Controlling the chromatin organization of vitamin D target genes by multiple vitamin D receptor binding sites

Journal of Steroid Biochemistry & Molecular Biology 103 (2007) 338–343

Controlling the chromatin organization of vitamin D target genes by multiple vitamin D receptor binding sites Carsten Carlberg ∗ , Thomas W. Dunlop, Anna Saram¨aki, Lasse Sinkkonen, Merja Matilainen, Sami V¨ais¨anen Department of Biochemistry, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland Received 30 November 2006

Abstract An essential prerequisite for the direct modulation of transcription by 1␣,25-dihydroxy vitamin D3 (1␣,25(OH)2 D3 ) is the location of at least one activated vitamin D receptor (VDR) protein close to the transcription start site of the respective primary 1␣,25(OH)2 D3 target gene. This is achieved through the specific binding of VDR to a 1␣,25(OH)2 D3 response element (VDRE). Although these elements are well characterized in vitro, the function of VDREs in living cells in the context of chromatin is still largely unknown. To resolve this issue, approximately 8 kB of the promoter regions of the primary 1␣,25(OH)2 D3 target genes CYP24, cyclin C and p21(Waf1/Cip1) were screened by chromatin immunoprecipitation (ChIP) assays for VDR binding sites using antibodies against VDR and its partner proteins. This approach identified three to four functional VDREs per gene promoter. In parallel, in silico screening of the extended gene areas (i.e. 10 kB of promoter, introns, exons and 10 kB of the downstream region) of all six members of the insulin-like growth factor binding protein (IGFBP) gene family was performed. Gel shift, reporter gene and ChIP assays identified in total 10 functional VDREs in the genes IGFBP1, IGFBP3 and IGFBP5. Taken together, both screening approaches suggest that a reasonable proportion of all VDR target genes, if not all, are under the control of multiple VDREs. © 2007 Elsevier Ltd. All rights reserved. Keywords: Vitamin D; Vitamin D receptor; Gene regulation; Chromatin; Response element; Nuclear receptor

1. The nuclear receptor superfamily Nuclear receptors represent an important transcription factor family [1]. The 48 human members of this superfamily are the best characterized genes of approximately 3000 mammalian genes that are involved in transcriptional regulation [2]. Nuclear receptors modulate genes that affect processes as diverse as reproduction, development, inflammation and general metabolism. They have a modular structure onto which Abbreviations: 1␣,25(OH)2 D3 , 1␣,25-dihydroxy vitamin D3 ; ChIP, chromatin immunoprecipitation; CoA, co-activator; CoR, co-repressor; CYP, cytochrome P450; DR, direct repeat; ER, everted repeat; IGFBP, insulin-like growth factor binding protein; LBD, ligand-binding domain; PPAR, peroxisome proliferator-associated receptor; RE, response element; RXR, retinoid X receptor; TSS, transcription start site; VDR, vitamin D3 receptor; VDRE, 1␣,25(OH)2 D3 response element ∗ Corresponding author. Tel.: +358 17 163062; fax: +358 17 2811510. E-mail address: [email protected] (C. Carlberg). 0960-0760/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2006.12.044

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 (CoA) 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 (LBD), 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 LBD consists of 250–300 amino acids in 11–13 ␣-helices [3]. Ligand binding causes a conformational change within the LBD, whereby, at least in the case of endocrine nuclear receptors, helix 12, the most carboxy-terminal ␣-helix, closes the ligand-binding pocket via a “mouse-trap like” intramolecular folding event [4]. The LBD is also involved in a variety of interactions

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with nuclear proteins, such as other members of the nuclear receptor superfamily and co-regulator proteins. Nuclear receptors mostly regulate transcription by binding directly to specific response elements (REs), either as a monomers, homo- or heterodimers. They can also indirectly influence transcription through other DNA-binding transcription factors. Vitamin D receptor (VDR), peroxisome proliferators receptors (PPARs) and a number of other nuclear receptors function as heterodimers with one of the three retinoid X receptors (RXRs) ␣, ␦ and ␥ [5]. An essential prerequisite for the direct modulation of transcription by nuclear receptor ligands is the location of at least one activated nuclear receptor protein close to transcription start site (TSS) of the respective primary target gene. This is achieved, in most cases, through the specific binding of the DNAbinding domain of the nuclear receptor 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) [6]. Heterodimeric RXR-nuclear receptor complexes bind to two core binding motifs in a direct repeat (DR)-type orientation with 1–5 intervening nucleotides [7,8]. The spacing nucleotide number depends on the particular RXR binding partner. For example, PPARs bind to DR1-type REs [9], whilst VDR binds to DR3-type REs [10]. In addition, VDR also binds to DR4-type REs along with other members of the superfamily [11]. It should also be noted that, effective VDR binding has also observed on everted repeat (ER)-type REs with 6–9 spacing nucleotides (ER6, ER7, ER8, ER9) [12,13].

2. Chromatin and cofactors The major protein constituents of chromatin are the four different histones that form a nucleosome, on to 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 [14]. 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. More than 10 specific modifications of histones are known to date, but it has been found that the acetylation of the lysine at position 8 of histone 4 correlates most strongly with the activation of chromatin on a promoter preceeding the initiation of transcription [15]. Therefore, in most cases, the histones associated with active regions of promoters have higher a degree of acetylation at certain positions than in repressed or silent regions. Presently, most studies on transcriptional regulation have concentrated on isolated promoter regions or proximal promoters, where binding sites of nuclear receptors and other transcription factors have been localized [16]. When nuclear receptors are bound to REs in the regulatory regions of their target genes, they recruit positive and

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negative co-regulatory proteins, referred to as CoAs [17] and co-repressors (CoRs) [18], respectively. In a simplified view of nuclear receptor signaling, in the absence of ligand, the nuclear receptor interacts with CoR proteins, such as NCoR, SMRT and Alien, which in turn associate with histone deacetylases leading to a locally increased chromatin packaging [19,20]. The binding of ligand induces the dissociation of the CoR and the association of a CoA of the p160-family, such as SRC-1, TIF2 or RAC3 [21]. Some CoAs have histone acetylase activity or are complexed with proteins harboring such activity and this results in the net effect of local chromatin relaxation [22]. In a subsequent step, ligand-activated nuclear receptors change rapidly from interacting with the CoAs of the p160-family to those of mediator complexes, such as Med1 [23]. The mediator complexes which consist of approximately 15–20 proteins build a bridge to the basal transcription machinery [24]. In this way ligand-activated nuclear receptors execute two tasks, the modification of chromatin and the regulation of transcription. Most co-regulators are not exclusive to nuclear receptors and are used in a similar manner by other transcription factors. Based on their mode of action, co-regulators can be classified into two main groups [25]. The first group contains factors that covalently modify histones, such as acetylation/deacetylation and methylation/demethylation, a process that follows a precise, combinatorial histone code [14]. The second group of co-regulators includes ATP-dependent chromatin remodeling factors that modulate promoter accessibility to transcription factors and to the basal transcriptional machinery [26]. The complex network of co-regulators can be viewed as defining a co-factor code being characterized by distinct patterns of co-regulator recruitment and by their regulated enzymatic activities. The histone code can therefore be considered to be in a continuum with the co-factor code, as histones are crucial targets for the enzymatic activities of co-regulators but also have a key role in specifying co-regulator recruitment on the basis of the reading of the histone code. Cell- and time-specific expression patterns of some coregulators can produce distinct modalities of nuclear receptor transcriptional activity due to differences in the relative CoR and CoA protein levels. This aspect may have some diagnostic and therapeutic value in different types of cancer [27]. However, the switch between gene repression and activation is more complex than a simple alternative recruitment of two different regulatory complexes. 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 CoAs and CoRs to ligandbound and -unbound nuclear receptor, respectively. Other co-regulators appear to bind in a ligand-independent fashion. Additionally, ourselves and others have observed that both CoAs and CoRs simultaneously associated with promoter regions participating in ligand-dependent responses. Therefore, repression and activation is more likely achieved

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by a series of sequential events that are mediated by multiple enzymatic activities that are promoter and cell-type specific. Transcriptional regulation is a highly dynamic event of rapid association and dissociation of proteins and their modification, 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 [16]. This study revealed detailed and coordinated patterns of co-regulator recruitment and preferential selectivity for factors that have similar enzymatic activities. Interestingly, some co-regulators seemed to be redundant and different family members were equally capable of being recruited alternately to the promoter.

3. In silico screening for nuclear receptor REs Statistically, the nuclear receptor consensus binding motif RGKTSA should be found, on average, in every 256 bp of genomic DNA. Furthermore, dimeric assemblies of such hexamers should show up as DRs every 65,536 bp and as ERs every 32,768 bp in a random sequence. Therefore, an in silico screen of the human genome would identify for every nuclear receptor, on average, 50,000–100,000 putative strong REs. In addition, natural REs often contain variations from the consensus binding motif and therefore the number of putative weaker binding sites may be an order of magnitude larger. Since nuclear receptor proteins have an abundance of, at most, a few thousand molecules per cell, a realistic number of nuclear receptor target genes per tissue should be closer to this number. If we also consider the fact that many nuclear receptor target genes appear to have more than one functional RE for any given nuclear receptor, it could be expected that the real number of nuclear receptor target genes in any cell type is much less than the number of nuclear receptor molecules. These calculations make it obvious that not every putative nuclear receptor RE is used in nature in any cell at any given time. The most obvious reason is that most of these sequences are covered by nucleosomes in some repressed way, so that they are not accessible to the nuclear receptor. This applies in particular to those sequences that are either contained within interspersed sequences, or located isolated from binding sites for other transcription factors or lie outside of insulator sequences marking the border of chromatin loops [28]. This perspective strongly discourages the idea that isolated nuclear receptor REs 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. An interesting 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 [29] acting as a link to the nuclear scaffold. By contacting CoA proteins and HATs, Runx2/Cbfa1

seems to mediate the opening of chromatin locally, which allows the efficient binding of VDR-RXR heterodimers to this decondensed region to occur. A detailed knowledge about the individual bindingsequence preference of a given nuclear receptor is of critical importance for an effective prediction of a nuclear receptor RE’s functionality. Experimental studies, such as series of gel shifts with a large number of natural REs [30–33], and systematic screens to check different consensus variants are essential to create specific affinity matrices that include information about the relative binding strength of a nuclear receptor within a dimeric complex. In a nuclear receptor affinity matrix not only its hexameric core binding sequences but also its preference for 5 -flanking dinucleotides should be taken into account [34,35]. Multidisciplinary efforts to develop tools and characterize the regulatory component of the human genome, such as the ENCODE project [36], are improving the conditions. Moreover programs, such as TRANSFAC [37], have the capability to identify larger numbers of REs for different transcription factors making the identification of potential complex REs possible. However, even these programs are unable to predict chromatin condensation states and nucleosome positioning, which are essential pieces of information in determining the likelihood that a given RE lies in an accessible region of a promoter. Therefore, general parameters, such as nucleosome positioning, interspecies homology screening of regulatory sequences as well as the binding sites of all other transcription factors and their cell-specific expression patterns have to be included in more effective versions of in silico screening software to more efficiently predict the in vivo functionality of REs.

4. The VDR and its target genes The biologically most active vitamin D3 metabolite, 1␣,25(OH)2 D3 , mediates its genomic effects via the VDR, which is the only nuclear protein that binds the nuclear hormone with high affinity (Kd = 0.1 nM) [6]. 1␣,25(OH)2 D3 is known to be essential for mineral homeostasis and skeletal integrity due its regulation of Ca2+ -transporting proteins and bone-forming enzymes, such as calbindin D9K , osteocalcin and osteopontin [38]. The VDR is widely expressed and can detected in nearly all human tissues suggesting a broader role for the receptor. Interestingly, 1␣,25(OH)2 D3 has also been shown to have cell-specific anti-proliferative, pro-differentiating and pro-apoptotic effects [39]. The cyclindependent kinase inhibitor p21(waf1/cip1) gene was suggested first by Jiang et al. [40] to be a key gene for understanding the anti-proliferative action of 1␣,25(OH)2 D3 . Another interesting 1␣,25(OH)2 D3 -target gene is cyclin C. The cyclin C-CDK8 complex was found to be associated with the RNA polymerase II basal transcriptional machinery [41] and is considered as a functional part of those mediator protein complexes that are involved in gene repression [42]. The fact that the cyclin C gene, being located in chromosome

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6q21, is deleted in a subset of acute lymphoblastic leukemias, suggests its involvement in tumorigenesis [43]. Catabolism of vitamin D3 metabolites is initiated by the widely expressed enzyme, 25(OH)D3 -24-hydroxylase, which is encoded by the gene CYP24. This gene, whose steady state mRNA expression level is very low in the absence of ligand, is up to 1000-fold induced by stimulation with 1␣,25(OH)2 D3 [44]. Most other known primary 1␣,25(OH)2 D3 target genes, such as cyclin C and p21(waf1/cip1) , often show an inducibility of 2-fold or less after short-term treatment with 1␣,25(OH)2 D3 [45,46]. However, both genes have 10,000- to 100,000-fold higher basal expression levels compare to that of the CYP24 gene. Therefore, when the relative levels are taken into account, 2- to 20-fold more cyclin C and p21(waf1/cip1) than CYP24 mRNA molecules are produced after induction with 1␣,25(OH)2 D3 . To understand the regulation of the CYP24, cyclin C and p21(waf1/cip1) genes by 1␣,25(OH)2 D3 in more detail, we analyzed 7.1–8.4 kB of their promoters using chromatin immunoprecipitation (ChIP) assays, using in each case a set of 20–25 overlapping promoter regions [44,47,48]. The spatio-temporal, 1␣,25(OH)2 D3 -dependent chromatin changes in the three gene promoters was 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 CYP24 and cyclin C genes and three VDRE containing regions in the p21(waf1/cip1) gene promoter. However, most of them are simultaneously under the control of other transcription factors, such as p53 in case of the p21(waf1/cip1) gene [48] and therefore possess significant basal levels of transcription. Consequently, the fold induction afforded by 1␣,25(OH)2 D3 stimulation is much less than for the CYP24 gene, despite the large numbers of new mRNA molecules which may be produced. An alternative approach was performed with the six members of the insulin-like growth factor binding protein (IGFBP) gene family, where first an in silico screen was performed, which was then followed by the analysis of candidate 1␣,25(OH)2 D3 -responsive sequences by gel shift, reporter gene and re-ChIP assays [49]. Induction of gene expression was confirmed independently using quantitative real-time PCR techniques. By using this approach, the genes IGFBP1, 3 and 5 were demonstrated to be primary 1␣,25(OH)2 D3 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 in the cell lines used. 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 [50], but involved up to 10 kB of flanking sequences as well as intronic and intergenic sequences. Therefore, this approach revealed candidate VDREs that are

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located more than 30 kB distant from their target gene TSS. Based on the present understanding of enhancers, DNA looping and chromatin units being flanked by insulators or matrix attachment sites these distances are not limiting [51]. Although, individual REs have been shown to be able to induce transactivation on their own, multiple, complex VDREs, as now observed in the primary VDR target genes discussed above [44,47–49], seem to favor the prominent response of the gene’s transcription to VDR. Even if the in vitro DNA-binding affinity of VDR-RXR heterodimers to these VDREs differs (compare [44,47–49]), at the chromatin level all VDRE-containing promoter regions show comparable association strength with VDR and RXR. Each of the multiple 1␣,25(OH)2 D3 -responsive promoter regions is able to contact independently the basal transcriptional machinery. This suggests that the simultaneous communication of the individual promoter regions with the basal transcription machinery occurs through a discrete three-dimensional organization of the promoter within the nucleus of a cell. Such an arrangement would therefore allow the close contact of distant regions [52]. This could provide an alternative explanation for different transcription factors complexes that were recruited over time via the estrogen receptor to the proximal promoter of the trefoil factor-1 gene [16]. A time-dependent association and dissociation of the chromatin regions carrying the different REs via dynamic chromatin looping of a promoter may then result in a rather different composition of the protein complexes associated with the TSS.

5. Cross talk between VDR and PPAR signaling Recently, we found that the human PPARδ gene is a primary 1␣,25(OH)2 D3 target with a potent DR3-type VDRE in close proximity to the TSS [53]. PPAR␦ and VDR are widely expressed and an apparent overlap in the physiological action of both receptors is their involvement in the regulation of cellular growth, particularly in neoplasms. Prostate and breast cancer cells display a spectrum of sensitivities to the anti-proliferative action of 1␣,25(OH)2 D3 [54] and the levels of expression of a number of genes, including that of VDR [55], CYP24 [56] and the CoR SMRT [27], have been used as markers for the efficacy of the treatment of a cancer with VDR agonists. Epidemiological studies have linked the incidence of prostate and breast cancer to low serum levels of 25(OH)D3 as a result of diet and environment [57,58] and the initiation or progression of these cancers seems to relate to reduced dietary intake and/or cellular resistance to the anti-proliferative effects of 1␣,25(OH)2 D3 . High PPARδ expression in tumor seems to be positive for the prognosis of the respective cancer [59]. In this way the upregulation of PPARδ expression can be considered as an supportive action and part of the mechanism of the anti-proliferative action of 1␣,25(OH)2 D3 and its synthetic analogues. Another potential area of overlap for these two nuclear receptors is metabolism and nutrition. PPARs sense fatty

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acid derivatives where VDR is known to interact with some specific bile acids, oxidized derivatives of cholesterol. Both nuclear receptors are highly expressed in the colon, an organ with an important role in absorbing nutrients from the diet. Therefore, an interaction between these two nuclear receptors in the gut may have important consequences for the body’s ability to handle material acquired from the diet. Therefore, micro- and macronutrient sensing nuclear receptors, such as VDR and the PPARs, are central topics in nutrigenomics [60].

6. Conclusions We have began to establish the intricate network of interactions which are required to mobilize a single gene’s expression in response to 1␣,25(OH)2 D3 . However a much larger challenge lies ahead when we will 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 E.U.funded Marie Curie Research Training Network, NUCSYS (www.uku.fi/nucsys).

Acknowledgement The Academy of Finland, the Finnish Cancer Organisation and the Juselius Foundation supported the work.

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