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3 Molecular Actions of Vitamin D: The Vitamin D Receptor

3 Molecular Actions of Vitamin D: The Vitamin D Receptor

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4



Vitamin D Receptor and Interaction with DNA



81



that this putative receptor was indeed able to bind 1,25(OH)2D3. In addition to their

presence in kidney, intestine, bone and parathyroid cells in several species, including mammals, VDRs were also discovered in tissues such as pancreas, placenta,

pituitary and mammary gland, ovary, testis and heart [6]. VDRs have also been

found in a large series of tumoral cells.

The VDR is expressed in low concentrations in target tissues and cell lines, ranging from a few to thousands of copies per cell. Although the receptor concentration

in cells may be a primary determinant of the magnitude of the cellular response to

1,25(OH)2D3, myriad additional factors also play a significant and possibly more

important role, including vitamin D-degrading enzymes (such as CYP24A1), the

presence and activities of vitamin D-binding protein (DBP) or intracellular vitamin

D-binding proteins, specific characteristics of the gene target itself, post-translational

modifications of the VDR, vitamin D-response element (VDRE)-binding proteins

and finally a mixture of comodulators that act downstream of the VDR but participate directly in the regulation of gene expression. In any case, examples of a strong

correlation between cellular VDR levels and biological response are described.

VDR abundance is also regulated transcriptionally. As described below, acquired

disturbances in the VDR number and/or functionality related to many of these VDRrelated processes have been induced in uremic conditions [61].

The most important characteristic of the VDR is its capacity to bind 1,25(OH)2D3

with high affinity and selectivity. The VDR displays an equilibrium dissociation

constant (KD) of ca. 10−10 M for the natural ligand 1,25(OH)2D3 and binds its precursors and less active metabolites with significantly lower affinities [6]. Both the

25-hydroxyl and the 1α-hydroxyl group on the 1,25(OH)2D3 molecule contribute to

specific and high-affinity binding. In the late 1970s it was discovered that the VDR

could bind to DNA [62], and the subsequent identification of specific DNA-binding

sites (VDRE’s) within gene promoters, enhancers or repressors led to a deeper

understanding of such binding. Interestingly, the molecular actions of 1,25(OH)2D3

are largely identical to those of its receptor, but VDR has additional ligandindependent functions [63]. Thus, in addition to increased “affinity” for non-specific

DNA following ligand binding, VDR displays “affinity” for DNA in the absence of

1,25(OH)2D3 [6]. VDR may also function unliganded, obviating the need for local

generation of 1,25(OH)2D3. For instance, VDR but not vitamin D is required to

sustain the mammalian hair cycle [the hairless (Hr) corepressor could function as a

surrogate VDR “ligand”], and calbindin induction by VDR does not require vitamin

D in brain (unlike in intestine, kidney and bone). However, the ability of VDR to

function unliganded is difficult to justify physicochemically and thus it has been

suggested that VDR may bind non-vitamin D ligands in order to exert its extraosseous actions [16]. Consequently, several additional nutritional lipids have been identified as candidate low-affinity VDR ligands that may function locally in high

concentrations; examples include omega-3 and omega-6 polyunsaturated fatty acids

(PUFAs), lithocolic or arachidonic acids, vitamin E derivatives and others [16].

High local concentrations of PUFAs could occur in select cells or tissues and may

partially explain the chemoprotective nature of diets rich in PUFAs, plus their cardioprotective and anti-inflammatory influences [16].



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Finally, other studies have suggested that the human VDR may comprise several

forms, and several polymorphisms have been linked to bone mineral density disorders as well as to various other diseases [6, 64]. Thus, VDR polymorphisms could be

involved in the development of secondary hyperparathyroidism in CKD patients or

different response patterns secondary to tissue-specific effects of the VDR response

to vitamin D metabolites [64, 65]. The BsmI genotype, as well as others such as the

ApaI, TaqI and FokI restriction length polymorphisms, can also affect PTH levels,

the response of hemodialysis patients to IV 1,25(OH)2D3, and the need for parathyroidectomy [66–69] . Moreover, experimental studies in human primary osteoblasts

and human parathyroid glands yielded opposite results with regard to haplotype

response to 1,25(OH)2D3 in osteoblasts and parathyroid glands [65]. Overall, the

results support the proposed significant role for VDR polymorphisms in bone and

parathyroid gland behavior, the observation of different patterns probably being due

to tissue-specific effects of the VDR response to 1,25(OH)2D3 [64, 65]. Studies investigating the relationship between VDR genotypes and left ventricular hypertrophy

have revealed a highly significant association with the BsmI Bb heterozygous genotype [43]. However, none of these polymorphism-derived data seems particularly

robust and no useful direct clinical contribution has been proven [70]. As a matter of

fact, none of the VDR polymorphisms showed differences in response to 1,25(OH)2D3

in parathyroid glands in culture [66, 71], and therefore any implications for treatment

are still to be considered very preliminary [66]. Consequently, adaptation of treatment algorithms on the basis of the allelic status of the individual patient is not currently recommended since the relevance of VDR polymorphisms are still a matter of

debate due to the poorly reproducible clinical correlations [66, 70].



4.4



VDR Structure



The initial cloning of the glucocorticoid and estrogen receptors represented the initial step in the characterization of this intracellular nuclear receptor gene family.

Some receptors can act as silencers of transcription in the absence of ligands or the

presence of antagonists. Agonists induce an alteration in the structure of the nuclear

receptor, which allows interaction with target gene promoters.

These receptors comprise of distinct regions or domains, leading to their designation as A, B, C, D, E and F [72]. The highly conserved DNA-binding domain is

designated the C domain. The domain structure of the 427-AA human VDR is

depicted in Figs. 4.1 and 4.2 [6, 16], with the two major functional units, the DNAbinding domain and the ligand-binding domain (LBD). The DNA-binding domain

(66 amino acids, from residues 24 to 89) is represented by the N-terminal classical

(Cys2-Cys2)2 two Zn-finger motifs, serving for DNA binding within the major

groove of genomic DNA. The LBD is represented in the multifunctional C-terminal

domain (approximately 200 amino acids). The A/B domains are relatively very short

compared with other members of the nuclear receptor family (Fig. 4.1). The C region

comprises the highly conserved DNA-binding domain, which mediates binding of



4



Vitamin D Receptor and Interaction with DNA



DNA-binding

domain



A/B



N-terminal -



C



83



Hinge



Multifunctional

ligand binding

domain



D



E/F



-C-terminal



1,25-vitD5-binding

•heterrodimerization

•nuclear localization

•phosphorylation





Fig. 4.1 Schematic structure of human VDR



Comodulator binding



Zn



N1



24



Zn



T A

89



DNA binding domain



Hinge

159



E1



Heptad repeats AF-2



201



C

427



Ligand binding domain



Fig. 4.2 Domains in human VDR. DNA-binding domain with zinc fingers on the left side. Ligandbinding domain on the right side. AF-2 Activation Function 2 (Adapted from Haussler et al. [16])



VDR to specific regions of the DNA flanking regions of the target genes. The E/F

domain contains the ligand 1,25(OH)2D3-binding region of the VDR and serves as an

important and highly complex protein-protein interface for a series of additional

interacting proteins essential to receptor activity (RXR heterodimerization and transcriptional comodulator binding) (Fig. 4.2) [16]. This region also harbors a highly

structured ligand-dependent activation function, termed activation function 2 (AF2), and hormone binding results in the creation of a functional AF-2 (see Fig. 4.2)

[16]. The E region of the VDR encodes a multifunctional domain that exerts indirect

control over the DNA binding as well as transcription-modifying properties of the

VDR. The presumed change in nuclear receptor structure in the AF-2 region that

occurs in response to hormone binding appears to play a key role in its ability to

attract DNA-binding partners (e.g. it acts as a major interface for dimerization with

RXR) and recruit protein complexes (coactivators or corepressors) vital for transcriptional activation (Fig. 4.2) [6, 16, 73]. The F domain is absent in the VDR.

The DNA-binding domain of the VDR is evolutionarily highly homologous to

other steroid receptors [74]. This domain comprises two similar modules, each consisting of a Zn-coordinated finger structure. The first Zn finger, the amino-terminal

module, is responsible for directing specific DNA binding in the major groove of

the DNA-binding site. In order to bind DNA and regulate gene expression, transcription factors require nuclear translocation, and nuclear localization depends on

various arrangements of basic amino acids in short stretches within the primary

amino acid sequence [75]. The second Zn finger, the carboxy-terminal module,



84

Fig. 4.3 1,25(OH)2D3

(1,25-dihydroxyvitamin D3

or calcitriol)-induced

heterodimerization

between VDR and the

retinoid X receptor (RXR).

1,25(OH)2D3 (ligand)

binds to VDR,

heterodimerizes with the

RXR and binds to a DR-3

type of vitamin D-response

element (VDRE) in the

promoter region of

1,25(OH)2D3-responsive

genes (Adapted from

Dowd and MacDonald

[76b])



J. Bover et al.



1,25(OH)2 D3



V

D

R

Heterodimerization

R V

X D

R R

DNA binding

R V

X D

R R

VDRE



RNA

Polymerase II



serves as a dimerization interface for interaction with the previously mentioned

partner proteins [6, 76] (see Fig. 4.2). VDR binding 1,25(OH)2D3 results in the generation of RXR heterodimers for specific DNA binding and coactivator docking for

transcriptional activation [8, 16, 76b] (Figs. 4.2 and 4.3). At physiologic receptor

concentrations and ionic strength, binding of VDR to VDREs will occur only when

both 1,25(OH)2D3 and RXR are present [16, 77]. Furthermore, it has been found

that in an in vitro transcription system containing native chromatin, inclusion of

RXR is essential for transactivation of 1,25(OH)2D3 via VDR [16, 78]. The key

event in this process is the binding of a ligand (e.g. 1,25(OH)2D3) to the VDR

(Fig. 4.4) [16].

Several steps are apparently set in motion by the ligand-binding event. The general fold of nuclear receptor LBD comprises a three-layered α-helical sandwich, and

LBDs undergo major conformational changes upon ligand binding [79]. The X-ray

crystallographic structure of the VDR-LBD was originally considered to consist of

12-α helices (and a set of β-sheets) [80] but recently it has been suggested that it in

fact contains 15-α helices [16]. VDR-LBD is a sandwich-like structure that presents

VDR surfaces for heterodimerization with RXR as well as for transactivation via

interaction with coactivators (see Fig. 4.2 and below) [8]. Coactivator interfaces in

VDR consist of portions of helices H3, H5 and the previously mentioned H12 (constituting the AF-2 domain), as well as a region immediately N-terminal of the Zn

fingers [8, 16] (Fig. 4.2). There is a pocket in the middle layer of the sandwich fold

[81]. The presence of 1,25(OH)2D3 ligand in the VDR-binding pocket causes a dramatic conformational change in the position of the most carboxy-terminal α-helix at

the C-terminus of the VDR. As a result, the ligand-binding pocket is brought, via a

“mouse-trap like” intramolecular folding, to the “closed” position, after which it



4



Vitamin D Receptor and Interaction with DNA



N



85



DBD



H-12 AF-2 C

LBD



a VDR activation



1,25(OH)2D3



Coactivator

P



RXR



VDR



VDRE



VDRE



Corepressor



b VDR repression



VDR



1,25(OH)2D3



RXR



VDRE



VDRE



Fig. 4.4 1,25(OH)2D3 (1,25-dihydroxyvitamin D3 or calcitriol): potential mechanisms of gene

activation and repression through VDR. (a) Upper panel: RXR-VDR activation after 1,25(OH)2D3

and coactivator binding, phosphorylation and docking on a positive vitamin D-response element

(VDRE) (i.e. osteocalcin gene). (b) Lower panel: VDR-RXR inactivation after 1,25-(OH)2 D3 and

corepressor binding, dephosphorylation and docking in reverse polarity on a negative vitamin

D-response element (VDRE) (i.e. PTH gene) (Adapted from Haussler et al. [16])



can serve in its AF-2 role as part of a platform for coactivator binding [2, 16, 82]

(Fig. 4.4). In other words, H12 folds onto the core of LBD, forming an hydrophobic

cleft together with other surface-exposed residues which accommodate the “nuclear

receptor box” of coactivators (see below) [73]. Ligand-intensified heterodimerization, VDRE docking, and coactivator recruitment by VDR appear functionally

inseparable in effecting 1,25(OH)2D3-elicited gene transcription. The binding of

VDR conformationally influences its RXR heteropartner and appears to cause the

RXR-AF2 region to pivot into an active position (Fig. 4.4) [16]. The RXR of the

heterodimer may now be endowed with the potential to bind additional comodulators [16].

Since the first determination of the crystal structure of the LBD complexed with

1,25(OH)2D3 was reported in 2000 [80], several dozens of crystal structures accommodating various ligands have been presented. Almost all of them display the

canonical active conformation observed in the VDR-LBD/1,25(OH)2D3 complex,

and they all have quite similar ligand binding pocket architectures within the LBD

described above. The ligand binding pocket refers to the inner surface of the



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J. Bover et al.



VDR-LBD forming a cavity that is created by a network of approximately 40 mostly

non-polar amino acids [83, 84]. The fact that VDR-LBD has many α-helices and is

a densely-packed structure means that its structural modification by ligand binding

is difficult [81]. Several analogs modulating this structure are currently opening a

new perspective for the development of VDR ligands exhibiting specific biological

activities by focusing on the loop region facing the LBP because loop regions in

protein tertiary structure are in general more flexible than regions composed of

α-helices and/or β-sheets [81].



4.5



Vitamin D Response Elements and VDR Function



In 1989, the sequence elements in the human osteocalcin gene conferring basal

activation and inducible response of this gene promoter to hormonal 1,25(OH)2D3

were described [85]. Since then, in addition to classical targets, many genes have

been explored for their sensitivity to vitamin D. Recently, at least 50 VDREs residing in or near vitamin D target genes that are directly modulated in their expression

by 1,25(OH)2D3 (and possibly other VDR ligands) have been identified [16],

although microarray studies suggest that the number of vitamin D-regulated genes

is in fact far greater. In some cases VDREs can be even 100 kb and more, either upor downstream from the transcription start site.

A typical “optimal” VDRE is characterized by a direct repeat of two hexanucleotide

half-elements (e.g. repeats of AGGTCA resembling the estrogen-responsive element)

that are separated by a spacer of three nucleotides (DR3) [2, 16, 86, 87]. Although the

sequence of the spacer is not generally conserved, it has recently been suggested that

it may modulate DNA binding and transactivation by the VDR in cellular response to

natural and synthetic ligands [88]. Thus, the multiple sequence variations in natural

VDREs may provide a range of affinities for the VDR/RXR heterodimer, thereby

enabling these elements to respond to different concentrations of the receptors or their

ligands [2, 16, 88]. It is also possible that other VDRE sequences (e.g. DR4, DR6,

everted repeats) induce unique conformations in the VDR/RXR complex, promoting

association of the heterodimer with distinct subsets of coactivators or permitting differential actions in the context of diverse tissues [2, 88, 89].

Every transcriptionally responsive primary VDR target gene must contain at

least one VDRE in its promoter region and these VDREs are often located relatively

close to the transcription start site of these genes (Fig. 4.5) [2]. VDRE clusters are

also described (e.g. for CYP24A1, the most responsive known primary 1,25(OH)2D3

target gene), the strong responsiveness being explained by the presence of two DR3

type VDREs that are separated by less than 100 bp and are located in close proximity to the transcription start site (Fig. 4.5) [2]. The concept of multiplicity and

remoteness to VDREs has also evolved, leading to the identification of novel VDREs

at some distance from the transcription start site [2, 16]. Remote VDREs may be

juxtapositioned with more proximal VDREs by way of DNA looping in chromatin,

creating a single platform to support the transcription machine [16]. Thus, distal



4



Vitamin D Receptor and Interaction with DNA



Fig. 4.5 Schematic

structure of a chromatin unit

(region between two matrix

attachment regions which

often contains only one

gene). DNA looping should

enable DNA sequences

within the same unit to be

located near the basal

transcriptional machinery.

VDRE vitamin D-response

element (Adapted from

Carlberg [2])



87



VDRE

V R

D X

R R

Translation start site

RNA

Polymerase II



AGT



TATA



R

X

R



V

D

R



VDRE



sequences can also serve as VDREs and even sequences downstream of the

transcription start site may act as functional VDR-binding sites [2]. In fact, most

primary VDR target genes use multiple VDREs for full functionality [2]. For

instance, in genes possessing multiple VDREs, such as RANKL, the chromatin

looping permits simultaneous binding of multiple factors in a supercomplex at the

promoter [16]. Typically, these VDREs are arranged in the proximity of binding

sites for other transcription factors into collections of neighboring sites, so-called

modules or enhancers [2]. It has been demonstrated that modules of transcription

factors that act on focused regions are far more effective than individual factors at

isolated locations [2].

1,25(OH)2D3 may also induce gene repression to genes encoding, for instance,

PTH, PTHrP and CYP27B1 (also repressed by FGF23). This negative feedback

loop limits the stimulation of CYP27B1 by PTH under low-calcium conditions,

serving to restrict the bone-resorbing effects of PTH in anticipation of 1,25(OH)2D3

increases in intestinal Ca absorption and bone resorption; as a result, hypercalcemia

is prevented. Expression profiling using whole genome microarrays indicates that a

similar number of genes are down- and up-regulated by 1,25(OH)2D3 [90]. Liganddependent repression of gene transcription by VDR/RXR probably shares some

molecular features with induction, but it is evident that it is much more complex

mechanistically since it seems to occur via multiple routes. In general, the mechanisms of down-regulation by 1,25(OH)2D3 also appear dependent upon the binding

of a VDR agonist. It is clear that only genes showing basal activity can be downregulated and that such genes exhibit basal activity because of other transcription

factors bound to their promoters [2]. Several different models have attempted to



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explain the down-regulation of genes, all of which posit that VDR counteracts the

activity of specific transcription factors. For instance, for the physiologically important down-regulation of the CYP27B1 gene by 1,25(OH)2D3, a negative VDRE has

been proposed to interact directly with a prebound transcriptional activator termed

VDIR [2, 6]. The VDR suppresses VDIR-activated expression but active epigenetic

mechanisms are also involved [6]. Thus, CYP27B1 is repressed by 1,25(OH)2D3

with a regulation influenced by epigenetic demethylation [91] in order to limit the

production of 1,25(OH)2D3. In an outstanding example of a biological counteregulatory feed-back loop, PTH is repressed by 1,25(OH)2D3 and Ca, whereas FGF23 is

induced by 1,25(OH)2D3 and P; the consequence is protection of mammals against

hypercalcemia and hyperphosphatemia, respectively, avoiding ectopic calcification.

Obviously, Ca, P and 1,25(OH)2D3 also inhibit the activities of VDR-dependent

enzymes such as CYP27B1 and the catalytic CYP24A1 [92].

In situations in which the activating transcription factors are other nuclear receptors or transcription factors that bind to composite nuclear receptor response

elements, VDR might simply compete for DNA-binding sites. Similarly, VDR

could compete for binding to partner proteins such as RXR or for common coactivators [such as steroid receptor coactivator-1 (SRC-1) or CBP, see below] [2]. The

recruitment of nuclear receptor corepressors to alter the chromatin structure in the

vicinity of the target gene is a further possibility (see below) [16]. Repressor functions may be carried out by “negative VDREs”, where liganded VDR is apparently

conformed in such a way that it binds corepressors rather than coactivators.

Experiments suggest that the DNA binding of the VDR to down-regulated genes

such as the PTH gene does not involve RXR [93], although neither is it a uniform

mechanism. Because it appears that non-consensus nucleotides in negative VDREs

occur in either or both half-elements, it has been proposed that base-pair changes

may suffice to drive RXR-VDR into reverse polarity on the negative VDRE [16]. On

the other hand, VDR may also be prone to protein-phosphatase rather than proteinkinase activity in this conformation, again favoring corepressor attraction [16, 94].

Furthermore, the regulatory region of primary 1,25(OH)2D3 may contain both negative and positive VDREs (e.g. human myc gene) [2]; liganded VDR thus transactivates or represses numerous target genes by binding to positive or negative VDRE

present in promoters, enhancers or suppressors of these genes [95, 96]. Finally, it is

interesting that classical steroidal nuclear receptors exist as complexes in the cytoplasm with heat shock factor proteins and therefore their transcriptional actions are

largely mediated by ligand-induced release from this complex and shuttling into the

nucleus [97]. The VDR differs from such receptors by being located in the nucleus

even in the absence of ligand and controls gene expression by switching between

repressing and activating states, in accordance with availability of ligand [97]. Thus,

the distribution of genomic binding sites, or so-called cistrome, of the VDR is biologically significant in both the absence and the presence of even low levels of

ligand [97].

A growing number of transcriptional/epigenetic factors that coregulate transcriptional functions of nuclear receptors have been identified, and further novel coregulators, particularly those involved in transrepression, are expected to be found [98].



4



Vitamin D Receptor and Interaction with DNA



89



A review of the regulation of the VDR gene itself by environmental, genetic and

epigenetic mechanisms, rather than the way in which VDR regulates other genes, is

beyond the scope of this chapter, and we encourage the reader to read recent publications on the subject [99].



4.5.1



Retinoid X Receptor and Coregulators



Nuclear receptor-binding sites can be categorized into three groups: (1) palindromic

half-sites that interact with homodimeric receptors for the sex-steroids; (2) directly

repeated half-sites with variable spacing that interact with heterodimeric receptors for

1,25(OH)2D3, retinoic acid, thyroid hormone and other ligands; and (3) single halfsites that mediate the actions of monomeric receptors (e.g. nerve growth factor IB)

[100]. The length of the spacer itself is a primary determinant of nuclear receptor

selectivity for 1,25(OH)2D3, retinoic acid, etc. Thus, the VDR preferred half-sites are

separated by 3 bp (the previously mentioned DR3) [87], in contrast to the retinoic

acid receptor (2 or 5 bp) and the thyroid receptor (4 bp). The DR3 percentage differs

significantly in the analyzed cellular models, ranging between 38.2 % in macrophages

and 9 % in B cells [84]. Interestingly, in every cell type investigated, the top 200 VDR

sites show a DR3 rate exceeding 60 %, e.g., DR3 type motifs are found preferentially

at highly ligand-responsive VDR loci and may be the first to be addressed by a therapeutic intervention with a synthetic VDR ligand [84].

It has already been mentioned that high-affinity binding of the VDR to DNA

in vitro requires the DNA-binding domain and the LBD. Moreover, high-affinity

VDRE binding requires the ability of the receptor to form dimers, and in the case of

VDR it was a heterodimer with a “nuclear accessory factor”, as it was initially

termed. This was later identified as the RXR [6, 101], a previously cloned member

belonging also to the nuclear receptor family. Both VDR and RXR subunits are

necessary not only for high-affinity DNA binding, but also for the activation process

itself [8, 16] (see Figs. 4.2 and 4.4). Only this liganded VDR/RXR can penetrate the

deep groove of DNA and recognize VDREs in the DNA sequence of vitamin-Dregulated genes. Target genes recognized by the combined Zn fingers of the two

receptors and their T-box and A-box C-terminal extensions (see Fig. 4.2) [8] obviously encode proteins which determine the diverse 1,25(OH)2D3 functions mentioned in this chapter and elsewhere in the book [16].

This binding to the VDRE (DR3-VDRE actually) must follow a defined polarity

(RXR binds to the upstream 5′-half-element and the VDR binds to the downstream

3′-half-element of the VDREs oriented in the DNA sense strand) [6] (see Fig. 4.3).

In this way, the interaction of the liganded VDR/RXR and a VDRE confers target

gene selectivity and ultimately exerts an influence on the rate of RNA polymerase

II-directed transcription [86]. Moreover, genomic studies have indicated that the

VDR is not bound in advance (“prebound”) to sites on vitamin D target genes;

rather it is induced to bind owing to 1,25(OH)2D3-mediated activation and RXR

heterodimerization [6]. On the other hand, significant binding of the VDR and its



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J. Bover et al.



partner RXR has been observed at various sites (e.g. bone cells) in the absence of

1,25(OH)2D3. Whether the “prebound” VDR/RXR heterodimer manifests activity

is unknown [6]. In any case, in the absence of ligand, the VDR/RXR complex

associates with corepressors (see below) at enhancer and promoter regions to

silence gene expression. The binding of ligand induces formation of a coactivator

complex (see below), leading to target gene transactivation via direct and indirect

mechanisms [97].

With respect to the involvement of coregulators or comodulators (corepressors

and coactivators) in transactivation by the VDR and RXR (see Fig. 4.4) [16], it is to

be noted that more than 250 published coregulators are known to interact with nuclear

receptors and to modify their transactivation potential [86, 102]. Thus, a primary

function of the VDR/RXR heterodimer is the recruitment of coregulatory complexes

containing enzyme activities essential for the modulation of events associated with

gene products. Transactivation requires DNA -binding and ligand-intensified heterodimerization, VDRE docking by recognition of direct repeat responsive elements

in the promoters of regulated genes and creation of an intact functional AF-2 domain

as part of a platform for coactivator binding (Figs. 4.2 and 4.4) [8, 16]. We have

already mentioned that activation of the VDR involves a 1,25(OH)2D3-dependent

conformational change in the LBD.

The regulation of VDR-mediated transcription involves a series of temporally

coordinated macromolecular interactions between the VDR/RXR heterodimer and

other transcription factors [86]. Associations between the liganded VDR/RXR heterodimer and other transcriptional components can be classified into two general

categories, namely general transcription factors and the coregulatory proteins [86].

Interaction of VDR with general transcription factors [important examples of which

are TATA-binding protein associated factors (TAFs) and basal transcription factors

such as TFIIB [16, 86]; (see Fig. 4.2) [8] leads to direct contact with the transcriptional preinitiation complex (PIC), which may facilitate assembly or recruitment of

the PIC, thereby stimulating transcription by the RNA polymerase II [86]. The

liganded VDR is also linked to the transcriptional PIC by the likely sequential

recruitment of multiple coregulators, proteins which modulate, either positively or

negatively, the ability of nuclear receptors to regulate transcription. As mentioned

previously, coregulators are classified as coactivators and corepressors. Coactivator

proteins may augment transcription via any of several proposed mechanisms [86]:

(1) by acting as macromolecular bridges between the liganded receptor and the

transcriptional machinery (by recruiting components of the PIC, assembling the

PIC or promoting the stability of the complex); (2) by recruiting secondary coactivators which possess chromatin modification or remodeling activities, such as histone acetyl transferase (HAT), histone deacetylase, methyl transferase activities,

and other ATP-dependent alterations in plasticity of chromatin structure and rearrangement of nucleosomal arrays [103]; and (3) by increasing the rate of coupling

between RNA polymerase II-directed transcription and more downstream events,

e.g. transcription elongation and RNA processing [86]. While primary coactivators

interact directly with nuclear receptors, secondary coactivators interact with primary coactivators to regulate transcription [86].



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Vitamin D Receptor and Interaction with DNA



91



Corepresors

R V

X D

R R



Ac



VDRE



Coactivators

R V

X D

R R

VDRE



RNA pol II

TATA



Ac



Ac

Ac



Fig. 4.6 Comodulator activity (coactivators and corepressors) on VDR-mediated expression. The

VDR/RXR heterodimer is loosely bound to a vitamin D-response element (VDRE) in the absence

of ligand, interacting with corepressors which keep gene transcription repressed (partially keeping

histone proteins deacetylated). Upon ligand-binding, corepressor is released and replaced by

coactivators. Coactivators remodel chromatin and help in the recruitment of RNA polymerase II

and other components of the preinitiation complex. Ac Acetylation/deacetylation (Adapted from

Dowd and MacDonald [76b])



On the other hand, corepressors are usually considered to be proteins that interact with unliganded nuclear receptors, thereby repressing the basal expression of

hormone-responsive genes. These corepressors are distinct from other transcriptional repressors that interfere with nuclear receptors via different mechanisms.

They directly modify histones or recruit modifying enzymes (e.g. histone deacetylases), ensuring that chromatin is maintained in a tightly packed condition, thus

silencing transcription from the promoter. They also associate with antagonists

bound to the nuclear receptor to inhibit target expression [76b, 86] (Fig. 4.6). At

later stages, coregulators may also promote the recruitment or stability of the RNAprocessing machinery, enhancing the rate at which mature RNAs are made and subsequently translated [86].



4.5.1.1



Coactivators of VDR



Binding of 1,25(OH)2D3 to the VDR sets in motion a cascade of protein assembly

that ultimately leads to transcriptional activation of selected target genes. Ligandinduced coactivator recruitment to the VDR/RXR heterodimer acts as the seed for

the assembly of intricate multiprotein complexes. These complexes remodel the

chromatin structure and recruit core transcriptional machinery in an orderly and

sequential manner [86]. The transfection of many of these factors to cells strongly

enhances 1,25(OH)2D3-induced transcription [6], and all are needed for robust

VDR-mediated transcription. As mentioned previously, many of the factors (though

not all) interact with the same region of VDR, i.e. the C-terminal AF-2 motif [16]

(see Figs. 4.2 and 4.4). More than 50 nuclear proteins are known to interact with

VDR LBD [104]. Chromatin has an intrinsic repressive potential, the purpose of

which is conservation of the epigenetic landscape of a differentiated cell, i.e. by

default it restricts the access of transcription factors to promoter or enhancer regions,

leaving only approximately 50–100,000 accessible chromatin regions per cell type

[84, 105]. Stimulation with 1,25(OH)2D3 results in a significant increase in chromatin accessibility [106].



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3 Molecular Actions of Vitamin D: The Vitamin D Receptor

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