ABSTRACT: The vitamin D3 metabolite 1?,25-dihydroxyvitamin D3 [1,25(OH)2D3] is the exclusive high-affinity ligand of the vitamin D receptor (VDR), a transcription factor with direct effects on gene expression. Transcriptome- and epigenome-wide data obtained in THP-1 human monocytes are the basis of the chromatin model of vitamin D signaling. The model describes, how VDR's spatio-temporal binding profile provides key insight into the pleiotropic action of vitamin D. The transcription of some 300 primary target genes is significantly modulated through the action of genomic VDR binding sites in concert with the pioneer transcription factor PU.1 and the chromatin organizer CTCF. In parallel, the short-term vitamin D intervention study VitDbol (NCT02063334) was designed, in order to extrapolate insight into vitamin D signaling from in vitro to in vivo. Before and 24?h after a vitamin D3 bolus chromatin and RNA were prepared from peripheral blood mononuclear cells for epigenome- and transcriptome-wide analysis. The study subjects showed a personalized response to vitamin D and could be distinguished into high, mid, and low responders. Comparable principles of vitamin D signaling were identified in vivo and in vitro concerning target gene responses as well as changes in chromatin accessibility. In conclusion, short-term vitamin D supplementation studies represent a new type of safe in vivo investigations demonstrating that vitamin D and its metabolites have direct effects on the human epigenome and modulate the response of the transcriptome in a personalized fashion.
Project description:For many individuals, in particular during winter, supplementation with the secosteroid vitamin D3 is essential for the prevention of bone disorders, muscle weakness, autoimmune diseases, and possibly also different types of cancer. Vitamin D3 acts via its metabolite 1?,25-dihydroxyvitamin D3 [1,25(OH)2D3] as potent agonist of the transcription factor vitamin D receptor (VDR). Thus, vitamin D directly affects chromatin structure and gene regulation at thousands of genomic loci, i.e., the epigenome and transcriptome of its target tissues. Modifications of 1,25(OH)2D3 at its side-chain, A-ring, triene system, or C-ring, alone and in combination, as well as nonsteroidal mimics provided numerous potent VDR agonists and some antagonists. The nearly 150 crystal structures of VDR's ligand-binding domain with various vitamin D compounds allow a detailed molecular understanding of their action. This review discusses the most important vitamin D analogs presented during the past 10 years and molecular insight derived from new structural information on the VDR protein.
Project description:The vitamin D3 metabolite 1?,25-dihydroxyvitamin D3 (1,25(OH)2D3) activates at sub-nanomolar concentrations the transcription factor vitamin D receptor (VDR). VDR is primarily involved in the control of cellular metabolism but in addition modulates processes important for immunity, such as anti-microbial defense and the induction of T cell tolerance. Monocytes and their differentiated phenotypes, macrophages and dendritic cells, are key cell types of the innate immune system, in which vitamin D signaling was most comprehensively investigated via the use of next generation sequencing technologies. These investigations provided genome-wide maps illustrating significant effects of 1,25(OH)2D3 on the binding of VDR, the pioneer transcription factors purine-rich box 1 (PU.1) and CCAAT/enhancer binding protein ? (CEBPA) and the chromatin modifier CCCTC-binding factor (CTCF) as well as on chromatin accessibility and histone markers of promoter and enhancer regions, H3K4me3 and H3K27ac. Thus, the epigenome of human monocytes is at multiple levels sensitive to vitamin D. These data served as the basis for the chromatin model of vitamin D signaling, which mechanistically explains the activation of a few hundred primary vitamin D target genes. Comparable epigenome- and transcriptome-wide effects of vitamin D were also described in peripheral blood mononuclear cells isolated from individuals before and after supplementation with a vitamin D3 bolus. This review will conclude with the hypothesis that vitamin D modulates the epigenome of immune cells during perturbations by antigens and other immunological challenges suggesting that an optimal vitamin D status may be essential for an effective epigenetic learning process, in particular of the innate immune system.
Project description:The molecular basis of vitamin D signaling implies that the metabolite 1?,25-dihydroxyvitamin D3 (1,25(OH)2D3) of the secosteroid vitamin D3 activates the transcription factor vitamin D receptor (VDR), which in turn modulates the expression of hundreds of primary vitamin D target genes. Since the evolutionary role of nuclear receptors, such as VDR, was the regulation of cellular metabolism, the control of calcium metabolism became the primary function of vitamin D and its receptor. Moreover, the nearly ubiquitous expression of VDR enabled vitamin D to acquire additional physiological functions, such as the support of the innate immune system in its defense against microbes. Monocytes and their differentiated phenotypes, macrophages and dendritic cells, are key cell types of the innate immune system. Vitamin D signaling was most comprehensively investigated in THP-1 cells, which are an established model of human monocytes. This includes the 1,25(OH)2D3-modulated cistromes of VDR, the pioneer transcription factors PU.1 and CEBPA and the chromatin modifier CTCF as well as of the histone markers of promoter and enhancer regions, H3K4me3 and H3K27ac, respectively. These epigenome-wide datasets led to the development of our chromatin model of vitamin D signaling. This review discusses the mechanistic basis of 189 primary vitamin D target genes identified by transcriptome-wide analysis of 1,25(OH)2D3-stimulated THP-1 cells and relates the epigenomic basis of four different regulatory scenarios to the physiological functions of the respective genes.
Project description:The physiological functions of vitamin D are mediated by its metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) activating the transcription factor vitamin D receptor (VDR). In THP-1 human monocytes we demonstrated epigenome-wide effects of 1,25(OH)2D3 at 8979 loci with significantly modulated chromatin accessibility. Maximal chromatin opening was observed after 24 h, while after 48 h most sites closed again. The chromatin-organizing protein CTCF bound to 14% of the 1,25(OH)2D3-sensitive chromatin regions. Interestingly, 1,25(OH)2D3 affected the chromatin association of CTCF providing an additional mechanism for the epigenome-wide effects of the VDR ligand. The 1,25(OH)2D3-modulated transcriptome of THP-1 cells comprised 1284 genes, 77.5% of which responded only 24 h after stimulation. During the 1,25(OH)2D3 stimulation time course the proportion of down-regulated genes increased from 0% to 44.9% and the top-ranking physiological function of the respective genes shifted from anti-microbial response to connective tissue disorders. The integration of epigenomic and transcriptomic data identified 165 physiologically important 1,25(OH)2D3 target genes, including HTT and NOD2, whose expression can be predicted primarily from epigenomic data of their genomic loci. Taken together, a large number of 1,25(OH)2D3-triggered epigenome-wide events precede and accompany the transcriptional activation of target genes of the nuclear hormone.
Project description:The biologically active form of vitamin D3, 1?,25-dihydroxyvitamin D3 (1,25(OH)2D3), modulates innate and adaptive immunity via genes regulated by the transcription factor vitamin D receptor (VDR). In order to identify the key vitamin D target genes involved in these processes, transcriptome-wide datasets were compared, which were obtained from a human monocytic cell line (THP-1) and peripheral blood mononuclear cells (PBMCs) treated in vitro by 1,25(OH)2D3, filtered using different approaches, as well as from PBMCs of individuals supplemented with a vitamin D3 bolus. The led to the genes ACVRL1, CAMP, CD14, CD93, CEBPB, FN1, MAPK13, NINJ1, LILRB4, LRRC25, SEMA6B, SRGN, THBD, THEMIS2 and TREM1. Public epigenome- and transcriptome-wide data from THP-1 cells were used to characterize these genes based on the level of their VDR-driven enhancers as well as the level of the dynamics of their mRNA production. Both types of datasets allowed the categorization of the vitamin D target genes into three groups according to their role in (i) acute response to infection, (ii) infection in general and (iii) autoimmunity. In conclusion, 15 genes were identified as major mediators of the action of vitamin D in innate and adaptive immunity and their individual functions are explained based on different gene regulatory scenarios.
Project description:Herein, we described the development of two virtual screens to identify new vitamin D receptor (VDR) antagonists among nuclear receptor (NR) ligands. Therefore, a database of 14330 nuclear receptor ligands and their NR affinities was assembled using the online available "Binding Database". Two different virtual screens were carried out in conjunction with a reported VDR crystal structure applying a stringent and less stringent pharmacophore model to filter docked NR ligand conformations. The pharmacophore models were based on the spatial orientation of the hydroxyl functionalities of VDR's natural ligands 1,25(OH2)D3 and 25(OH2)D3. The first virtual screen identified 32 NR ligands with a calculate free energy of VDR binding of more than -6.0 kJ/mol. All but nordihydroguaiaretic acid (NDGA) are VDR ligands, which inhibited the interaction between VDR and coactivator peptide SRC2-3 with an IC50 value of 15.8 µM. The second screen identified 162 NR ligands with a calculate free energy of VDR binding of more than -6.0 kJ/mol. More than half of these ligands were developed to bind VDR followed by ER?/? ligands (26%), TR?/? ligands (7%) and LxR?/? ligands (7%). The binding between VDR and ER? ligand H6036 as well as TR?/? ligand triiodothyronine and a homoserine analog thereof was confirmed by fluorescence polarization.
Project description:For a global understanding of the physiological impact of the nuclear hormone 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) the analysis of the genome-wide locations of its high affinity receptor, the transcription factor vitamin D receptor (VDR), is essential. Chromatin immunoprecipitation sequencing (ChIP-seq) in GM10855 and GM10861 lymphoblastoid cells, undifferentiated and lipopolysaccharide-differentiated THP-1 monocytes, LS180 colorectal cancer cells and LX2 hepatic stellate cells revealed between 1000 and 13,000 VDR-specific genomic binding sites. The harmonized analysis of these ChIP-seq datasets indicates that the mechanistic basis for the action of the VDR is independent of the cell type. Formaldehyde-assisted isolation of regulatory elements sequencing (FAIRE-seq) data highlight accessible chromatin regions, which are under control of 1,25(OH)2D3. In addition, public data, such as from the ENCODE project, allow to relate the genome-wide actions of VDR and 1,25(OH)2D3 to those of other proteins within the nucleus. For example, locations of the insulator protein CTCF suggest a segregation of the human genome into chromatin domains, of which more than 1000 contain at least one VDR binding site. The integration of all these genome-wide data facilitates the identification of the most important VDR binding sites and associated primary 1,25(OH)2D3 target genes. Expression changes of these key genes can serve as biomarkers for the actions of vitamin D3 and its metabolites in different tissues and cell types of human individuals. Analysis of primary tissues obtained from vitamin D3 intervention studies using such markers indicated a large inter-individual variation for the efficiency of vitamin D3 supplementation. In conclusion, a genome-wide (over)view on the genomic locations of VDR provides a broader basis for addressing vitamin D's role in health and disease.
Project description:Vitamin D3 has transcriptome- and genome-wide effects and activates, via the binding of its metabolite 1?,25-dihydroxyvitamin D3 to the transcription factor vitamin D receptor (VDR), several hundred target genes. Using samples from a 5-month vitamin D3 intervention study (VitDmet), we recently reported that the expression of 12 VDR target genes in peripheral blood mononuclear cells (PBMCs) as well as 12 biochemical and clinical parameters of the study participants are significantly triggered by vitamin D3. In this study, we performed a more focused selection of further 12 VDR target genes and demonstrated that changes of their mRNA expression in PBMCs of VitDmet subjects significantly correlate with alterations of 25-hydroxyvitamin D3 serum levels. Network and self-organizing map analysis of these datasets together with that of the other 24 parameters was followed by relevance calculations and identified changes in parathyroid hormone serum levels and the expression of the newly selected genes STS, BCL6, ITGAM, LRRC25, LPGAT1 and TREM1 as well as of the previously reported genes DUSP10 and CD14 as the most relevant parameters for describing vitamin D responsiveness in vivo. Moreover, parameter relevance ranking allowed the segregation of study subjects into high and low responders. Due to the long intervention period the vitamin D response was not too prominent on the level of transcriptional activation. Therefore, we performed in the separate VitDbol trial a short-term but high dose stimulation with a vitamin D3 bolus. In PBMCs of VitDbol subjects we observed direct transcriptional effects on the selected VDR target genes, such as an up to 2.1-fold increase already one day after supplementation onset. In conclusion, both long-term and short-term vitamin D3 supplementation studies allow monitoring the vitamin D responsiveness of human individuals and represent new types of human in vivo vitamin D3 investigations.
Project description:Genome- and transcriptome-wide data has significantly increased the amount of available information about primary 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) target genes in cancer cell models, such as human THP-1 myelomonocytic leukemia cells. In this study, we investigated the genes G0S2, CDKN1A and MYC as master examples of primary vitamin D receptor (VDR) targets being involved in the control of cellular proliferation. The chromosomal domains of G0S2 and CDKN1A are 140-170 kb in size and contain one and three VDR binding sites, respectively. This is rather compact compared to the MYC locus that is 15 times larger and accommodates four VDR binding sites. All eight VDR binding sites were studied by chromatin immunoprecipitation in THP-1 cells. Interestingly, the site closest to the transcription start site of the down-regulated MYC gene showed 1,25(OH)2D3-dependent reduction of VDR binding and is not associated with open chromatin. Four of the other seven VDR binding regions contain a typical DR3-type VDR binding sequence, three of which are also occupied with VDR in macrophage-like cells. In conclusion, the three examples suggest that each VDR target gene has an individual regulatory scenario. However, some general components of these scenarios may be useful for the development of new therapy regimens.
Project description:The transdifferentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells has been implicated in the context of vascular calcification. We investigated the roles of vitamin D receptor (Vdr) and runt-related transcription factor 2 (Runx2) in the osteoblastic differentiation of VSMCs in response to vitamin D3 using in vitro VSMCs cultures and in vivo in Vdr knockout (Vdr(-/-)) and Runx2 carboxy-terminus truncated heterozygous (Runx2(+/?C)) mice. Treatment of VSMCs with active vitamin D3 promoted matrix mineral deposition, and increased the expressions of Vdr, Runx2, and of osteoblastic genes but decreased the expression of smooth muscle myosin heavy chain in primary VSMCs cultures. Immunoprecipitation experiments suggested an interaction between Vdr and Runx2. Furthermore, silencing Vdr or Runx2 attenuated the procalcific effects of vitamin D3. Functional cooperation between Vdr and Runx2 in vascular calcification was also confirmed in in vivo mouse models. Vascular calcification induced by high-dose vitamin D3 was completely inhibited in Vdr(-/-) or Runx2(+/?C) mice, despite elevated levels of serum calcium or alkaline phosphatase. Collectively, these findings suggest that functional cooperation between Vdr and Runx2 is necessary for vascular calcification in response to vitamin D3.