Genome-wide analyses of gene expression profile identify key genes and pathways involved in skeletal response to phosphate and 1,25-dihydroxyvitamin D in vivo.

Phosphate (P) is an essential element involved in various biological actions, such as bone integrity, energy production, cell signaling and molecular component. P homeostasis is modulated by 4 main tissues; intestine, kidney, bone, and parathyroid gland, where 1,25-dihydroxyvitamin D (1,25(OH)D), parathyroid hormone and fibroblast growth factor 23 (FGF23) are produced and/or have an influence. In bone, serum P level modulates the production of FGF23 which then controls not only P excretion but also vitamin D metabolism in kidney in an endocrine manner.

A parathyroid hormone/salt-inducible kinase signaling axis controls renal vitamin D activation and organismal calcium homeostasis.

The renal actions of parathyroid hormone (PTH) promote 1,25-vitamin D generation; however, the signaling mechanisms that control PTH-dependent vitamin D activation remain unknown. Here, we demonstrated that salt-inducible kinases (SIKs) orchestrated renal 1,25-vitamin D production downstream of PTH signaling. PTH inhibited SIK cellular activity by cAMP-dependent PKA phosphorylation.

Rapid genomic changes by mineralotropic hormones and kinase SIK inhibition drive coordinated renal Cyp27b1 and Cyp24a1 expression via CREB modules.

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 (1,25(OH)D), and reciprocal regulations for Cyp24a1. This coordinated genomic regulation results in production of endocrine 1,25(OH)D, which, together with PTH and FGF23, controls mineral homeostasis. However, how these events are coordinated is unclear.

Deletion of a putative promoter-proximal Tnfsf11 regulatory region in mice does not alter bone mass or Tnfsf11 expression in vivo.

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.

Genomic Mechanisms Governing Mineral Homeostasis and the Regulation and Maintenance of Vitamin D Metabolism.

Our recent genomic studies identified a complex kidney-specific enhancer module located within the introns of adjacent (M1) and (M21) genes that mediate basal and PTH induction of , as well as suppression by FGF23 and 1,25-dihydroxyvitamin D [1,25(OH)D]. 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 expression in the kidney.

A Control Region Near the Fibroblast Growth Factor 23 Gene Mediates Response to Phosphate, 1,25(OH)2D3, and LPS In Vivo.

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.

A chromatin-based mechanism controls differential regulation of the cytochrome P450 gene in renal and non-renal tissues.

Cytochrome P450 family 27 subfamily B member 1 (CYP27B1) and CYP24A1 function to maintain physiological levels of 1,25-dihydroxyvitamin D (1,25(OH)D) in the kidney. Renal and expression levels are transcriptionally regulated in a highly reciprocal manner by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and 1,25(OH)D In contrast, regulation in nonrenal target cells (NRTCs) is limited to induction by 1,25(OH)D Herein, we used ChIP-Seq analyses of mouse tissues to identify regulatory regions within the gene locus. We found an extended region downstream of 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).

Targeted genomic deletions identify diverse enhancer functions and generate a kidney-specific, endocrine-deficient pseudo-null mouse.

Vitamin D is terminally bioactivated in the kidney to 1α,25-dihydroxyvitamin D (1,25(OH)D) 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)D Our recent genomic studies in the mouse have revealed a complex kidney-specific enhancer module within the introns of adjacent methyltransferase-like 1 () and that mediate basal and PTH-induced expression of and FGF23- and 1,25(OH)D-mediated repression. Gross deletion of these segments in mice has severe effects on regulation and skeletal phenotype but does not affect expression in nonrenal target cells (NRTCs). Here, we report a bimodal activity in the intronic enhancer with components responsible for PTH-mediated induction and 1,25(OH)D-mediated repression and additional activities, including FGF23 repression, within the enhancers.

The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2.

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.

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

The vitamin D endocrine system regulates mineral homeostasis through its activities in the intestine, kidney, and bone. Terminal activation of vitamin D to its hormonal form, 1α,25-dihydroxyvitamin D (1,25(OH)D), 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, , are unknown.

The impact of VDR expression and regulation in vivo.

The vitamin D receptor (VDR) mediates the pleiotropic biological actions of 1,25-dihydroxyvitamin D (1,25(OH)D). 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.

The vitamin D receptor: contemporary genomic approaches reveal new basic and translational insights.

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.

Epigenetic Plasticity Drives Adipogenic and Osteogenic Differentiation of Marrow-derived Mesenchymal Stem Cells.

Terminal differentiation of multipotent stem cells is achieved through a coordinated cascade of activated transcription factors and epigenetic modifications that drive gene transcription responsible for unique cell fate. Within the mesenchymal lineage, factors such as RUNX2 and PPARγ are indispensable for osteogenesis and adipogenesis, respectively. We therefore investigated genomic binding of transcription factors and accompanying epigenetic modifications that occur during osteogenic and adipogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (MSCs).

Genomic Determinants of Vitamin D-Regulated Gene Expression.

Insight into mechanisms that link the actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to the regulation of gene expression has evolved extensively since the initial discovery of a nuclear protein known as the vitamin D receptor (VDR). Perhaps most important was the molecular cloning of this receptor which enabled its inclusion within the nuclear receptor gene family and further studies of both its structure and regulatory function. Current studies are now refocused on the vitamin D hormone's action at the genome, where VDR together with other transcription factors coordinates the recruitment of chromatin active coregulatory complexes that participate directly in the modification of gene output.

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

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.

Selective regulation of Mmp13 by 1,25(OH)D, PTH, and Osterix through distal enhancers.

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)D), parathyroid hormone (PTH), and several cytokines.

1,25-Dihydroxyvitamin D3 Controls a Cohort of Vitamin D Receptor Target Genes in the Proximal Intestine That Is Enriched for Calcium-regulating Components.

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.

Epigenetic histone modifications and master regulators as determinants of context dependent nuclear receptor activity in bone cells.

Genomic annotation of unique and combinatorial epigenetic modifications along with transcription factor occupancy is having a profound impact on our understanding of the genome. These studies have led to a better appreciation of the dynamic nature of the epigenetic and transcription factor binding components that reveal overarching principles of the genome as well as tissue specificity. In this minireview, we discuss the presence and potential functions of several of these features across the genome in osteoblast lineage cells.

Selective Distal Enhancer Control of the Mmp13 Gene Identified through Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Genomic Deletions.

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.

The parathyroid hormone-regulated transcriptome in osteocytes: parallel actions with 1,25-dihydroxyvitamin D3 to oppose gene expression changes during differentiation and to promote mature cell function.

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.

Profiling histone modifications by chromatin immunoprecipitation coupled to deep sequencing in skeletal cells.

Chromatin, tightly packaged genomic DNA, is reliant on posttranslational modification of histone N-terminal tails for accessibility of DNA by transcription factors to activate transcription. Each histone modification may denote permissible states for gene activation or repression. As cells undergo differentiation, as they do in the skeleton from multipotential precursors through osteoblasts and into osteocytes, their histone code may be altered to help accommodate these transitions.

Genomic determinants of gene regulation by 1,25-dihydroxyvitamin D3 during osteoblast-lineage cell differentiation.

The biological effects of 1α,25-dihydroxyvitamin D3 (1,25 (OH)2D3) on osteoblast differentiation and function differ significantly depending upon the cellular state of maturation. To explore this phenomenon mechanistically, we examined the impact of 1,25(OH)2D3 on the transcriptomes of both pre-osteoblastic (POBs) and differentiated osteoblastic (OBs) MC3T3-E1 cells, and assessed localization of the vitamin D receptor (VDR) at sites of action on a genome-scale using ChIP sequence analysis. We observed that the 1,25(OH)2D3-induced transcriptomes of POBs and OBs were quantitatively and qualitatively different, supporting not only the altered biology observed but the potential for a change in VDR interaction at the genome as well.

The osteoblast to osteocyte transition: epigenetic changes and response to the vitamin D3 hormone.

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.

The RUNX2 cistrome in osteoblasts: characterization, down-regulation following differentiation, and relationship to gene expression.

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.