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INVITED REVIEW

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Vitamin D receptor signaling and its therapeutic implications: Genome-wide and structural view1 Carsten Carlberg and Ferdinand Molnár

Abstract: Vitamin D3 is one of the few natural compounds that has, via its metabolite 1␣,25-dihydroxyvitamin D3 (1,25(OH)2D3) and the transcription factor vitamin D receptor (VDR), a direct effect on gene regulation. For efficiently applying the therapeutic and disease-preventing potential of 1,25(OH)2D3 and its synthetic analogs, the key steps in vitamin D signaling need to be understood. These are the different types of molecular interactions with the VDR, such as (i) the complex formation of VDR with genomic DNA, (ii) the interaction of VDR with its partner transcription factors, (iii) the binding of 1,25(OH)2D3 or its synthetic analogs within the ligand-binding pocket of the VDR, and (iv) the resulting conformational change on the surface of the VDR leading to a change of the protein–protein interaction profile of the receptor with other proteins. This review will present the latest genome-wide insight into vitamin D signaling, and will discuss its therapeutic implications. Key words: vitamin D, vitamin D analogs, chromatin immunoprecipitation, crystal structure, VDR partner proteins, vitamin D signaling in vivo. Résumé : La vitamine D3 appartient a` la courte liste de composés naturels qui exerce, par l’intermédiaire de son métabolite, la 1␣,25-dihydroxyvitamine D3 (1,25(OH)2D3), et de son récepteur (VDR), un facteur de transcription, un effet direct sur la régulation génique. Afin d’exploiter de manière efficace le potentiel thérapeutique et préventif de la 1,25(OH)2D3 et ses analogues synthétiques, les étapes clés de la signalisation de la vitamine D doivent être comprises. Le VDR est engagé dans différents types d’interactions moléculaires comme (i) la formation d’un complexe impliquant le VDR et l’ADN génomique, (ii) l’interaction du VDR avec des facteurs de transcription partenaires, (iii) la liaison de la 1,25(OH)2D3 ou de ses analogues synthétiques dans la poche de liaison du ligand du VDR et (iv) le changement de conformation a` la surface du VDR qui en résulte et qui mène a` un changement du profil d’interaction protéine–protéine du récepteur avec d’autres protéines. Cet article de synthèse présentera un aperçu des données les plus récentes a` l’échelle du génome de la signalisation de la vitamine D et il discutera de ses implications thérapeutiques. [Traduit par la Rédaction] Mots-clés : vitamine D, analogues de la vitamine D, immunoprécipitation de chromatine, structure cristalline, protéines partenaires du VDR, signalisation de la vitamine D in vivo.

Introduction The secosteroid vitamin D3 is a pleiotropic signaling molecule that was already being used by early unicellular organisms to protect their DNA against UV-B irradiation (Holick 2011). Even in humans, vitamin D3 is still linked to UV-B, since its energy is essential to convert 7-dehydrocholesterol in the skin into previtamin D3. The step-wise depigmentation of human skin that occurred when Homo sapiens moved from equatorial regions in Africa to higher latitudes in Asia and Europe, demonstrates that the need for endogenous vitamin D3 production acted as an evolutionary driver (Hochberg and Templeton 2010). Vitamin D3 started to get used as a signaling molecule when the first fish developed bones, for which calcium uptake needed to be regulated (Bouillon and Suda 2014). Today, the endocrinology of vita-

min D3 is still tightly connected with calcium homeostasis and bone formation. It is undisputed that a sufficient vitamin D status is essential for bone health, such as in the prevention of osteoporosis (Institute-of-Medicine 2011), but in parallel tissues, the greatest fear for a side-effect that might be caused by overdosing with natural and synthetic vitamin D analogs is calcification (Cheskis et al. 2006). However, the physiological impact of vitamin D is broader than just controlling proper bone mineralization (Carlberg 2014b), as it is involved in the regulation of cellular growth and differentiation (Feldman et al. 2014) as well as innate and adaptive immunity (Chun et al. 2014). This provides vitamin D not only with a therapeutic and disease-preventive potential in osteoporosis, but also in different types of cancer, in several infectious diseases such as tuberculosis, and in autoimmune diseases, such as multiple sclerosis (Holick 2007).

Received 4 October 2014. Accepted 27 December 2014. Abbreviations: 1,25(OH)2D3, 1␣,25-dihydroxyvitamin D3; 25(OH)D3, 25-hydroxyvitamin D3; AR, androgen receptor; ChIP, chromatin immunoprecipitation; ChIP-seq, ChIP coupled with massive parallel sequencing; CYP24A1, cytochrome P450, family 24, subfamily A, polypeptide 1; DBD, DNA-binding domain; DR3, direct repeat spaced by 3 nucleotides; ER, estrogen receptor; ESRRB, estrogen-related receptor ␤; FAIRE-seq, formaldehyde-assisted isolation of regulatory elements sequencing; FXR, farnesoid X receptor; GABPA, GA binding protein transcription factor, alpha subunit 60 kDa; GR, glucocorticoid receptor; HDAC, histone deacetylase; HOMER, Hypergeometric Optimization of Motif EnRichment; IGV, Integrative Genomics Viewer; LBD, ligand-binding domain; LBP, ligand-binding pocket; PBMC, peripheral blood mononuclear cell; PRMT10, protein arginine methyltransferase 10; PXR, pregnane X receptor; RAR, retinoid acid receptor; RXR, retinoid X receptor; SH2B1, SH2B adaptor protein 1; SPI1, spleen focus forming virus (SFFV) proviral integration oncogene; TR, thyroid hormone receptor; TsA, trichostatin A; TSS, transcription start site; VDR, vitamin D receptor. C. Carlberg. School of Medicine, Institute of Biomedicine, University of Eastern Finland, POB 1627, FI-70211 Kuopio, Finland. F. Molnár. School of Pharmacy, Institute of Biopharmacy, University of Eastern Finland, FI-70211 Kuopio, Finland. Corresponding author: Carsten Carlberg (e-mail: carsten.carlberg@uef.fi). 1This Invited Review is part of a Special Issue entitled “Pharmacology of vitamins and beyond: Vitamin D.” Can. J. Physiol. Pharmacol. 93: 311–318 (2015) dx.doi.org/10.1139/cjpp-2014-0383

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Fig. 1. Molecular basis of vitamin D signaling. The complex of vitamin D receptor (VDR; in green) with its partner transcription factor retinoid X receptor (RXR; in blue) bound to DNA is shown in the central panel. (A) The details of the contact of the DNA-binding domains of VDR and RXR with DNA (based on Protein Database (PDB) file 1YNW). The sequence logo of a DR3-type VDR binding site is shown on the bottom. (B) Demonstration of the heterodimerization of the ligand-binding domains (LBDs) of VDR and RXR (based on a model derived from 1RK3 cryo-electron microscopic studies with full length receptors (Orlov et al. 2012)). (C) VDR’s ligand-binding pocket filled with a 1,25(OH)2D3 molecule (based on PDB file 1DB1). The key amino acids contacting the 3 hydroxyl groups (in red) of 1,25(OH)2D3 are indicated. (D) Detail of the surface of the VDR LBD (in green), where a rather minor movement of helix 12 (in red) after ligand binding allows the binding of the LXXLL motif of the co-activator TRAP220 (also called MED1, in orange; based on PDB file 1RK3).

Vitamin D acts as a pre-hormone that is converted in 2 hydroxylation steps via 25-hydroxyvitamin D3 (25(OH)D3) into its biologically active metabolite 1,25(OH)2D3 (Norman 2008). The latter molecule is the only natural high-affinity ligand to the VDR (Haussler et al. 1997). VDR is not only one of approximately 1900 human transcription factors (Vaquerizas et al. 2009), but it also belongs to the nuclear receptor superfamily (Evans and Mangelsdorf 2014). Like other endocrine nuclear receptors, such as the receptors for estrogen (ER), testosterone (AR), and cortisol (GR), which specifically bind lipophilic molecules the size of cholesterol (Carlberg and Molnár 2012), VDR is also directly activated by 1,25(OH)2D3. VDR is rather ubiquitously expressed, i.e., tissues and cell types that are not related to calcium homeostasis and bone formation, such as cancer cells and cells of the immune system, are also responsive to 1,25(OH)2D3 (Wang et al. 2012). The molecular actions of 1,25(OH)2D3 are largely identical to those of its receptor, but VDR also has additional ligand-independent functions (Polly et al. 2000). The different types of molecular interactions of the VDR (Fig. 1) are the mechanistic core of vitamin D signaling. These are (i) the protein–DNA interaction of VDR’s DNA-binding domain (DBD) with genomic sequences, (ii) the protein–protein interaction of VDR with its partner proteins, (iii) the protein–ligand interaction of 1,25(OH)2D3 or its synthetic analogs with the ligand-binding pocket (LBP) within the ligand-binding domain (LBD), and (iv) the resulting conformational changes on the surface of the LBD, which change the protein–protein interaction profile of the receptor with nuclear adaptor proteins, such as co-repressors, co-

activators, and the Mediator complex. The latter proteins attract (i) chromatin remodeling complexes that re-arrange the local positioning of nucleosomes, and (ii) chromatin modifying enzymes that read, write, or erase post-translational marks to histone proteins, such as the transfer of acetyl and methyl groups of the local nucleosomes. These epigenomic changes facilitate looping of the VDR-marked genomic regions, i.e., 1,25(OH)2D3-inducible enhancers, towards the basal transcriptional machinery that has assembled on accessible transcription start sites (TSSs) (Carlberg and Campbell 2013). This assembly of enhancer and TSS regions with RNA polymerase II, other nuclear adaptor proteins, and ligandactivated VDR finally leads to an increase or decrease in the transcription of primary vitamin D target genes. In this review, we will provide more details on vitamin D signaling by describing the latest (epi)genome-wide and structural insight, and will discuss its therapeutic implications.

Genome-wide VDR binding Nuclear receptors, such as VDR, are characterized by a highly conserved DBD, which is formed by 2 zinc fingers specifically contacting the sequence RGKTSA (R = A or G, K = G or T, S = C or G) within the major groove of genomic DNA (Shaffer and Gewirth 2002) (Fig. 1A). Like most other transcription factors, VDR increases its DNA binding affinity and specificity via heterodimerization with a partner transcription factor (Fig. 1B). In most studies, this protein partner turned out to be the nuclear receptor retinoid X receptor (RXR) (Sone et al. 1991; Carlberg et al. 1993), but VDR has also been shown to form homodimers (Cheskis and Freedman Published by NRC Research Press

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Fig. 2. Different modes of vitamin D receptor (VDR) interacting with genomic DNA. The Integrative Genomics Viewer (IGV) browser (Robinson et al. 2011) was used to visualize the binding of VDR in unstimulated LX2 cells and cells that were stimulated with the 1,25(OH)2D3 analog MC903 (Ding et al. 2013). The input lane serves as a negative control. (A) Example of a VDR binding site close to the transcription start site (TSS) of the CYP24A1 gene, which is drastically up-regulated by the treatment with VDR ligand. At this locus a DR3-type sequence was identified below the peak summit, providing strong evidence that VDR binds DNA as a heterodimer with retinoid X receptor (RXR). (B) Example of a site close to the TSS of the PRMT10 gene. At this site, VDR binding does not change after ligand stimulation and no DR3-type sequence is found below the peak summit. (C) Example of a DR3-free site close to the TSS of the SH2B1 gene where ligand treatment leads to a significant reduction of VDR binding. The DNA binding mode of VDR to the sites in (B) and (C) are not known, and it can only be speculated that VDR eithers bind directly in a complex with an alternative partner transcription factor, or indirectly, i.e., piggybacks onto an undefined “carrier” transcription factor.

1994) and complexes with other nuclear receptors, such as retinoid acid receptor (RAR) (Schräder et al. 1993) and thyroid hormone receptor (TR) (Schräder et al. 1994). In-vitro experiments have determined the optimal binding site of VDR–RXR heterodimers as a direct repeat of 2 hexameric nuclear receptor binding motifs spaced by 3 nucleotides (DR3, see the sequence logo on the bottom of Fig. 1A) (Umesono et al. 1991; Shaffer and Gewirth 2004). Nowadays, a more appropriate approach to determine the association of VDR with its genomic targets is to apply the chromatin immunoprecipitation (ChIP) method (Orlando 2000). When combined with massive parallel sequencing (ChIP-seq) (Park 2009), this technique can monitor, genome-wide, all binding sites of a transcription factor in living cells. For human VDR, public ChIPseq data are available from the following cell lines: (i) GM10855 and GM10861 (B cells) (Ramagopalan et al. 2010), (ii) THP-1 (monocytes) (Heikkinen et al. 2011), (iii) lipopolysaccharide-differentiated THP-1 (macrophages) (Tuoresmäki et al. 2014), (iv) LS180 (colon cancer) (Meyer et al. 2012), and (v) LX2 (hepatic stellate cells) (Ding et al. 2013). A harmonized re-analysis of all these ChIP-seq datasets resulted in 23 409 non-overlapping VDR binding loci (Tuoresmäki et al. 2014), of which more than 70% are unique for one of the analyzed cellular models. Importantly, de novo search of the genomic sequence below VDR peak summits (±100 bp) confirmed DR3-type elements as the most abundant motifs. However, motif

screening algorithms, such as HOMER (Heinz et al. 2010) at a moderate score of 7, could only identify DR3-type elements at 2686 VDR peaks (11.5% of total) (Tuoresmäki et al. 2014). This DR3 percentage differs significantly between the analyzed cellular models, ranging between 38.2% in macrophages and 9.0% in B cells. Interestingly, in every cell type investigated, the top 200 VDR sites (ranked by binding strength) show a DR3 rate of more than 60%, i.e., DR3-type motifs are found preferentially at highly ligand-responsive VDR loci, such as that close to the TSS of the gene cytochrome P450, family 24, subfamily A, polypeptide 1 (CYP24A1, Fig. 2A). Therefore, the genes that are controlled by this type of VDR binding site seem to be the prime targets of vitamin D, and may be the first to be addressed by a therapeutic intervention with a synthetic VDR ligand. This finding also implies that the mechanistic basis of VDR’s action is independent of the cell type and the total number of identified binding sites (Carlberg 2014a). Taken together, human cells have between 1000 and 10 000 genomic VDR binding loci. This is clearly more than the 100–500 primary 1,25(OH)2D3 target genes per cell type. Not only in vitro, but also in living cells, DR3-type sites are the preferred VDR binding motifs, but such an element is far from being found at all VDR loci. Nevertheless, VDR sites with a DR3-type motif show more pronounced responsiveness to ligands, and may be the preferred targets for a therapy with 1,25(OH)2D3 analogs. Published by NRC Research Press

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VDR partner proteins

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Although the most prominent genomic VDR binding sites preferentially carry DR3-type sequences on which VDR heterodimerizes with RXR, there are a significant number of important VDR loci (some 40% of the top 200 and 80%–90% of the lower ranking sites) where no DR3-type element is available. Interestingly, at the latter sites, VDR ligands have either no significant effect on VDR binding, such as shown for the site close to the TSS of the gene protein arginine methyltransferase 10 (PRMT10, Fig. 2B), or even reduce VDR association, such as demonstrated for the gene SH2B adaptor protein 1 (SH2B1, Fig. 2C). It is not yet known how VDR associates at such sites with DNA, but it is most likely that either VDR binds (i) directly to DNA by using a different protein partner than RXR, or (ii) indirectly piggybacks onto a DNA-binding “carrier” protein (Fig. 2). In both scenarios the specific DNA binding site would be different to a DR3type sequence, and either composed of a single nuclear receptor binding motif and the motif of the partner protein, or the specific motif of the carrier protein. Interestingly, HOMER motif screening of the VDR ChIP-seq datasets from hematopoietic cells found significant enrichment of binding sites for the transcription factors spleen focus forming virus (SFFV) proviral integration oncogene (SPI1, also called PU.1), estrogen-related receptor ␤ (ESRRB, also called NR3B2), and GA binding protein transcription factor, alpha subunit 60 kDa (GABPA) (Tuoresmäki et al. 2014). SPI1 is a well-known pioneer factor (Zaret and Carroll 2011) that is known to work together with VDR in monocytic differentiation (Novershtern et al. 2011). The lack of a dominant non-DR3 binding sequence below VDR peak summits suggests that there is no single alternative protein partner of the receptor, but rather a larger set of proteins (Tuoresmäki et al. 2014). These VDR partnering proteins will have a cell-specific expression pattern and may explain some of the cell-specific actions of VDR and its natural ligand 1,25(OH)2D3. Importantly, VDR already binds to a number of its genomic targets, such as those close to the genes PRMT10 and SH2B1 (Figs. 2B and 2C) in the absence of ligand. Since these ligand-independent genomic VDR loci have a clearly lower DR3 rate than liganddependent sites (Heikkinen et al. 2011), they associate preferentially with proteins related to gene repression, such as is demonstrated for the example of the MYC gene (Toropainen et al. 2010). Although so far no direct protein–protein interaction between VDR and SPI1, ESRRB, or GABPA has been reported, the concept of alternative VDR binding modes has the potential to modulate VDR signaling via the activation of signal transduction pathways that affect VDR’s alternative protein partners. However, in the case of the well-characterized VDR–RXR heterodimers, there is no significant effect of the RXR ligand 9-cis retinoid acid when VDR is not ligandactivated at the same time, i.e., VDR–RXR heterodimers are nonpermissive (Schräder et al. 1995; Schulman et al. 1997). In summary, VDR ChIP-seq data indicated that there are alternative VDR binding motifs to VDR–RXR heterodimer complexes on DR3-type sites. However, we do not know the identity of these alternative VDR partner proteins. Therefore, further investigations are necessary before a potential therapeutic impact of these proteins on vitamin D signaling can be evaluated.

nuclear receptors pregnane X receptor (PXR) and farnesoid X receptor (FXR), which share this evolutionary precursor with VDR (Makishima 2005). Bile acids are cholesterol derivatives and therefore structurally related to vitamin D3 and its metabolites, but they bind with inverse orientation to the LBP (Fig. 3A) (Masuno et al. 2013). They are positioned within the LBP by the same 3 pairs of polar amino acids, but owing to their more rigid structure they need to use 2 or more water molecules to contact these fixation points. However, non-polar amino acids also stabilize VDR ligands within the LBP via hydrophobic interactions. Interestingly, when comparing the complexes of 1,25(OH)2D3 and 3-keto lithocholic acid within the LBP (Fig. 3B), it becomes obvious that additional hydrophobic interactions contribute to 1,25(OH)2D3 aliphatic sidechain stabilization (namely L223 and Y397). All interactions are relatively conserved, but there is a difference in the position of the interacting amino acids with respect to ligand orientation with the 2 types of natural ligands. Therefore, the finding on the reverse binding mode of the bile acids may stimulate the design of new types of 1,25(OH)2D3 analogs. Further evidence for the flexibility of the LBP are provided by a comparison of the crystal structures of VDR’s LBD complexed with 1,25(OH)2D3 and 3 structurally, rather divergent synthetic analogs (Fig. 4). The goal of the development of synthetic 1,25(OH)2D3 analogues is to improve their biological profile for a therapeutic application in one of the pleiotropic functions of the natural hormone in bone disorders, such as osteoporosis, and in hyperproliferative diseases, such as psoriasis and different types of cancer (Bouillon et al. 1995). Preferential sides for a modification of 1,25(OH)2D3 are the aliphatic side chain, the A-ring, the CD-rings, and the triene system (Carlberg et al. 2012). One strategy for the design of 1,25(OH)2D3 analogs is the addition of extra carbons in form of aliphatic, cyclic, or even aromatic structures. A prominent example is the 2 side-chain analog Gemini (1,25-dihydroxy-21-(3methyl-3-hydroxy-butyl)-cholecalciferol) (Herdick et al. 2000), which has a significantly increased volume (25%) but still fits into the LBP (Fig. 4B). One of the 2 side chains of Gemini takes the same location as in the natural hormone, whereas for the second side chain, an extra subcavity is opened within the LBP (Vaisanen et al. 2003). A second analog design strategy is to aim for an increased LBP binding affinity via replacing hydrogens by more electronegative atoms, such as fluorines. Moreover, side-chain fluorinations often make the compounds resistant to degradation by CYP24A1. An example of this is the semi-steroidal D-ring analogue CD578 (Fig. 4C) (Eelen et al. 2008), which is missing the C-ring within the secosteroid structure. The bis-aromatic molecule CD3938 exemplifies a third strategy, the design of pure nonsteroidal ligands (Fig. 4D) (Ciesielski et al. 2012). The 4 structurally divergent VDR ligands all fit to the LBP and demonstrate the flexibility of the latter. Taken together, VDR’s LBD binds the natural ligand 1,25(OH)2D3 with high affinity, but also with lower affinity, and in inverse orientation bile acids. The LBP has a dynamic structure that can adapt in its volume to the structure of a rather divergent set of synthetic analogs.

Ligand binding to the VDR

Interaction of VDR with chromatin components

Resolving the crystal structure of the VDR’s LBD complexed with 1,25(OH)2D3 (Rochel et al. 2000) created a new level in the understanding of the molecular mechanisms of the receptor. The inner surface of the VDR–LBD forms a cavity with a volume of ⬃700 Å3 (Molnár et al. 2006), which is created by a network of around 40 mostly non-polar amino acids (Fig. 1C). Within this LBP there are 3 pairs of polar amino acids, each of which fixes one of the 3 hydroxyl groups of the natural VDR ligand 1,25(OH)2D3 via hydrogen bonds (Fig. 3A). From an evolutionary point of view, bile acids such as lithocholic acid were the first ligands of a VDR precursor molecule. Bile acids are still major natural ligands to the

The binding of VDR ligands to the LBP induces minor but significant conformational changes to the surface of the LBD that result in an exchange of protein–protein interaction partners, such as co-activator and co-repressor proteins (Carlberg 2003). Agonistic VDR ligands have to effect both an efficient dissociation of co-repressor proteins from the LBD surface as well as a specific association with co-activator proteins, in order to lead to transcriptional activation. More than 50 nuclear proteins are known to interact with VDR’s LBD (reviewed in (Molnar 2014)), which include different components of the Mediator complex, which builds a bridge to the basal transcription machinery assembled on Published by NRC Research Press

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Fig. 3. Binding mode of 1,25(OH)2D3 and bile acid to the vitamin D receptor (VDR) ligand binding pocket (LBP). (A) 1,25(OH)2D3 (in green; based on Protein Database (PBD) file 1RK3) and the bile acid 3-keto lithocholic acid (in blue; based on PBD file 3W5P) in 2 different orientations. Hydrogen bonds (broken lines) between polar amino acids of the LBP and hydroxyl groups of the ligands or stabilizing water molecules are indicated. The data are based on X-ray analysis rat VDR crystal structures. (B) The same structures in a more schematic plane display indicating that (i) in part, different amino acids of the LBP are involved in contacting the ligands, and (ii) lithocholic acid fills the LBP in an inverse orientation to all other tested VDR ligands. The illustrations were created with the software PyMOL (The PyMOL Molecular Graphics System, version 1.3, Schrödinger).

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Fig. 4. Shapes of vitamin D receptor (VDR) ligand binding pocket (LBP) in a complex with most divergent ligands. The central panel shows the position of the LBP (in grey) inside a zebrafish VDR (in green). (A) Details of the shape of the LBP complexed with the natural VDR ligand 1,25(OH)2D3 in 2 different orientations. (B–D) The LBP in the same orientation but complexed with (B) the 2 side-chain analog Gemini, (C) the fluorinated D-ring analog CD578, and (D) the bis-aromatic nonsteroidal analog CD3938. Broken boxes indicate regions of major (in blue) and minor (in red) deviations between the LBP shapes. The Protein Database identifiers of the respective crystal structures are indicated in brackets behind the VDR ligand name.

TSS regions. VDR-interacting co-activators and co-repressors act as adaptor proteins that connect the receptor with chromatin modifying enzymes, such as histone acetyltransferases and histone deacetylases (HDACs), respectively. This allows the VDR to initiate specific changes to its local chromatin environment. In contrast, HDAC inhibitors, such as trichostatin A (TsA), achieve a less specific but more global activation of chromatin (Marks et al. 2001; Monneret 2005). Interestingly, 1,25(OH)2D3 and TsA act synergistically in their antiproliferative effect on malignant and non-malignant human breast cancer cell lines (Liu et al. 1996; Warrener et al. 2003; Malinen et al. 2008) and in their global gene-regulatory effect in THP-1 cells (Seuter et al. 2013a). Chromatin has an intrinsic repressive potential with the purpose to conserve the epigenetic landscape of a differentiated cell, i.e., by default it largely restricts the access of transcription factors to promoter and enhancer regions, leaving only in the order of 50–100 000 accessible chromatin regions per cell type (ENCODEProject-Consortium et al. 2012). VDR-mediated histone modifications can change this pattern of accessible chromatin regions. Experimentally, this can be monitored genome-wide by using the Formaldehyde-Assisted Isolation of Regulatory Elements sequencing (FAIRE-seq) method (Giresi et al. 2007). A detailed FAIRE-seq time course in 1,25(OH)2D3-stimulated THP-1 cells demonstrated that some 87% of VDR binding sites co-localize with open chromatin (Seuter et al. 2013b). At approximately 20% of these loci, a stimulation with 1,25(OH)2D3 leads to a significant increase in chromatin accessibility (Seuter et al. 2013b). These sites are strong indications for the location of primary 1,25(OH)2D3 target genes within the same genomic region. Thus, approaches to categorize VDR binding sites are useful. For example, VDR loci that (i) carry a DR3-type sequence, (ii) show ligand-stimulated VDR association, (iii) co-locate with ligand-induced chromatin opening, and (iv) are conserved between several cellular systems, may play a more im-

portant role in mediating the functions of 1,25(OH)2D3 than the vast majority of other VDR sites that lack most of these properties. In summary, the therapeutic effect of 1,25(OH)2D3 on, for example, the control of cellular proliferation can be combined with that of chromatin modifiers, such as HDAC inhibitors. The molecular basis of this synergy is related to the fact that 1,25(OH)2D3 dynamically controls the epigenetic state of chromatin at approximately 1 in 6 of all genome-wide VDR binding sites. Vitamin D signaling in vivo The endocrinology of vitamin D3 is designed not to imply any fast change in its physiological effects (DeLuca 2004; Norman 2008). When the production of vitamin D3 in UV-B exposed skin or its intake of from diet or supplements is sufficient, the serum 25(OH)D3 level, i.e., the indicator of the vitamin D3 status of the human body (Hollis 2005), should be optimal. Seasonal variations in sun exposure cause changes in serum 25(OH)D3 concentrations by a factor of 2–3, but only in the order of weeks and months (Virtanen et al. 2011). This is in clear contrast to cell-culture experiments, where often pharmacological doses of 1,25(OH)2D3 are applied in the time frame of a few hours to days. This comparison brings to question how far results from in-vitro experiments, such as most of those described above, represent the physiological reality in vivo. T cells isolated from 9 human individuals with variant serum 25(OH)D3 concentrations were the first primary human cells that were analyzed by VDR ChIP-seq (Handel et al. 2013). Interestingly, the number of identified VDR loci varied between 200 and 7000, and correlated with the 25(OH)D3 levels of the individuals. In all, 9 subjects, together with more than 14 000 unique VDR loci were identified, of which, however, only 3.1% co-localized with a DR3type sequence. An alternative approach was the study of peripheral blood mononuclear cells (PBMCs) and adipose tissue biopsies from 71 elderly, pre-diabetic individuals, who were supplemented Published by NRC Research Press

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over 5 months with vitamin D3 (Carlberg et al. 2013). The changes in the mRNA expression of 12 primary 1,25(OH)2D3 target genes at the start and the end of the vitamin D3 intervention correlated with the alteration in 25(OH)D3 levels, but only for some 60% of the study participants (Ryynänen et al. 2014; Wilfinger et al. 2014). These individuals are considered as “responders”. The remaining individuals may not have benefited from the vitamin D3 supplementation because they were already at their individual optimal vitamin D status at the start of the intervention (Carlberg et al. 2013). Taken together, an insufficient vitamin D status can contribute to a large variety of diseases, such as osteoporosis, cancer, or autoimmune diseases, reflecting the pleiotropic profile of the micronutrient/pre-hormone. Genome- and transcriptome-wide studies using preferentially easily accessible samples, such as blood cells, from human individuals, allow (i) a molecular profiling of their vitamin D status, and (ii) their classification concerning responsiveness to vitamin D3.

Conclusions Historically, vitamin D and its metabolite 1,25(OH)2D3 were understood to control calcium homeostasis and bone formation; but at present, the genome-wide actions of the vitamin/nuclear hormone have been studied in most detail in the cells of the hematopoietic system. This emphasizes the impact of 1,25(OH)2D3 on the function of innate and adaptive immunity. There are preliminary indications that the core actions of 1,25(OH)2D3 and its receptor VDR can be extrapolated from hematopoietic cells to other cell types (Carlberg et al. 2013), suggesting that the vitamin D status and the responsiveness of a human individual can be derived from the response of, for example, PBMCs. ChIP-seq and FAIRE-seq studies on VDR locations and accessible chromatin in living cells provided initial insight on the genome-wide actions of 1,25(OH)2D3 and its nuclear receptor. The large number of vitamin D-modulated genomic loci supported the view that vitamin D has pleiotropic effects, and suggests many possibilities for therapeutic applications, such as addressing different VDR partner proteins. In general, this could be (i) the use of appropriate doses of vitamin D3 for supplementing everyone to their individual optimal vitamin D status, (ii) the combination of vitamin D with chromatin modifying compounds, such as HDAC inhibitors, or (iii) the application of synthetic 1,25(OH)2D3 analogs. For designing and understanding the latter, crystal structure information on the analog-VDR LBD complex is indispensable. These data demonstrated the great flexibility of VDR’s LBP, harboring a large variety of structurally divergent secosteroidal and nonsteroidal 1,25(OH)2D3 analogs. Importantly, all known VDR–1,25(OH)2D3 analog crystal structures display the active LBD conformation, with helix 12 tightly packed towards the surface of the domain, i.e., the respective compounds will initiate vitamin D signaling in a very comparable way.

Acknowledgements C.C. thanks the Academy of Finland and the Juselius Foundation for support.

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