b1 and Cyp24a1 expression levels are transcriptionally regulated in a highly reciprocal manner by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and 1,25(OH)(2)D(3). In contrast, Cyp24a1 regulation in nonrenal target cells (NRTCs) is limited to induction by 1,25(OH)(2)D(3). Herein, we used ChIP-Seq analyses of mouse tissues to identify regulatory regions within the Cyp24a1 gene locus. We found an extended region downstream of Cyp24a1 containing a cluster of sites, termed C24-DS1, binding PTH-sensitive cAMP-responsive element–binding protein (CREB) and a cluster termed C24-DS2 binding the vitamin D receptor (VDR). VDR-occupied sites were present in both the kidney and NRTCs, but pCREB sites were occupied only in the kidney. We deleted each segment in the mouse and observed that although the overt phenotypes of both cluster deletions were unremarkable, RNA analysis in the C24-DS1–deleted strain revealed a loss of basal renal Cyp24a1 expression, total resistance to FGF23 and PTH regulation, and secondary suppression of renal Cyp27b1; 1,25(OH)(2)D(3) induction remained unaffected in all tissues. In contrast, loss of the VDR cluster in the C24-DS2–deleted strain did not affect 1,25(OH)(2)D(3) induction of renal Cyp24a1 expression yet reduced but did not eliminate Cyp24a1 responses in NRTCs. We conclude that a chromatin-based mechanism differentially regulates Cyp24a1 in the kidney and NRTCs and is essential for the specific functions of Cyp24a1 in these two tissue types.

Post-translational modifications are key regulators of protein function, providing signals that alter protein interactions and activity. Phosphorylation of estrogen receptor-α (ER) at serine 118 (pS118-ER) occurs in response to multiple stimuli and is involved in modulating ER-dependent gene transcription. While the effects of pS118 on ER-DNA interactions have been investigated at specific genomic sites, the genome-wide binding profile (cistrome) of pS118-ER remains to be studied. To determine and characterize the pS118-ER cistrome, chromatin immunoprecipitation sequencing (ChIP-seq) was performed on both pS118-ER and ER and their binding profiles were compared. A subset of ER sites was found to be occupied by pS118-ER and although these sites were not found to be enriched in promoter regions relative to all ER sites, the pS118-ER sites were associated with the active enhancer mark H3K27ac as well as genes upregulated, but not downregulated, by estrogen. Additionally, pS118-ER sites were found to be enriched in the DNA binding motif for GRHL2 as well as the estrogen response element (ERE) relative to all ER sites. Utilizing a DNA binding microarray, pS118-ER was found to be more commonly associated with direct DNA binding events compared to indirect binding events. These results suggest a role for pS118-ER at active enhancers and as a regulator of direct ER-DNA interactions. Prevention of this phosphorylation event could serve as a potential treatment for ER-positive breast cancers by reducing the transcriptional activity of ER and decreasing its occupancy on DNA.

Posttranslational modifications are key regulators of protein function, providing cues that can alter protein interactions and cellular location. Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) occurs in response to multiple stimuli and is involved in modulating ER-dependent gene transcription. While the cistrome of ER is well established, surprisingly little is understood about how phosphorylation impacts ER-DNA binding activity. To define the pS118-ER cistrome, chromatin immunoprecipitation sequencing was performed on pS118-ER and ER in MCF-7 cells treated with estrogen. pS118-ER occupied a subset of ER binding sites which were associated with an active enhancer mark, acetylated H3K27. Unlike ER, pS118-ER sites were enriched in GRHL2 DNA binding motifs, and estrogen treatment increased GRHL2 recruitment to sites occupied by pS118-ER. Additionally, pS118-ER occupancy sites showed greater enrichment of full-length estrogen response elements relative to ER sites. In an in vitro DNA binding array of genomic binding sites, pS118-ER was more commonly associated with direct DNA binding events than indirect binding events. These results indicate that phosphorylation of ER at serine 118 promotes direct DNA binding at active enhancers and is a distinguishing mark for associated transcription factor complexes on chromatin.

The cytokine RANKL is essential for osteoclast formation during physiological and pathological bone resorption. RANKL also contributes to lymphocyte production, development of lymph nodes and mammary glands, as well as other biological activities. Transcriptional control of the Tnfsf11 gene, which encodes RANKL, is complex and involves distant regulatory regions. Nevertheless, cell culture studies suggest that an enhancer region near the transcription start site is involved in the control of Tnfsf11 expression by hormones such as 1,25-(OH)2 vitamin D3 and parathyroid hormone, as well as the sympathetic nervous system. To address the significance of this region in vivo, we deleted the sequence between -510 to -1413 bp, relative to Tnfsf11 exon 1, from mice using CRISPR-based gene editing. MicroCT analysis of the femur and fourth lumbar vertebra of enhancer knockout mice showed no differences in bone mass compared to wild type littermates at 5 weeks and 6 months of age, suggesting no changes in osteoclast formation. RNA extracted from the tibia, fifth lumbar vertebra, thymus, and spleen at 6 months of age also showed no reduction in Tnfsf11 mRNA abundance between these groups. However, maximal stimulation of Tnfsf11 mRNA abundance in cultured stromal cells by PTH was reduced approximately 40% by enhancer deletion, while stimulation by 1,25-(OH)2 vitamin D3 was unaffected. The abundance of B and T lymphocytes in the bone marrow did not differ between genotypes. These results demonstrate that the region between -510 and -1413 does not contribute to Tnfsf11 expression, osteoclast support, or lymphocyte production in mice under normal physiological conditions but may be involved in situations of elevated parathyroid hormone.

Matrix metalloproteinase 13 (MMP13, collagenase-3) is a vital component for chondrocyte and osteoblast maturation, and is aberrantly expressed in numerous disease states. At the transcriptional level, Mmp13 is controlled by many different growth factors and hormones. Most notably, Mmp13 is regulated by the vitamin D hormone (1,25(OH)2D3), parathyroid hormone (PTH), and several cytokines. These activities occur through participation by the transcription factors VDR, RUNX2, FOS, JUN, and Osterix (OSX), respectively. Recently, we discovered that Mmp13 is regulated by elements quite distal to the transcriptional start site -10, -20, and -30kb upstream. These enhancers, along with minor contributions from the region proximal to the promoter, are responsible for the ligand inducible and, most strikingly, the basal activities of Mmp13 gene regulation. Here, we found that the actions of PTH and OSX do not occur through the -10kb VDR bound enhancer. Rather, the -30kb RUNX2 bound enhancer and the promoter proximal regions were essential for activity. Through RUNX2 deletion and OSX overexpression in cells, we showed a specific role for OSX in Mmp13 regulation. Finally, we created an in vivo CRISPR deleted -10kb enhancer mouse model. Despite normal bone density and growth, they fail to up-regulate Mmp13 in response to 1,25(OH)2D3. These data are consistent with those obtained through UAMS osteoblast cell culture and further define the specific roles of distal enhancers in the regulation of Mmp13.

The biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the vitamin D receptor (VDR), whose expression in bone cells is regulated positively by 1,25(OH)2D3, retinoic acid, and parathyroid hormone through both intergenic and intronic enhancers. In this report, we used ChIP-sequencing analysis to confirm the presence of these Vdr gene enhancers in mesenchyme-derived bone cells and to describe the epigenetic histone landscape that spans the Vdr locus. Using bacterial artificial chromosome-minigene stable cell lines, CRISPR/Cas9 enhancer-deleted daughter cell lines, transient transfection/mutagenesis analyses, and transgenic mice, we confirmed the functionality of these bone cell enhancers in vivo as well as in vitro. We also identified VDR-binding sites across the Vdr gene locus in kidney and intestine using ChIP-sequencing analysis, revealing that only one of the bone cell-type enhancers bound VDR in kidney tissue, and none were occupied by the VDR in the intestine, consistent with weak or absent regulation by the 1,25(OH)2D3 hormone in these tissues, respectively. However, a number of additional sites of VDR binding unique to either kidney or intestine were present further upstream of the Vdr gene, suggesting the potential for alternative regulatory loci. Importantly, virtually all of these regions retained histone signatures consistent with those of enhancers and exhibited unique DNase I hypersensitivity profiles that reflected the potential for chromatin access. These studies define mechanisms associated with hormonal regulation of the Vdr and hint at the differential nature of VDR binding activity at the Vdr gene in different primary target tissues in vivo.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) plays an integral role in calcium homeostasis in higher organisms through its actions in the intestine, kidney, and skeleton. Interestingly, although several intestinal genes are known to play a contributory role in calcium homeostasis, the entire caste of key components remains to be identified. To examine this issue, Cyp27b1 null mice on either a normal or a high calcium/phosphate-containing rescue diet were treated with vehicle or 1,25(OH)2D3 and evaluated 6 h later. RNA samples from the duodena were then subjected to RNA sequence analysis, and the data were analyzed bioinformatically. 1,25(OH)2D3 altered expression of large collections of genes in animals under either dietary condition. 45 genes were found common to both 1,25(OH)2D3-treated groups and were composed of genes previously linked to intestinal calcium uptake, including S100g, Trpv6, Atp2b1, and Cldn2 as well as others. An additional distinct network of 56 genes was regulated exclusively by diet. We then conducted a ChIP sequence analysis of binding sites for the vitamin D receptor (VDR) across the proximal intestine in vitamin D-sufficient normal mice treated with vehicle or 1,25(OH)2D3. The residual VDR cistrome was composed of 4617 sites, which was increased almost 4-fold following hormone treatment. Interestingly, the majority of the genes regulated by 1,25(OH)2D3 in each diet group as well as those found in common in both groups contained frequent VDR sites that likely regulated their expression. This study revealed a global network of genes in the intestine that both represent direct targets of vitamin D action in mice and are involved in calcium absorption.

Matrix metalloproteinase 13 (Mmp13, collagenase-3) plays an essential role in bone metabolism and mineral homeostasis. It is regulated by numerous factors, including BMP-2, parathyroid hormone, and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), through transcription factors such as Runt-related transcription factor 2 (RUNX2), CCAAT/enhancer-binding protein β (C/EBPβ), OSX, and vitamin D receptor (VDR). During osteoblast maturation, the basal expression of Mmp13 and its sensitivity to 1,25(OH)2D3 are strikingly increased. In this report, ChIP-sequencing analysis in mouse preosteoblasts revealed that the Mmp13 gene was probably regulated by three major enhancers located -10, -20, and -30 kb upstream of the gene promoter, occupied by activated VDR and prebound C/EBPβ and RUNX2, respectively. Initially, bacterial artificial chromosome clone recombineering and traditional mutagenesis defined binding sites for VDR and RUNX2. We then employed a CRISPR/Cas9 gene editing approach to delete the -10 and -30 kb Mmp13 enhancers, a region proximal to the promoter, and VDR or RUNX2. VDR-mediated up-regulation of Mmp13 transcription was completely abrogated upon removal of the -10 kb enhancer, resulting in a 1,25(OH)2D3-directed repression of Mmp13. Deletion of either the -30 kb enhancer or RUNX2 resulted in a complete loss of basal transcript activity and a ChIP-identified destabilization of the chromatin enhancer environment and factor binding. Whereas enhancer deletions only affected Mmp13 expression, the RUNX2 deletion led to changes in gene expression, a reduction in cellular proliferation, and an inability to differentiate. We conclude that the Mmp13 gene is regulated via at least three specific distal enhancers that display independent activities yet are able to integrate response from multiple signaling pathways in a model of activation and suppression.

Although localized to the mineralized matrix of bone, osteocytes are able to respond to systemic factors such as the calciotropic hormones 1,25(OH)2D3 and PTH. In the present studies, we examined the transcriptomic response to PTH in an osteocyte cell model and found that this hormone regulated an extensive panel of genes. Surprisingly, PTH uniquely modulated two cohorts of genes, one that was expressed and associated with the osteoblast to osteocyte transition and the other a cohort that was expressed only in the mature osteocyte. Interestingly, PTH's effects were largely to oppose the expression of differentiation-related genes in the former cohort, while potentiating the expression of osteocyte-specific genes in the latter cohort. A comparison of the transcriptional effects of PTH with those obtained previously with 1,25(OH)2D3 revealed a subset of genes that was strongly overlapping. While 1,25(OH)2D3 potentiated the expression of osteocyte-specific genes similar to that seen with PTH, the overlap between the two hormones was more limited. Additional experiments identified the PKA-activated phospho-CREB (pCREB) cistrome, revealing that while many of the differentiation-related PTH regulated genes were apparent targets of a PKA-mediated signaling pathway, a reduction in pCREB binding at sites associated with osteocyte-specific PTH targets appeared to involve alternative PTH activation pathways. That pCREB binding activities positioned near important hormone-regulated gene cohorts were localized to control regions of genes was reinforced by the presence of epigenetic enhancer signatures exemplified by unique modifications at histones H3 and H4. These studies suggest that both PTH and 1,25(OH)2D3 may play important and perhaps cooperative roles in limiting osteocyte differentiation from its precursors while simultaneously exerting distinct roles in regulating mature osteocyte function. Our results provide new insight into transcription factor-associated mechanisms through which PTH and 1,25(OH)2D3 regulate a plethora of genes important to the osteoblast/osteocyte lineage.

Skeletal cell differentiation is an orchestrated, multifaceted process involving transcription factors and epigenetic changes from mesenchymal progenitor cells. Osteoblastogenesis is driven by the expression and genomic binding of RUNX2. Throughout differentiation, secondary regulators such as ligand-activated vitamin D receptor (VDR) are recruited to the same genomic enhancers as RUNX2 to modulate the actions of RUNX2 for lineage-specific gene expression. The activities of these transcription factor-bound enhancers, or variable chromatin modifiers, are aided by dynamic epigenetic changes. Ligand-activated VDR has an exquisite influence on the epigenetic landscape surrounding those genes being regulated. In turn, this genomic landscape can adjust and react to varying levels of receptor to prioritize those genes most important to vitamin D stimulation. This bimodal control allows expression and regulation of VDR and its genetic program throughout osteogenesis. In this chapter, we will explore and review the relationship at the genomic level between vitamin D, mesenchymal differentiation, and epigenetics. Knowledge of the mechanisms of skeletal cell differentiation may increase the potential for new therapeutic control, diagnostic tools, and an understanding of skeletal disease progression.

The central role of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is to regulate calcium and phosphorus homeostasis through actions in the intestine, kidney, and bone. These actions are mediated by the vitamin D receptor (VDR), which acts in all target tissues to regulate the expression of genes that are integral to cellular response. Studies over the past decade or more have identified a number of target genes in a myriad of tissues, including the intestine and colon. Recent studies on a genome-wide scale have confirmed and extended old ideas and lead to new concepts as well. In the intestine, the actions of 1,25(OH)2D3 and its receptor are focused upon calcium and phosphate transport, xenobiotic metabolism, and growth regulation. This latter activity may be relevant to the antitumor effects of the hormone in colorectal cancer. Future studies are designed to further understand the actions of the vitamin D hormone in not only the intestine but also other tissues.

Highlights • This case report highlights a rare type of esophageal polyps (inflammatory fibroid polyp). • This case report presents a giant esophageal inflammatory fibroid polyp. • This case report represents the second reported case for giant inflammatory fibroid polyps that is originated from the upper esophagus., Introduction Benign inflammatory fibroid polyps (IFP) are rare submucosal tumors of the upper gastrointestinal tract. Rarely, they can develop in the esophagus, usually in the lower third. There are only 12 cases of giant IFP of the esophagus reported in literature and little is known about their origin, biological behavior and operative management. We present a patient with a giant benign IFP of the esophagus that originated from the upper esophagus. Case presentation The patient is a 59-year-old male who presented with dysphagia. Upper endoscopy and esophagram revealed a giant intraluminal esophageal mass with a pedicle in the upper esophagus. Resection of this mass was performed through a left cervical esophagotomy. Pathology confirmed IFP, On 2 year follow up, there was no recurrence of the mass. Discussion A giant IFP is defined as an IFP greater than 4 cm, commonly present in the distal esophagus. Pathology usually reveals vascularized fibrous stroma with elements of inflammatory infiltrate. This mass is slow-growing and asymptomatic until it grows to a large size. Common diagnostic studies include barium esophagram, upper endoscopy, and CT imaging. A key pre-operative work-up is to identify the location of the pedicle to plan out surgical approach and to avoid injuring the rich blood supply thus preventing a life threatening hemorrhage during the operation. Conclusion Giant IFPs are infrequent in clinical practice. Resection is indicated and usually performed by a surgical intervention or endoscopic removal. The pathogenesis of these polyps remains poorly understood due to the rarity of these lesions.

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.

The vitamin D receptor (VDR) is the single known regulatory mediator of hormonal 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in higher vertebrates. It acts in the nucleus of vitamin D target cells to regulate the expression of genes whose products control diverse, cell type-specific biological functions that include mineral homeostasis. In this Review we describe progress that has been made in defining new cellular sites of action of this receptor, the mechanisms through which this mediator controls the expression of genes, the biology that ensues, and the translational impact of this receptor on human health and disease. We conclude with a brief discussion of what comes next in understanding vitamin D biology and the mechanisms that underlie its actions.

In addition to osteoblast lineage cells, the TNF-like factor receptor activator of NF-κB ligand (RANKL) is expressed in both B and T cells and may play a role in bone resorption. Rankl gene (Tnfsf11) expression in mouse T cells is mediated through multiple distal elements marked by increased transcription factor occupancy, histone tail acetylation, and RNA polymerase II recruitment. Little is known, however, of the regulation of human TNFSF11 in T cells. Accordingly, we examined the consequence of T cell activation on the expression of this factor both in Jurkat cells and in primary human T cells. We then explored the mechanism of this regulation by scanning over 400 kb of DNA surrounding the TNFSF11 locus for regulatory enhancers using ChIP-chip analysis. Histone H3/H4 acetylation enrichment identified putative regulatory regions located between -170 and -220 kb upstream of the human TNFSF11 TSS that we designated the human T cell control region (hTCCR). This region showed high sequence conservation with the mouse TCCR. Inhibition of MEK1/2 by U0126 resulted in decreased RANKL expression suggesting that stimulation through MEK1/2 was a prerequisite. ChIP-chip analysis also revealed that c-FOS was recruited to the hTCCR as well. Importantly, both the human TNFSF11 D5a/b (RLD5a/b) enhancer and segments of the hTCCR mediated robust inducible reporter activity following TCR activation. Finally, SNPs implicated in diseases characterized by dysregulated BMD co-localized to the hTCCR region. We conclude that the hTCCR region contains a cell-selective set of enhancers that plays an integral role in the transcriptional regulation of the TNFSF11 gene in human T cells.

Initial research focused upon several known genetic targets provided early insight into the mechanism of action of the vitamin D hormone (1,25-dihydroxyvitamin D3 (1,25(OH)2D3)). Recently, however, a series of technical advances involving the coupling of chromatin immunoprecipitation (ChIP) to unbiased methodologies that initially involved tiled DNA microarrays (ChIP-chip analysis) and now Next Generation DNA Sequencing techniques (ChIP-seq analysis) has opened new avenues of research into the mechanisms through which 1,25(OH)2D3 regulates gene expression. In this review, we summarize briefly the results of this early work and then focus on more recent studies in which ChIP-chip and ChIP-seq analyses have been used to explore the mechanisms of 1,25(OH)2D3 action on a genome-wide scale providing specific target genes as examples. The results of this work have advanced our understanding of the mechanisms involved at both genetic and epigenetic levels and have revealed a series of new principles through which the vitamin D hormone functions to control the expression of genes. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

Osteocytes are derived from osteoblast lineage cells that become progressively embedded in mineralized bone. Development of the osteocytogenic cell line IDG-SW3 has enabled a temporal and mechanistic investigation of this process. Through RNA-sequencing analyses, we show that although substantial changes in gene expression occur during the osteoblast to osteocyte transition, the majority of the transcriptome remains qualitatively osteoblast like. Genes either up-regulated or expressed uniquely in the osteocyte include local and systemic factors such as Sost and Fgf23 as well as genes implicated in neuronal, muscle, vascular, or regulatory function. As assessed by chromatin immunoprecipitation coupled to high-throughput sequencing, numerous changes in epigenetic histone modifications also occur during osteocytogenesis; these are largely qualitative rather than quantitative. Specific epigenetic changes correlate with altered gene expression patterns that are observed during the transition. These genomic changes likely influence the highly restricted transcriptomic response to 1,25(OH)(2)D(3) that occurs during differentiation. VDR binding in osteocytes revealed an extensive cistrome co-occupied by retinoid X receptor and located predominantly at sites distal to regulated genes. Although sites of VDR binding were apparent near many 1,25(OH)(2)D(3)-regulated genes, the expression of others adjacent to VDR-binding sites were unaffected; lack of VDR binding was particularly prevalent at down-regulated genes. Interestingly, 1,25(OH)(2)D(3) was found to induce the Boc and Cdon coreceptors that are active in hedgehog signaling in osteocytes. We conclude that osteocytogenesis is accompanied by changes in gene expression that may be driven by both genetic and epigenetic components. These changes are likely responsible for the osteocyte phenotype and may contribute to reduced sensitivity to 1,25(OH)(2)D(3).

RUNX2 is a transcription factor that is first expressed in early osteoblast-lineage cells and represents a primary determinant of osteoblastogenesis. While numerous target genes are regulated by RUNX2, little is known of sites on the genome occupied by RUNX2 or of the gene networks that are controlled by these sites. To explore this, we conducted a genome-wide analysis of the RUNX2 cistrome in both pre-osteoblastic MC3T3-E1 cells (POB) and their mature osteoblast progeny (OB), characterized the two cistromes and assessed their relationship to changes in gene expression. We found that although RUNX2 was widely bound to the genome in POB cells, this binding profile was reduced upon differentiation to OBs. Numerous sites were lost upon differentiation, new sites were also gained; many sites remained common to both cell states. Additional features were identified as well including location relative to potential target genes, abundance with respect to single genes, the frequent presence of a consensus TGTGGT RUNX2 binding motif, co-occupancy by C/EBPβ and the presence of a typical epigenetic histone enhancer signature. This signature was changed quantitatively following differentiation. While RUNX2 binding sites were associated extensively with adjacent genes, the distal nature of the majority of these sites prevented assessment of whether they represented direct targets of RUNX2 action. Changes in gene expression, however, revealed an abundance of genes that contained RUNX2 binding sites and were regulated in concert. These studies establish a basis for further analysis of the role of RUNX2 activity and its function during osteoblast lineage maturation.

Forkhead Box O (FoxO) transcription factors and Sestrins (SESN) are highly conserved and related stress-responsive proteins that protect the organism against age-related pathologies. For FoxOs, growing evidence shows a crucial role in osteoblast function. Here we investigated the role of different FoxO and SESN isoforms in 1,25(OH)2D3-treated MC3T3-E1 osteoblasts. 1,25(OH)2D3 rapidly and strongly induced the expression of SESN1 and FoxO3a but down-regulated the expression of SESN3 and FoxO1. SESN2 and FoxO4 levels were hardly affected by 1,25(OH)2D3. Chromatin Immunoprecipitation (ChIP)-sequencing revealed significant VDR/RXR binding to a DR3-type VDRE in SESN1 but not in the genomic region where FoxO3a is located. Mutation of the SESN1 VDRE abolished responsiveness to 1,25(OH)2D3 in luciferase-based transfection assays. siRNA-mediated knock-down of SESN1, SESN3, FoxO1 or FoxO3a did not prevent 1,25(OH)2D3 from reducing the expression of cell cycle markers like Cyclin D1 and Cdc6 and from exerting its characteristic antiproliferative effect on MC3T3-E1 osteoblasts. Accordingly, the 1,25(OH)2D3-induced reduction in the number of S-phase cells was also maintained. The antiproliferative effect was still present in primary osteoblast in which all three FoxO isoforms were deleted (TKOpOB). Interestingly, both MC3T3-E1 osteoblasts in which FoxO1 was knocked-down and TKOpOBs accumulated significantly more reactive oxygen species (ROS) after treatment with 1,25(OH)2D3 than control cells. siRNA-mediated knock-down of individual SESN isoforms did not result in significant differences in ROS levels. In conclusion, 1,25(OH)2D3 directly and indirectly alters the expression levels of different FoxO and SESN isoforms in osteoblasts, presumably not to exert its antiproliferative action but to control ROS levels. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

The vitamin D endocrine system is essential for calcium metabolism and skeletal integrity. 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] regulates bone mineral homeostasis and acts directly on osteoblasts. In the present study we characterized the transcriptional regulation of the class 3 semaphorin (Sema3) gene family by 1,25(OH)(2)D(3) in osteoblastic cells. Class 3 semaphorins are secreted proteins that regulate cell growth, morphology and migration, and were recently shown to be involved in bone homeostasis. In ST2, MC3T3-E1 and primary calvarial osteoblast cell cultures we found that all members of the Sema3 gene family were expressed, and that Sema3e and Sema3f were the most strongly induced 1,25(OH)(2)D(3) target genes among the studied cell types. In addition, transcription of Sema3b and Sema3c was upregulated, whereas Sema3d and Sema3g was downregulated by 1,25(OH)(2)D(3) in different osteoblastic cells. Chromatin immunoprecipitation analysis linked to DNA sequencing (ChIP-seq analysis) revealed the presence of the vitamin D receptor at multiple genomic loci in the proximity of Sema3 genes, demonstrating that the genes are primary 1,25(OH)(2)D(3) targets. Furthermore, we showed that recombinant SEMA3E and SEMA3F protein were able to inhibit osteoblast proliferation. However, recombinant SEMA3s did not affect ST2 cell migration. The expression of class 3 semaphorins in osteoblasts together with their regulation by 1,25(OH)(2)D(3) suggests that these genes, involved in the regulation of bone homeostasis, are additional mediators for 1,25(OH)(2)D(3) signaling in osteoblasts.

CYP24A1 functions in vitamin D target tissues to degrade 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ). Thus, the concentration of this enzyme and the regulation of its expression is a primary determinant of the overall biological activity of 1,25(OH) 2 D 3 within cells. The principle regulator of CYP24A1 expression is 1,25(OH) 2 D 3 itself, which functions through the vitamin D receptor to upregulate the transcriptional activity of the Cyp24a1 gene. In this report, we explore the mechanism of this regulation using recently developed ChIP-chip and ChIP-seq techniques that permit an unbiased search for enhancer elements that participate in this transcriptional control. Our studies both confirm a regulatory region defined earlier and located proximal to the transcriptional start site (TSS) of mouse Cyp24a1 (−160 and −265 nt) and identify a novel intergenic region located downstream of the transcription unit that contains two enhancers (+35 and +37 kb) that facilitate 1,25(OH) 2 D 3 -dependent upregulation of Cyp24a1 expression. Interestingly, while C/EBPβ also binds under basal conditions to a site located immediately upstream of the Cyp24a1 promoter (−345 nt), occupancy by this factor is strikingly increased following 1,25(OH) 2 D 3 treatment. The locations and activities of these regulatory regions that mediate 1,25(OH) 2 D 3 actions were confirmed in mice in vivo. We conclude that the mechanism through which 1,25(OH) 2 D 3 induces the CYP24A1 enzyme, thereby autoregulating its own destruction, involves both promoter-proximal as well as downstream-distal enhancers. These findings highlight new concepts regarding the molecular mechanism of action of 1,25(OH) 2 D 3 and other hormonal regulators.

RANKL is a stromal cell-derived tumor necrosis factor (TNF)-like factor that plays a primary role in osteoclast formation and function. Recent studies suggest that 1,25(OH)(2) D(3) induces Rankl expression via vitamin D receptor (VDR) interaction at several enhancers located up to 76 kb upstream of the gene's transcriptional start site (TSS). In the current studies, we explored these interactions further using ChIP-chip and RNA analysis. We confirm VDR and RXR binding to the five enhancers described previously and identify two additional sites, one located within the Rankl coding region. We also show that RNA polymerase II is recruited to these enhancers, most likely through transcription factors TBP, TFIIB, and TAF(II) 250. Interestingly, the recruitment of these factors leads to the production of RNA transcripts, although their role at present is unknown. We also discovered that histone H4 acetylation (H4ac) marks many upstream Rankl enhancers under basal conditions and that H4ac is increased upon 1,25(OH)(2) D(3) treatment. Surprisingly, the hormone also induces C/EBPβ binding across the Rankl locus. C/EBPβ binding correlates directly with increased H4ac activity following 1,25(OH)(2) D(3) treatment. Finally, elevated H4ac is restricted to an extended region located between two potential insulator sites occupied by CTCF and Rad21. These data suggest a mechanism whereby 1,25(OH)(2) D(3) functions via the VDR and C/EBPβ to upregulate Rankl expression.

Receptor activator of NF-κB ligand (Rankl) is a TNF-like factor that induces the formation of osteoclasts responsible for bone resorption. Although T cell activation up-regulates this gene, the molecular mechanism of its transcriptional control remains unknown. We used ChIP-chip analysis in mouse primary T cells and a T cell hybridoma to define the regulatory enhancers responsible for this up-regulation and to characterize their properties. Elevated H3/H4 acetylation and increased RNA polymerase II density were evident at mRL-D5, a known enhancer located 76 kb upstream of the TSS, as well as at a cluster of regulatory sites located even further upstream between -123 to -156 kb, termed the T cell control region (TCCR). Based upon the ability of calcium signaling and MAPK inhibitors to block Rankl expression, we conducted further ChIP-chip analysis of the transcriptional mediators c-Fos, NF-κB, and Nfat. T cell activation induced c-Fos binding at the mRL-D5 enhancer and within the TCCR. The interaction of NF-κB was observed at the transcriptional start site and at mRL-D5. Both mRL-D5 and segments of the TCCR exhibited robust transcriptional activity in reporter assays, and site-specific mutagenesis of c-Fos and Nfat elements abrogated reporter activity, suggesting a role for both factors in the control of enhancer-mediated Rankl transcription. Finally, chromosome conformation capture analysis confirmed that mRL-D5 and segments of the TCCR were located in proximity to the Rankl gene promoter and thus potentially able to influence directly Rankl gene promoter activity. We conclude that both mRL-D5 and the TCCR represent control segments that play an integral role in Rankl expression in T cells.

High homocysteine (HCY) levels are a risk factor for osteoporotic fracture. Furthermore, bone quality and strength are compromised by elevated HCY owing to its negative impact on collagen maturation. HCY is cleared by cystathionine β-synthase (CBS), the first enzyme in the transsulfuration pathway. CBS converts HCY to cystathionine, thereby committing it to cysteine synthesis. A microarray experiment on MC3T3-E1 murine preosteoblasts treated with 1,25-dihydroxyvitamin D(3) [1,25(OH)(2) D(3) ] revealed a cluster of genes including the cbs gene, of which the transcription was rapidly and strongly induced by 1,25(OH)(2) D(3) . Quantitative real-time PCR and Western blot analysis confirmed higher levels of cbs mRNA and protein after 1,25(OH)(2) D(3) treatment in murine and human cells. Moreover, measurement of CBS enzyme activity and quantitative measurements of HCY, cystathionine, and cysteine concentrations were consistent with elevated transsulfuration activity in 1,25(OH)(2) D(3) -treated cells. The importance of a functional vitamin D receptor (VDR) for transcriptional regulation of cbs was shown in primary murine VDR knockout osteoblasts, in which upregulation of cbs in response to 1,25(OH)(2) D(3) was abolished. Chromatin immunoprecipitation on chip and transfection studies revealed a functional vitamin D response element in the second intron of cbs. To further explore the potential clinical relevance of our ex vivo findings, human data from the Longitudinal Aging Study Amsterdam suggested a correlation between vitamin D status [25(OH)D(3) levels] and HCY levels. In conclusion, this study showed that cbs is a primary 1,25(OH)(2) D(3) target gene which renders HCY metabolism responsive to 1,25(OH)(2) D(3).

The chromatin environment dictates activity throughout the genome. Post-translational modification of the N-terminal tails of histone proteins allow nucleosomes to shift, the chromatin to relax and genes to become activated. Histone modification events and markers will change in response to environmental stimuli; therefore they present a method for identification of sites of transcription factor activity. 1,25-Dihydroxyvitamin D3 induces the vitamin D receptor (VDR) to bind to DNA and activate transcription. These actions alter the chromatin environment and can be detected by increases or decreases in the histone modifications. In fact, in genomic loci with multiple enhancers, selective modulation of those enhancers after vitamin D3 stimulation can be readily detected by histone modifications. We provide an example of these actions on the Mmp13 gene locus where VDR binds selectively to an enhancer 10kb upstream of the transcriptional start site. This binding event is accompanied by an enhancer-selective increase in histone 3 lysine 9 acetylation (H3K9Ac). ChIP-seq analysis of histone modifications requires less genomic material than transcription factor ChIP-seq, thus proving advantageous to in vivo assays with limited cellular material. Therefore, histone modification activity alone may be used as a guide for discovering sites of VDR action for further biochemical analysis. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

The actions of the vitamin D hormone 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) are mediated by the vitamin D receptor (VDR), a ligand-activated transcription factor that functions to control gene expression. After ligand activation, the VDR binds directly to specific sequences located near promoters and recruits a variety of coregulatory complexes that perform the additional functions required to modify transcriptional output. Recent advances in transcriptional regulation, which permit the unbiased identification of the regulatory regions of genes, are providing new insight into how genes are regulated. Surprisingly, gene regulation requires the orchestrated efforts of multiple modular enhancers often located many kilobases upstream, downstream, or within the transcription units themselves. These studies are transforming our understanding of how 1,25(OH)(2)D(3) regulates gene transcription.