Key Words

1 INTRODUCTION

The biologically active form of vitamin D, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], acts as a ligand to the transcription factor vitamin D receptor (VDR). In order to directly activate a gene by 1,25(OH)2D at least one VDR molecule has to bind in sufficient vicinity to the gene’s transcription start site (TSS) (1). However, “vicinity” could in some cases be a distance of up to 100 kB, irrespective if upstream or downstream of the TSS. Moreover, there are a number of evidences that most primary VDR target genes use multiple VDR-binding sites, the so-called vitamin D response elements (VDREs), for their full functionality (2). The complete sequence of the human genome and also that of other mammalian species, such as chimp, dog, mouse, and rat, is now available, so that we are able to screen for all putative VDREs. However, the constant packaging of genomic DNA into chromatin provides a repressive environment, which in most cases denies the access to putative VDREs (3). Fortunately, new experimental techniques for genome-wide analyses of chromatin modifications and transcription factor binding, such as chromatin immunoprecipitation (ChIP)-chip and massively parallel sequencing, are now available (4). This will revolutionize our understanding of the genome-wide effects of the VDR and of the diversity of 1,25(OH)2D target genes as outlined in this chapter.

2 Vdr Is A Nuclear Receptor

2.1 The Nuclear Receptor Superfamily

Nuclear receptors are the best-characterized representatives of approximately 3,000 different mammalian proteins that are involved in transcriptional regulation in human tissues (5). They form a superfamily with 48 human members, of which the most have the special property to be ligand-inducible (6, 7). Nuclear receptors modulate genes that affect processes as diverse as reproduction, development, inflammation, and general metabolism. They can be classified based on ligand sensitivity (6), evolution of the nuclear receptor genes (8), and their physiological role as interpreted from tissue-specific expression patterns (9).

The ligand sensitivity approach suggests three classes of nuclear receptors. Class I contains the endocrine receptors with high-affinity hormonal lipids, such as the receptors for the steroid hormones estradiol (estrogen receptors α and β), progesterone (progesterone receptor), testosterone (androgen receptor), cortisol (glucocorticoid receptor), and aldosterol (mineralocorticoid receptor), for thyroid hormones (thyroid hormone receptors α and β), and for the biologically active forms of the fat-soluble vitamins A and D, all-trans retinoic acid (retinoid acid receptors α, β, and γ) and 1,25(OH)2D (VDR). In class II are adopted orphan receptors that bind to dietary lipids and xenobiotics with low affinity, such as peroxisome proliferator-activated receptors (PPARs) α, δ, and γ, constitutive androstane receptor, and pregnane X receptor. Finally, in class III orphan receptors are placed, such as estrogen-related receptors, for which a physiological ligand has not yet been identified.

When the sequences of nuclear receptors are compared on DNA and protein level, the grouping significantly differs from the ligand-centered view. For example, VDR is in the same group with PPARs and the highly ligand-sensitive estrogen receptors and the orphan estrogen-related receptors are together in another group.

On the basis of mRNA expression of all nuclear receptor genes in 39 different tissues in two different mouse strains, nuclear receptors are divided into clades with distinct physiological roles. In this classification, for example VDR is grouped to bile acid and xenobiotic metabolism based on its high expression in gastroentric tissues and PPARs are linked to lipid metabolism and energy homeostasis.

2.2 Modular Structure of Nuclear Receptors

Nuclear receptors have a modular structure, onto which certain functions can be ascribed. The amino-terminus is of variable length and sequence in the different family members. It contains a transactivation domain, termed AF-1, which is recognized by co-activator proteins and/or other transcription factors, often in a ligand-independent fashion. The central DNA-binding domain has two zinc-finger motifs that are common to the entire family. The carboxy-terminal ligand-binding domain, whose overall architecture is well conserved between the various family members, nonetheless diverges sufficiently to guarantee selective ligand recognition as well as accommodate the broad spectrum of nuclear receptor ligand structures. The ligand-binding domain consists of 250–300 amino acids in 11–13 α-helices (10). Ligand binding causes a conformational change within the ligand-binding domain, whereby, at least in the case of endocrine nuclear receptors, helix 12, the most carboxy-terminal α-helix (also called AF-2 domain), closes the ligand-binding pocket via a “mouse-trap like” intramolecular folding event (11). The ligand-binding domain is also involved in a variety of interactions with nuclear proteins, such as other members of the nuclear receptor superfamily and co-regulator proteins.

2.3 The VDR

VDR is an endocrine member of the nuclear receptor superfamily (7), because it is the only nuclear protein that binds 1,25(OH)2D with high affinity (K d = 0.1 nM) (12, 13). VDR has been shown to form homodimers (14, 15) and heterodimers with thyroid hormone receptors (16, 17) and retinoid acid receptors (18), but by far the strongest binding partner of VDR is one of the three retinoid X receptors (RXRs) α, β, and γ (19). In mammals, the highest VDR expression is found in metabolic tissues, such as intestine and kidney, as well as in skin and the thyroid gland, but moderate expression is found in nearly all tissues (9). Moreover, the receptor is also expressed in many malignant tissues (20). Mice lacking a functional VDR gene develop alopecia (likewise found in many patients with mutations in the VDR) (21); these mice also exhibit a defect in epidermal differentiation. Moreover, VDR-null mice also show an increased susceptibility to tumor formation (22). More details on the receptor have been provided in the previous chapter.

3 Vdr-Binding Sites

3.1 DR3-Type VDREs

The binding of VDR–RXR heterodimers is achieved through the specific binding of the DNA-binding domain of the VDR to the major grove of a hexameric DNA sequence, referred to as core-binding motif, with the consensus sequence RGKTSA (R = A or G, K = G or T, S = C or G) (13) (Fig. 1). Numerous studies (for example (14, 23) have confirmed Umesomo’s suggestion (24) that VDR–RXR heterodimers bind well to response elements (REs) that are formed by a direct repeat of the hexameric sequences with three spacing nucleotides. These DR3-type REs are therefore widely accepted as the classical structure of a VDRE.

Fig. 1.
figure 11_1_144707_2_En

VDR–RXR heterodimer on VDREs: VDR–RXR heterodimer binding to different types of VDREs is schematically depicted.

Full size image

Every 1,25(OH)2D target gene has to contain at least one VDRE in its promoter region and the first VDREs have been identified rather close to the TSS of the genes. The strongest DR3-type VDREs has been identified within the rat atrial natriuretic factor promoter (25), the mouse and pig osteopontin promoter (26, 27), and the chicken carbonic anhydrase II promoter (28). A number of other DR3-type VDREs have been published and were based on their in vitro binding affinity categorized into different classes (29). The REs with the lowest affinity show a significant deviation from the RGKTSA consensus and may not be functional. However, it is possible that these VDREs may gain responsiveness to 1,25(OH)2D in their natural promoter context through the help of flanking partner proteins (see also Section 3.4.). Moreover, the functionality of a 1,25(OH)2D responding gene will also depend on a potential cooperative action of two or more VDREs, such as in the case of the 24-hydroxylase (CYP24A1) gene (30). Nevertheless, VDR–RXR heterodimers form identical complexes on all DR3-type VDREs and display no significant differences in their interaction with a given co-activator or co-repressor protein (29).

3.2 Other Types of VDREs

There are also other VDRE structures, such as direct repeats with four intervening nucleotides (DR4). Effective VDR binding has also been observed on everted repeat (ER)-type REs with 6–9 spacing nucleotides (ER6, ER7, ER8, ER9) (17, 31) (Fig. 1). A VDRE classification according to the affinity for VDR–RXR heterodimers suggests that the degree of deviation from the core-binding motif consensus sequence RGKTSA (32) is proportional to the loss of in vitro functionality (29). Interestingly, the DR4-type RE of the rat pit1 gene (33), which contains two perfect core-binding motifs, was found to be stronger than any known natural DR3-type VDRE (29). However, one has to consider that a DR4-type REs is also recognized by the heterodimeric complexes of thyroid hormone receptor, constitutive active receptor and pregnane X receptor and other orphan nuclear receptors with RXR (19, 34), whereas the same complexes bind to DR3-type REs less tightly than VDR–RXR heterodimers. The competitive situation on DR4-type REs may therefore be the reason why in vivo VDR–RXR heterodimers still prefer DR3-type REs. Moreover, VDR–RXR heterodimers bind to DR4-type REs in the same conformation as to DR3-type REs (34), i.e., there seem to be no differential action of VDR on these elements due to a differential complex formation with RXR.

The VDRE of the human osteocalcin promoter was the first identified natural binding site for the VDR (35, 36). It was initially described as a DR6-type structure but later a third cryptic hexamer was identified at a distance of 3 nucleotides, so that the whole VDRE is more likely a complex DR6/DR3-type RE (see Section 3.4.). The DR6 part of the VDRE has been shown to bind VDR homodimers (14) and VDR–RAR heterodimers (18), whereas the DR3 part weakly binds VDR–RXR heterodimers. Other examples of a DR6-type VDREs that bind VDR homodimers and VDR–RAR heterodimers have been identified in the promoters of mouse fibronectin (37), rat CYP24A1 (38), and human phospholipase C (39). Their functionality remain to be determined.

3.3 Chromatin and Co-factors

The major protein constituents of chromatin are the four different histones that form a nucleosome, around which DNA is wound. Covalent modifications of the lysines at the amino-terminal tails of these histone proteins neutralize their positive charge and thus their attraction for the negatively charged DNA backbone is diminished (40). As a consequence, the association between the histone and the DNA becomes less stable. This influences the degree of chromatin packaging and regulates the access of transcription factors to their potential binding sites. When nuclear receptors are bound to REs in the regulatory regions of their target genes, they recruit positive and negative co-regulatory proteins, referred to as co-activators (41) and co-repressors (42), respectively.

In a simplified view of nuclear receptor signaling, in the absence of ligand, the nuclear receptor interacts with co-repressor proteins, such as NCoR1, SMRT, hairless, and Alien, which in turn associate with histone deacetylases leading to a locally increased chromatin packaging (43, 44). The binding of ligand induces the dissociation of the co-repressor and the association of a co-activator of the p160 family, such as SRC-1, TIF2, or RAC3 (45). Some co-activators have histone acetytransferase (HAT) activity or are complexed with proteins harboring such activity and this results in the net effect of local chromatin relaxation (46). In a subsequent step, ligand-activated nuclear receptors change rapidly from interacting with the co-activators of the p160 family to those of the mediator complex, such as Med1 (47). The mediator complex, which consists of approximately 15–20 proteins, builds a bridge to the basal transcriptional machinery (48). In this way ligand-activated nuclear receptors execute two tasks, the modification of chromatin and the regulation of transcription.

Cell- and time-specific patterns of relative protein expression levels of some co-regulators can distinctly modulate nuclear receptor transcriptional activity. This aspect may have some diagnostic and therapeutic value in different types of cancer (49). Concerning skin cancer it was postulated that the stoichiometric ratio between co-activators of the p160 family and Med1 might regulate a 1,25(OH)2D-dependent balance between proliferation and differentiation of keratinocytes (50). However, the switch between gene repression and activation is more complex than a simple alternative recruitment of two different regulatory complexes (51). Most co-regulators are co-expressed in the same cell type at relatively similar levels, which raises the possibility of their concomitant recruitment to a specific promoter. This has been resolved by the mutually exclusive binding of co-activators and co-repressors to ligand-bound and ligand-unbound nuclear receptors, respectively. Therefore, repression and activation are more likely achieved by a series of sequential multiple enzymatic reactions that are promoter and cell-type specific. Transcriptional regulation is a highly dynamic event of rapid association and dissociation of proteins and their modifications, including proteolytic degradation and de novo synthesis. A pattern of recruitment and release of cohorts of co-regulatory complexes was demonstrated on a single region of the trefoil factor-1 promoter in breast cancer cells (52). This study revealed detailed and coordinated patterns of co-regulator recruitment and preferential selectivity for factors that have similar enzymatic activities. Interestingly, similar cyclic behavior was also observed for the VDR (53).

3.4 VDREs in the Chromatin Context

It is assumed that matrix attachment regions (MARs) subdivide genomic DNA into units of an average length of 100 kB containing the coding region of at least one gene(54) (Fig. 2). DNA looping should be able to bring any DNA site within the same chromatin unit close to the basal transcriptional machinery that is assembled on the TSS. This model suggests that also very distant sequences can serve as VDREs and that even sequences downstream of the TSS could serve as functional VDR-binding sites.

Fig. 2.
figure 11_2_144707_2_En

1,25(OH)2D responsive chromatin units: A chromatin unit carrying two genes and six VDREs is schematically depicted. The loop may be formed by some 100 kB of genomic sequence, which is kept together by a matrix attachment region (MAR) complex. For clarity the some 500 nucleosome covering this genomic region are not shown. In this scenario VDREs 4 and 5 do sub-looping to the basal transcriptional machinery (Pol II) on the TSS (arrow) of gene 2 and induce transcription. Similarly all six VDREs have the potential to activate genes 1 and 2, i.e., both genes are supposed to be primary VDR target genes.

Full size image

Due to its optimized 5ʹ-flanking dinucleotide and core-binding motif sequences the DR4-type RE of the rat pit-1 gene is the most efficient known VDRE in vitro (29, 34). However, the chromatin in the region of the pit-1 gene promoter containing this RE seems to be closed in the adult rat, so that the responsiveness of the gene to 1,25(OH)2D is lower than expected (46). This indicates that a high in vitro binding affinity of VDR–RXR heterodimers for a VDRE is not sufficient for responsiveness to 1,25(OH)2D. When the promoter region that contains the VDRE is covered by condensed chromatin, VDR–RXR heterodimers are unable to bind there. This makes sufficiently decondensed chromatin an essential prerequisite for a functional VDRE. Chromatin decondensation is achieved by the activity of HATs, which are recruited to their local chromatin target by co-activator proteins. In turn, these co-activators are transiently attracted to a promoter region by ligand-activated nuclear receptors and other active transcription factors. Therefore, the more transcription factor binding sites a given genomic region has and the more of these transcription factors are expressed in the respective cell, the higher is the chance that this area of the promoter gets locally decondensed. One example is the VDRE of the rat osteocalcin gene, which is flanked on both sides with a binding site for the transcription factor Runx2/Cbfa1 (55). By contacting co-activator proteins and HATs Runx2/Cbfa1 seems to mediate the opening of chromatin locally, which allows efficient binding of VDR–RXR heterodimers to this decondensed region to occur. This mechanism suggests that VDREs are better targets for VDR–RXR heterodimers, if other transcription factors are bound to the same chromatin region. In this respect, promoter context and cell-specific expression of other transcription factors may be of greater importance to VDRE functionality and specificity than its in vitro binding profile.

The DR6/DR3 and ER9/DR3 structures of the VDREs of the human and rat osteocalcin genes, respectively, are the first examples of complex VDREs. These two complex VDREs show only limited homology to each other, although they are derived from orthologous genes. This suggests that for an important primary 1,25(OH)2D responding gene, such as osteocalcin, there may be limited evolutionary pressure for a specific VDRE structure. It seems to be more important to guarantee an efficient binding of the VDR to a promoter in competition with the tight packaging of nucleosomes. It is interesting to note that the complex VDRE of the human osteocalcin gene is overlaid by a binding site of the transcription factor AP-1 (56), which provides the RE with an increased activity. These types of REs are also observed for other nuclear receptors and often referred to as composite REs (57). Another interesting example of a complex/composite VDRE has been reported in the mouse c-fos promoter (58). Within this VDRE three hexameric core-binding motifs are forming a DR7/DR7 structure, which contains an internal binding site for the transcription factor NF-1. Additional examples are the VDRE of the human and mouse fibronectin gene, which contains an internal binding site for the transcription factor CREB (37), or the VDRE of the rat bone sialo protein, which also seems to bind the general transcription factor TBP (59).

3.5 Negative VDREs

Expression profiling using microarray technology indicates that comparable numbers of genes are down-regulated by 1,25(OH)2D as are up-regulated by the hormone (60). In general, the mechanisms of the down-regulation of genes by 1,25(OH)2D are much less understood, but they also seem to require the binding of an agonist to the VDR. It is obvious that only genes, which show basal activity, can be down-regulated, i.e., these genes exhibit basal activity due to other transcription factors binding to their promoter. There are several different models that attempt to explain, how 1,25(OH)2D and the VDR can mediate down-regulation of genes, but the common theme is that VDR counteracts the activity of specific transcription factors. For the physiologically important down-regulation of the 25-dihydroxyvitamin D 3 1 α -hydroxylase (CYP27B1) gene by 1,25(OH)2D a negative VDRE located at position –0.5 kB has been proposed, where VDR–RXR heterodimers do not bind directly, but via the transcription factor VDR-interacting repressor (VDIR, also called TCF3) (61). In addition, two positive VDREs are located –2.6 and –3.2 kB upstream from the TSS and modulate the cell-specific activity of the negative VDRE (62). Association of VDR–RXR heterodimers to TCF3-binding sites may also occur through ligand-dependent chromatin looping from more distal regions that directly bind the VDR (62). In situations where these activating transcription factors are other nuclear receptors or transcription factors that bind to composite nuclear receptor REs, VDR could simply compete for DNA-binding sites (63, 64). In a similar way, VDR could also compete for binding to partner proteins, such as RXR, or for common co-activators, such as SRC-1 or CBP (65). In all these situations the down-regulating effects of the VDR should be of general impact, i.e., the mechanism could apply to other genes in the same way. So far, however, no general down-regulating effects of 1,25(OH)2D have been reported.

The concept that multiple VDREs (see also Fig. 2) together with other transcription factor binding sites regulate primary 1,25(OH)2D responding genes (see Section 5.2.) suggests that a promoter may contain both negative and positive VDREs. The activities of the different VDREs are determined by the promoter context and may not be simultaneously active. One might imagine that prior to stimulation with 1,25(OH)2D only the negative VDREs bind the VDR and recruit co-repressors. This would actively condense the chromatin on a particular promoter region. The addition of ligand induces the release of co-repressor proteins and reduces chromatin density. The VDR may then be transiently released from the negative VDRE and bind to a positive VDRE, which may be uncovered through 1,25(OH)2D-dependent local nucleosome acetylation. The VDR then interacts with the mediator protein complex on this positive VDRE leading to transient transcriptional activation. After a certain period of time, newly synthesized, unliganded VDR again binds to the negative VDRE, which initiates chromatin closing and inactivation of the positive VDRE (62). In this or even more complex scenarios, the balance between negative and positive VDREs could explain the time course of the activation of primary 1,25(OH)2D responding genes.

4 Vdr Target Genes

4.1 Classical VDR Targets

The most striking effect of severe vitamin D deficiency is rickets. Rickets can also by inflicted by mutations in the CYP27B1 or the VDR gene. 1,25(OH)2D is essential for adequate Ca2+ and Pi absorption from the intestine and hence for bone formation (66). Liganded VDR has been shown to induce expression of the gene encoding for the major Ca2+ channel in intestinal epithelial cell, TRPV6, by direct binding to a VDRE at -1.2 kB from the TSS (67). The phosphate co-transporter NaP(i)-IIb gene was also found to be induced by 1,25(OH)2D but no VDREs have yet been identified for this gene (68). 1,25(OH)2D also down-regulates the expression of the PTH gene that opposes 1,25(OH)2D in regulation of serum Ca2+ and Pi levels, but up-regulates the FGF23 gene, which, like PTH, lowers serum Pi levels (69). The induction of the RANKL gene by liganded VDR via multiple distant VDREs (up to 70 kB from the TSS) leads to stimulation of osteoclast precursors to fuse and form new osteoclasts, resulting in enhanced resorption of bone (70).

4.2 VDR Targets in Cell Cycle Regulation

The main anti-proliferative effect of 1,25(OH)2D on cells is a cell cycle block at the G1 phase. This can be explained by changed expression of multiple cell cycle regulator genes. Among the first targets described, expression of cyclin-dependent kinase inhibitors (CDKIs) p21 (CDKN1A) and p27 were found to be up-regulated by ligand treatment (71, 72). For the CDKN1A gene a VDRE in the proximal promoter was characterized, thus establishing CDKN1A as a direct 1,25(OH)2D target gene (73). Later on it has been questioned, whether CDKN1A truly is a primary target or a secondary target via up-regulation of TGFβ or of the insulin-like growth factor binding protein (IGFBP) 3, and whether the described VDRE is truly functional ( 74, 75). However, by screening 7 kB of the CDKN1A promoter with overlapping ChIP regions three novel regions with 1,25(OH)2D-induced VDR enrichment, two of which harvested also p53 were identified (76). The direct role of the characterized 1,25(OH)2D-responsive regions on regulation of CDKN1A expression is illustrated by the their association with VDR-RNA polymerase II complexes as well as by their ligand-dependent looping to the CDKN1A TSS (51). Also other CDKIs, such as p15, p16, p18, and p19, show transcriptional response to 1,25(OH)2D, but for p16 the response is secondary as it can be blocked by inhibition of protein synthesis (73, 77). In addition, cyclin E, cyclin D1, and CDK2 were found to be down-regulated by 1,25(OH)2D (74). It remains to be elucidated, whether these effects are primary and occur via functional VDREs on regulatory regions of these genes.

Another interesting 1,25(OH)2D-target gene is cyclin C (CCNC). The cyclin C-CDK8 complex was found to be associated with the RNA polymerase II basal transcriptional machinery (78) and is considered as a functional part of those mediator protein complexes that are involved in gene repression (79). The fact that the CCNC gene, being located in chromosome 6q21, is deleted in a subset of acute lymphoblastic leukemias, suggests its involvement in tumorigenesis (80). In addition, GADD45A and members of the IGFBP gene family respond to 1,25(OH)2D (81, 82). GADD45A plays an essential role in DNA repair and GADD45 proteins displace cyclin B1 from Cdc2 and thus inhibit the formation of M phase-promoting factor that is essential for G2/M transition (83). GADD45A has been shown to be a direct transcriptional target of 1,25(OH)2D with a functional VDRE within the fourth exon of the gene (84). IGFBPs modulate the activity of the circulating insulin-like growth factors (IGF) I and II. The IGFBP-3 gene was first discovered to be up-regulated by 1,25(OH)2D and contains a functional VDRE (85). Later also IGFBP-1 and IGFBP-5 have been characterized as primary 1,25(OH)2D target genes (82). Another interesting primary 1,25(OH)2D target is the PPARD gene, which carries a potent DR3-type VDRE in close proximity to its TSS (86). PPARδ and VDR proteins are widely expressed and an apparent overlap in the physiological action of the two nuclear receptors is their involvement in the regulation of cellular growth, particularly in neoplasms. High PPARD expression in tumor seems to be positive for the prognosis of the respective cancer (87).

Overall, 1,25(OH)2D restricts cell cycle progression in several phases via multiple and partially redundant targets on parallel pathways, that when combined, provide robustness for its anti-proliferative effect.

4.3 Relative Expression of VDR Target Genes

The steady-state mRNA expression levels of some VDR target genes, such as that of the CYP24A1 gene, are very low in the absence of ligand, but are up to 1,000-fold induced by stimulation with 1,25(OH)2D (88). Most other known primary 1,25(OH)2D target genes, such as CCNC and CDKN1A, often show an inducibility of twofold or less after short-term treatment with 1,25(OH)2D (89, 90). However, both genes have 10,000- to 100,000-fold higher basal expression levels compared to that of the CYP24A1 gene. Therefore, when the relative levels are taken into account, 2- to 20-fold more CCNC and CDKN1A than CYP24A1 mRNA molecules are produced after induction with 1,25(OH)2D.

5 Vdr Target Gene Analysis

5.1 Transcriptome Analysis

There are a number of modern methods for the identification and characterization of VDR target genes. The effect of 1,25(OH)2D on the mRNA expression, i.e., 1,25(OH)2D-induced changes of the transcriptome, has been assayed by multiple microarray experiment in cellular models (either an established cell line or primary cells) or in in vivo models (mostly rodents). In case the focus is on the identification of primary VDR target genes, the stimulation times are short (2–6 h), but when the overall physiological effects are the center of the study, longer treatment times are used (24–72 h). For a limited number of putative VDR target genes quantitative PCR can be applied, but for a whole genome perspective on VDR signaling, microarrays have to be used.

A few years ago mostly cDNA arrays with an incomplete number of genes were used and rather short lists of VDR target genes from colon (90), prostate (49, 91 – 93), breast (89), and osteoblasts (94, 95). In squamous cell carcinoma more than 900 genes respond to 1,25(OH)2D after 12 h treatment in the presence of the protein synthesis inhibitor cycloheximide (96). However, the number of overlapping VDR target genes in these lists was low. Since the setups of these microarray analyses were different in treatment times and probe sets, this suggests that most VDR target genes reply to 1,25(OH)2D in a very tissue-specific fashion and may have only a rather transient response. However, on the basis of these results the total number of convincing primary 1,25(OH)2D target genes is in the order of 250. In addition, secondary 1,25(OH)2D-responding genes contribute to the physiological effects of 1,25(OH)2D, but their induction is delayed by a few hours or even days and are probably mediated by primary 1,25(OH)2D-responding gene products, such as transcription factors or co-regulator proteins (90). For a more detailed meta-analysis of VDR target genes, standardized microarray procedures performed on whole genome chips from Affymetrix, Illumina or other commercial suppliers are essential. Results from such approaches will be published soon.

5.2 ChIP Analysis

For a detailed analysis of the regulatory regions of primary VDR target genes since a couple of years the method of ChIP became the golden standard. For the genes CYP24A1 (88), CYP27B1 (62), CCNC (97), and CDKN1A (76) 7.1–8.4 kB of their promoter regions were investigated by using in each case a set of 20–25 overlapping genomic region. The spatio-temporal, 1,25(OH)2D-dependent chromatin changes in the four gene promoters were studied by ChIP assays with antibodies against acetylated histone 4, VDR, RXR, and RNA polymerase II. Promising promoter regions were then screened in silico for putative VDREs, whose functionality was analyzed sequentially with gel shift, reporter gene, and re-ChIP assays. This approach identified four VDREs for both the CYP24A1 and CCNC genes, three in the CDKN1A promoter and two in the CYP27B1 gene. However, most of them are simultaneously under the control of other transcription factors, such as p53 in case of the CDKN1A gene (76), and therefore possess significant basal levels of transcription.

An alternative approach to the identification of primary 1,25(OH)2D target genes was performed with the six members of the IGFBP gene family. Here first an in silico screen was performed, which was then followed by the analysis of candidate 1,25(OH)2D-responsive sequences by gel shift, reporter gene, and re-ChIP assays (82). Induction of gene expression was confirmed independently using quantitative PCR. By using this approach, the genes IGFBP1, IGFBP3, and IGFBP5 were demonstrated to be primary 1,25(OH)2D target genes. The in silico screening of the 174 kB of genomic sequence surrounding all six IGFBP genes identified 15 candidate VDREs, 10 of which were shown to be functional in ChIP assays. Importantly, the in silico screening approach was not restricted to regulatory regions that comprise only maximal 2 kB of sequence up- and downstream of the TSS, as in a recent whole genome screen for regulatory elements (98), but involved up to 10 kB of flanking sequences as well as intronic and intergenic sequences. In a similar approach the 5-lipoxygenase (ALOX5) gene has been analyzed and confirmed to be a primary 1,25(OH)2D target gene. From the 22 putative VDREs identified in the whole ALOX5 gene sequence (–10 kB to +74 kB) by in silico screening, at least two have been validated to be functional in vitro and in the living cells. One of these VDREs is located far downstream of the TSS (+42 kB) and is one of the strongest known VDREs of the human genome (99). No functional VDRE had been reported for the ALOX5 gene before, since previous studies had been restricted to the proximal promoter region (100, 101). Therefore, this approach revealed candidate VDREs that are located more than 30 kB distant from their target gene’s TSS. Based on the present understanding of enhancers, DNA looping and chromatin units being flanked by insulators or MARs, these distances are not limiting (102).

5.3 ChIP-Chip Assays

The combination of ChIP assays with hybridization of the resulting chromatin fragments on microarrays, the so-called ChIP-chip assays, provides an additional step for a larger scale analysis of VDR target genes. The ChIP-chip technology has been applied for the analysis of the VDR gene itself (103), the intestinal calcium ion channel gene TRPV6 (67), the Wnt signaling co-regulator LRP5 (104), and the TNF-like factor RANKL that promotes the formation of calcium-resorbing osteoclasts (105). For all those genes a number of VDR-associated chromatin regions were identified, some of which were far upstream of the gene’s TSS. These studies confirmed that many, if not all, VDR target genes have multiple VDR-associated regions. However, not all of these VDREs may be functional, i.e., they may not contact the gene’s TSS via DNA looping. Therefore, it is necessary to apply an additional method, the so-called chromosome-conformation-capture (3C) assay. So far, 3C assays confirmed the functionality of the VDREs in the CYP27B1 (62) and the CDKN1A (51) genes.

The next step in genome-wide association studies will be massively parallel sequencing of genomic fragments obtained after ChIP assays, also referred to as ChIP-Seq, with antibodies against VDR and its partner proteins. Results will be published soon.

5.4 In Silico Screening of VDREs

The specificity of VDR for its DNA-binding sites allows constructing a model to describe the VDRE properties that can be used to predict potential binding sites in genomic sequences. For this the VDR-binding preference, often expressed as position weight matrix, has to be described on the basis of experimental data, such as series of gel shift assays with a large number of natural binding sites (106 – 109). However, VDR–RXR heterodimers do not only recognize a pair of the nuclear receptor consensus binding motifs AGGTCA, but also a number of variations to it. Dependent on the individual position weight matrix description this leads to a prediction of VDREs every 1,000–10,000 bp of genomic sequence. This probably contains many false-positive predictions, which is mainly due to scoring methodology and the limitations that are imposed by the available experimental data. Wang et al. combined microarray analysis and in silico genome-wide screens for DR3- and ER6-type VDREs (96). This approach identified several novel VDREs and VDR target genes, but most of the VDREs await a confirmation by ChIP and 3C assays.

In a position frequency matrix the quantitative characteristics of a transcription factor, i.e., its relative binding strength to a number of different binding sites, are neglected, since simply the total number of observations of each nucleotide is recorded for each position. Moreover, in the past there was a positional bias of transcription factor binding sites upstream in close vicinity to the TSS. This would be apparent from the collection of identified VDREs (13), but is in contrast with a multi-genome comparison of nuclear receptor binding site distribution (98) and other reports on wide-range associations of distal regulatory sites (110).

Internet-based software tools, such as TRANSFAC (111), screen DNA sequences with databases of matrix models. The accuracy of such methods can be improved by taking the evolutionary conservation of the binding site and that of the flanking genomic region into account. Moreover, cooperative interactions between transcription factors, i.e., regulatory modules, can be taken into account by screening for binding site clusters. The combination of phylogenetic footprinting and position weight matrix searches applied to orthologous human and mouse gene sequences reduces the rate of false predictions by an order of magnitude, but leads to some reduction in sensitivity (112). Recent studies suggest that a surprisingly large fraction of regulatory sites may not be conserved but yet are functional, which suggests that sequence conservation revealed by alignments may not capture some relevant regulatory regions (113).

The recently published classifier method for the in silico screening of transcription factor binding sites (114) showed at the example of PPAR–RXR heterodimers, how a set of in vitro binding preferences of the three PPAR subtypes can be used as an experimental data set. Single nucleotide variants were sorted into three classes, where in class I the PPAR subtypes are able to bind the sequence with a strength of 75 ± 15% of that of the consensus PPAR-binding site, in class II with 45 ± 15%, and in class III with 15 ± 15%. Additional 130 PPREs were sorted on the basis of counting increasing number of variations from the consensus and taking into account the single nucleotide variant binding strength. Those variants that alone decrease the binding only modestly (class I) could be combined with even three deviations from consensus still resulting in more than 20% binding relative to consensus. Other combinations resulted in faster loss of binding detailed in 11 categories, where such combinations still resulted in more than 1% relative binding. The main advantage, when comparing the classifier to position weight matrix methods, is a clear separation between weak PPREs and those of medium and strong strength (114). With this method the gene-dense human chromosome 19 (63.8 MB, 1,445 known genes) and its syntenic mouse regions (956 genes have known orthologs) were screened. Twenty percent of genes of chromosome 19 were found to contain a strong RE and additional 4% have more than two medium REs or one proximal medium RE. These numbers suggest a total of 4,000–5,000 targets for PPARs in the human genome. Presently, the same approach is used for a genome-wide screening of VDREs and the results will be published soon. First results already indicated that the number of putative VDR target genes is in the same order as that of possible PPAR targets. Certainly not all sites will be accessible and the human genome also contains weak binding sites that could gain function via interaction with other transcription factors.

In effect, these approaches and tools are still insufficient and there has to be a focus on the creation of bioinformatics resources that include more directly the biochemical restraints to regulate gene transcription. One important aspect is that most putative transcription factor binding sites are covered by nucleosomes, so that they are not accessible to the transcription factor. This repressive environment is found in particular for those sequences that are either contained within interspersed sequences, are located isolated from transcription factor modules, or lie outside of insulator sequences marking the border of chromatin loops (115). This perspective strongly discourages the idea that isolated, simple VDREs may be functional in vivo. In turn, this idea implies that the more transcription factor binding sites a given promoter region contains and the more of these transcription factors are expressed, the higher is the chance that this area of the promoter becomes locally decondensed.

6 Conclusions

The sequencing of the complete human genome and the genome of other species, i.e., the availability of all regulatory sequences, enable a more mature understanding of the diversity of 1,25(OH)2D target genes. Perhaps the idea of simple isolated VDREs should shift to the concept of complex VDREs, of which a simple DR3-, DR4- or ER-type VDRE represents the core. Depending on the temporal presence of cell-specific transcription factors, these complex REs may act positively or negatively in respect to 1,25(OH)2D. The coordinated action of these different types of VDREs could explain the individual response of target genes to 1,25(OH)2D.

Methods incorporating both experimental- and informatics-derived evidence to arrive at a more reliable prediction of VDR targets and binding modules can bring all available data together with the aim to predict the outcome in a specific context. We envision that in future the emphasis will shift from target genes to target regulatory modules to alter a physiological response and from individual genes to whole genome response. Therefore, a much larger challenge lies ahead when we would be confronted with the higher order of regulated networks of genes, where the sum effect of ligand treatment may reveal itself. In an effort to study this, we have started applying systems biology to the field of nuclear receptor biology, through an EU-funded Marie Curie Research Training Network, NUCSYS (http://www.uku.fi/nucsys).