Vitamin D Receptor Binding Sites Research Articles

Vitamin D receptor attenuate ischemia-reperfusion kidney injury via inhibiting ATF4

Activating transcription factor 4 (ATF4) is one of the key effectors of endoplasmic reticulum stress (ERS), ATF4/CHOP pathway-mediated ERS plays an important role in the progression of acute kidney disease (AKI). We have previously reported that Vitamin D receptor (VDR) exert renoprotection in rodent AKI models. However, whether ATF4, as well as ERS, is involved in the protective effect of VDR in ischemia-reperfusion (I/R) induced AKI is unknown. Herein, we showed that VDR agonist paricalcitol and VDR overexpression alleviated I/R-induced renal injury and cells apoptosis with decreased ATF4 and attenuated ERS, while VDR deletion significantly resulted in further increased ATF4, more drastic ERS and renal injury in I/R mice models. In addition, paricalcitol remarkably reduced Tunicamycin (TM) induced ATF4 and ERS with attenuated renal injury, while VDR deletion aggravated the above changes in TM mice models. Moreover, overexpression of ATF4 partially abolished the effect of paricalcitol against TM-induced ERS and apoptosis, while inhibition of ATF4 enhanced the protective effect of paricalcitol. Bioinformatics analysis indicated potential VDR binding sites on ATF4 promotor sequence which were further confirmed by ChIP-qPCR and dual-luciferase reporter gene assay. In conclusion, VDR attenuated I/R-induced AKI by suppressing ERS partly via transcriptional regulation of ATF4.

PD07-11 VITAMIN D HAS A STRONGER IMPACT ON PROSTATE TUMOR GENE EXPRESSION IN BLACK MEN

You have accessJournal of UrologyCME1 May 2022PD07-11 VITAMIN D HAS A STRONGER IMPACT ON PROSTATE TUMOR GENE EXPRESSION IN BLACK MEN Neil Mistry, Cordero McCall, Moray Campbell, and Adam Murphy Neil MistryNeil Mistry More articles by this author , Cordero McCallCordero McCall More articles by this author , Moray CampbellMoray Campbell More articles by this author , and Adam MurphyAdam Murphy More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002526.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Black men have a higher risk of prostate cancer (PCa) mortality and also have a higher prevalence of vitamin D deficiency. The VITAL clinical trial showed that patients randomized to 2000 IU of vitamin D3 had a significant reduction in advanced cancers compared to placebo (1.7% vs. 2.1%; HR, 0.83; P = .04). Black men also trended towards lower PCa mortality in that trial. Thus, we sought to investigate prostate cancer responsiveness to vitamin D by evaluating differentially expressed genes (DEG) in Black vs. non-Black men’s prostate tumors in high and low vitamin D states. METHODS: We enrolled 169 men in a cross-sectional study evaluating serum vitamin D status and adverse pathology at radical prostatectomy. We selected 78 men. RNA sequencing was done using punch biopsies of >70% epithelium in the dominant tumor nodule and contralateral benign tissue to assess DEG between Black and non-Black tumors. ChIP-seq for vitamin D receptor (VDR) binding sites was used on RC43T (Black PCa cell line) and LNCaP to identify vitamin D responsive genes. Samples were stratified by vitamin D status (above vs. below race-specific median) and race (Black vs non-Black). RESULTS: There were 57 Black and 21 non-Black men. There were 39 lower risk tumors (Gleason score ≤ 3+4) and 39 higher risk tumors (GS ≥ 3+4 with tertiary 5). In a high vitamin D state, we found 2,101 DEGs in Black tumors vs normal tissue (Figure 1a) compared to only 15 DEGs in non-Black tumors vs normal tissue (Figure 1b). A direct comparison of Black vs non-Black tumors in a high vitamin D state demonstrated 434 DEGs (Figure 1c). When comparing tumors with above vs below-median vitamin D in all patients, we found 2,992 DEGs. Of these, 1,746 DEGs contained VDR binding sites and were found in RC43T while only 10 DEGs with VDR binding sites were found in LNCaP (Figure 1d). CONCLUSIONS: Vitamin D directly impacts gene expression in prostate cancer. Tumors in Black men express more genes that have VDR binding sites than tumors in non-Black men. Due to this unique relationship, Vitamin D may have a role in chemoprevention for clinically localized prostate cancer in Black men. Source of Funding: None © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e103 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Neil Mistry More articles by this author Cordero McCall More articles by this author Moray Campbell More articles by this author Adam Murphy More articles by this author Expand All Advertisement PDF DownloadLoading ...

Vitamin D receptor, STAT3, and TET2 cooperate to establish tolerogenesis.

The active form of vitamin D, 1,25-dihydroxyvitamin D3, induces a stable tolerogenic phenotype in dendritic cells (DCs). This process involves the vitamin D receptor (VDR), which translocates to the nucleus, binds its cognate genomic sites, and promotes epigenetic and transcriptional remodeling. In this study, we report the occurrence of vitamin D-specific DNA demethylation and transcriptional activation at VDR binding sites associated with the acquisition of tolerogenesis invitro. Differentiation to tolerogenic DCs associates with activation of the IL-6-JAK-STAT3 pathway. We show that JAK2-mediated STAT3 phosphorylation is specific to vitamin D stimulation. VDR and the phosphorylated form of STAT3 interact with each other to form a complex with methylcytosine dioxygenase TET2. Most importantly, pharmacological inhibition of JAK2 reverts vitamin D-induced tolerogenic properties of DCs. This interplay among VDR, STAT3, and TET2 opens up possibilities for modulating DC immunogenic properties in clinics.

Vitamin D Receptor, STAT3 and TET2 Cooperate to Establish Tolerogenesis

The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), induces stable tolerogenic phenotype in dendritic cells (DCs). This process involves the vitamin D receptor (VDR), which translocates to the nucleus, binds its cognate genomic sites and promotes epigenetic and transcriptional remodeling. In this study, we report the occurrence of vitamin D-specific DNA demethylation and transcriptional activation at VDR binding sites associated with the acquisition of tolerogenesis. Tolerogenic properties of DCs are acquired together with activation of the IL6-JAK-STAT3 pathway. In fact, ChIP-seq analysis shows that VDR directly binds the IL6 gene, and JAK2-mediated STAT3 phosphorylation is specific to vitamin D stimulation. VDR and the phosphorylated form of STAT3 interact with each other to form a complex with methylcytosine dioxygenase TET2. Most importantly, pharmacological inhibition of STAT3 phosphorylation reverts vitamin D-induced tolerogenic properties of DCs. This novel interplay between VDR, STAT3 and TET2 opens up possibilities for modulating DC immunogenic properties in the clinics.

High levels of PIWI-interacting RNAs are present in the small RNA landscape of prostate epithelium from vitamin D clinical trial specimens.

BackgroundVitamin D, a hormone that acts through the nuclear vitamin D receptor (VDR), upregulates antitumorigenic microRNA in prostate epithelium. This may contribute to the lower levels of aggressive prostate cancer (PCa) observed in patients with high serum vitamin D. The small noncoding RNA (ncRNA) landscape includes many other RNA species that remain uncharacterized in prostate epithelium and their potential regulation by vitamin D is unknown.MethodsLaser capture microdissection (LCM) followed by small‐RNA sequencing was used to identify ncRNAs in the prostate epithelium of tissues from a vitamin D‐supplementation trial. VDR chromatin immunoprecipitation‐sequencing was performed to identify vitamin D genomic targets in primary prostate epithelial cells.ResultsIsolation of epithelium by LCM increased sample homogeneity and captured more diversity in ncRNA species compared with publicly available small‐RNA sequencing data from benign whole prostate. An abundance of PIWI‐interacting RNAs (piRNAs) was detected in normal prostate epithelium. The obligate binding partners of piRNAs, PIWI‐like (PIWIL) proteins, were also detected in prostate epithelium. High prostatic vitamin D levels were associated with increased expression of piRNAs. VDR binding sites were located near several ncRNA biogenesis genes and genes regulating translation and differentiation.ConclusionsBenign prostate epithelium expresses both piRNA and PIWIL proteins, suggesting that these small ncRNA may serve an unknown function in the prostate. Vitamin D may increase the expression of prostatic piRNAs. VDR binding sites in primary prostate epithelial cells are consistent with its reported antitumorigenic functions and a role in ncRNA biogenesis.

Modulation of vitamin D signaling by the pioneer factor CEBPA

The myeloid master regulator CCAAT enhancer-binding protein alpha (CEBPA) is known as a pioneer factor. In this study, we report the CEBPA cistrome of THP-1 human monocytes after stimulation with the vitamin D receptor (VDR) ligand 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) for 2, 8 and 24 h. About a third of the genomic VDR binding sites co-located with those of CEBPA. In parallel, the binding strength of 5% of the CEBPA cistrome, i.e. some 1500 sites, is significantly (p < 0.001) affected by 1,25(OH)2D3. Transcriptome-wide analysis after CEBPA silencing indicated that the pioneer factor enhances both the basal expression and ligand inducibility of 70 vitamin D target genes largely involved in lipid signaling and metabolism. In contrast, CEBPA suppresses 82 vitamin D target genes many of which are related to the modulation of T cell activity by monocytes. The inducibility of the promoter-specific histone marker H3K4me3 distinguishes the former class of genes from the latter. Moreover, prominent occupancy of the myeloid pioneer factor PU.1 on 1,25(OH)2D3-sensitive CEBPA enhancers mechanistically explains the dichotomy of vitamin D target genes. In conclusion, CEBPA supports vitamin D signaling concerning actions of the innate immune system, but uses the antagonism with PU.1 for suppressing possible overreactions of adaptive immunity.

Vitamin D Genomics: From In Vitro to In Vivo.

The vitamin D3 metabolite 1α,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.

Vitamin D receptor-targeted treatment to prevent pathological dedifferentiation of pancreatic β cells under hyperglycaemic stress

Dedifferentiation has been identified as one of the causes of β-cell failure resulting in type 2 diabetes (T2D). This study tested whether increasing vitamin D receptor (VDR) expression prevents dedifferentiation of β cells in a high-glucose state in vitro. Culturing a mouse insulinoma cell line (MIN6) in a high-glucose environment decreased VDR expression. However, increased VDR following vitamin D3 (VD3) treatment improved insulin release of early-passage MIN6 and insulin index of db/- (heterozygous) islets to levels seen in normal functional islets. Treatment with VD3, its analogues and derivatives also increased the expression of essential transcription factors, such as Pdx1, MafA and VDR itself, ultimately increasing expression of Ins1 and Ins2, which might protect β cells against dedifferentiation. VD3 agonist lithocholic acid (LCA) propionate was the most potent candidate molecule for protecting against dedifferentiation, and an e-pharmacophore mapping model confirmed that LCA propionate exhibits a stabilizing conformation within the VDR binding site. This study concluded that treating db/+ islets with a VD3 analogue and/or derivatives can increase VDR activity, preventing the pathological dedifferentiation of β cells and the onset of T2D.

ETS transcription factor family member GABPA contributes to vitamin D receptor target gene regulation

Binding motifs of the ETS-domain transcription factor GABPA are found with high significance below the summits of the vitamin D receptor (VDR) cistrome. VDR is the nuclear receptor for the biologically most active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). In this study, we determined the GABPA cistrome in THP-1 human monocytes and found that it is comprised of 3822 genomic loci, some 20% of which were modulated by 1,25(OH)2D3. The GABPA cistrome showed a high overlap rate with accessible chromatin and the pioneer transcription factor PU.1. Interestingly, 23 and 12% of persistent and transient VDR binding sites, respectively, co-localized with GABPA, which is clearly higher than the rate of secondary VDR loci (4%). Some 40% of GABPA binding sites were found at transcription start sites, nearly 100 of which are of 1,25(OH)2D3 target genes. On 593 genomic loci VDR and GABPA co-localized with PU.1, while only 175 VDR sites bound GABPA in the absence of PU.1. In total, VDR sites with GABPA co-localization may control some 450 vitamin D target genes. Those genes that are co-controlled by PU.1 preferentially participate in cellular and immune signaling processes, while the remaining genes are involved in cellular metabolism pathways. In conclusion, GABPA may contribute to differential VDR target gene regulation.

1α,25-Dihydroxyvitamin D3 up-regulates IL-34 expression in SH-SY5Y neural cells.

Vitamin D supplementation is regarded as a novel approach to treat Alzheimer's disease, but the underlying mechanism remains elusive. The cytokine IL-34 provides strong neuroprotective and survival signals in brain injury and neurodegeneration and could be an immunological mediator for the vitamin D-induced protection. The aim of this study was to investigate whether human IL-34 is up-regulated in neuronal cells by the hormonally active form of vitamin D, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3]. We found that IL-34 was detectable in a variety of cell lines and its expression was strongly induced in SH-SY5Y neural cells in a dose- and time-dependent manner by 1α,25(OH)2D3 through the vitamin D receptor (VDR). Furthermore, we identified the core promoter of IL-34 gene and a VDR binding site (CGCCCT) that was required for 1α,25(OH)2D3-induced IL-34 expression. These findings suggest that the induction of IL-34 expression by 1α,25(OH)2D3 may constitute a mechanism that explains the protective function of vitamin D in Alzheimer's disease.

Association between genetic variation within vitamin D receptor-DNA binding sites and risk of basal cell carcinoma.

An increasing number of studies have reported a protective association between vitamin D and cancer risk. The vitamin D endocrine system regulates transcriptional programs involved in inflammation, cell growth and differentiation through the binding of vitamin D receptor (VDR) to specific VDR elements. However, limited attention has been given to the role of variation within VDR binding sites in the development of basal cell carcinoma (BCC). Across 2,776 previously identified VDR binding sites, we identified 2,540 independent single-nucleotide polymorphisms (SNPs) and examined their associations with BCC risk in a genome-wide association meta-analysis totaling 17,187 BCC cases and 287,054 controls from two data sets. After multiple testing corrections, we identified two SNPs at new loci (rs16917546 at 10q21.1: odds ratio (OR) = 1.06, p = 3.16 × 10-7 and rs79824801 at 12q13.3: OR = 1.10, p = 1.88 × 10-5 ) for the first time as independently related to BCC risk in meta-analysis; and both SNPs were nominally significant in two data sets. In addition, the SNP rs3769823 within VDR binding site at a previously reported BCC susceptibility locus (2q33.1, rs13014235) also exhibited a significant association (OR = 1.12, p = 3.99 × 10-18 ). A mutually adjusted model suggested that rs3769823 explained the signal in this region. Our findings support the hypothesis that inherited common variation in VDR binding sites affects the development of BCC.

Epigenomic PU.1-VDR crosstalk modulates vitamin D signaling

The ETS-domain transcription factor PU.1 acts as a pioneer factor for other transcription factors including nuclear receptors. In this study, we report that in THP-1 human monocytes the PU.1 cistrome comprises 122,319 genomic sites. Interestingly, at 6498 (5.3%) of these loci PU.1 binding was significantly modulated by the vitamin D receptor (VDR) ligand 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). In most cases 1,25(OH)2D3 increased PU.1 association, which correlated strongly with VDR co-location and overlap ratios for canonical DR3-type VDR binding sites. Genome-wide 6488 sites associating both with PU.1 and VDR as well as 5649 non-VDR overlapping, 1,25(OH)2D3-sensitive PU.1 loci represent the PU.1-VDR crosstalk and can be described by four gene regulatory scenarios, each. Chromatin accessibility was the major discriminator between these models. The location of the PU.1 binding loci in open chromatin coincided with a significantly smaller mean distance to the closest 1,25(OH)2D3 target gene. PU.1 knockdown indicated that the pioneer factor is relevant for the transcriptional activation of 1,25(OH)2D3 target genes but its impact differed in magnitude and orientation. In conclusion, PU.1 is an important modulator of VDR signaling in monocytes, including but also exceeding its role as a pioneer factor, but we found no evidence for a direct interaction of both proteins.

Integration of VDR genome wide binding and GWAS genetic variation data reveals co-occurrence of VDR and NF-\u03baB binding that is linked to immune phenotypes

BackgroundThe nuclear hormone receptor superfamily acts as a genomic sensor of diverse signals. Their actions are often intertwined with other transcription factors. Nuclear hormone receptors are targets for many therapeutic drugs, and include the vitamin D receptor (VDR). VDR signaling is pleotropic, being implicated in calcaemic function, antibacterial actions, growth control, immunomodulation and anti-cancer actions. Specifically, we hypothesized that the biologically significant relationships between the VDR transcriptome and phenotype-associated biology could be discovered by integrating the known VDR transcription factor binding sites and all published trait- and disease-associated SNPs. By integrating VDR genome-wide binding data (ChIP-seq) with the National Human Genome Research Institute (NHGRI) GWAS catalog of SNPs we would see where and which target gene interactions and pathways are impacted by inherited genetic variation in VDR binding sites, indicating which of VDR’s multiple functions are most biologically significant.ResultsTo examine how genetic variation impacts VDR function we overlapped 23,409 VDR genomic binding peaks from six VDR ChIP-seq datasets with 191,482 SNPs, derived from GWAS-significant SNPs (Lead SNPs) and their correlated variants (r2 > 0.8) from HapMap3 and the 1000 genomes project. In total, 574 SNPs (71 Lead and 503 SNPs in linkage disequilibrium with Lead SNPs) were present at VDR binding loci and associated with 211 phenotypes. For each phenotype a hypergeometric test was used to determine if SNPs were enriched at VDR binding sites. Bonferroni correction for multiple testing across the 211 phenotypes yielded 42 SNPs that were either disease- or phenotype-associated with seven predominately immune related including self-reported allergy; esophageal cancer was the only cancer phenotype. Motif analyses revealed that only two of these 42 SNPs reside within a canonical VDR binding site (DR3 motif), and that 1/3 of the 42 SNPs significantly impacted binding and gene regulation by other transcription factors, including NF-κB. This suggests a plausible link for the potential cross-talk between VDR and NF-κB.ConclusionsThese analyses showed that VDR peaks are enriched for SNPs associated with immune phenotypes suggesting that VDR immunomodulatory functions are amongst its most important actions. The enrichment of genetic variation in non-DR3 motifs suggests a significant role for the VDR to bind in multimeric complexes containing other transcription factors that are the primary DNA binding component. Our work provides a framework for the combination of ChIP-seq and GWAS findings to provide insight into the underlying phenotype-associated biology of a given transcription factor.

Vitamin D receptor is a novel transcriptional regulator for Axin1

BackgroundAxin1 is a scaffold protein in the β-catenin destruction complex, which, if disrupted, contributes to pathogenesis of various human diseases, including colorectal carcinogenesis and inflammatory bowel diseases (IBD). We have previously demonstrated that Salmonella infection promotes the degradation and plasma sequestration of Axin1, leading to bacterial invasiveness and inflammatory responses. Vitamin D and the vitamin D receptor (VDR) appear to be important regulators of IBD and colon cancer. Although VDR and Axin1 are all involved in intestinal inflammation, it remains unclear whether these processes are related or function independently. In the current study, we hypothesize that VDR is an important regulator for the maintenance of physiological level of Axin1. MethodsUsing the intestinal epithelial conditional VDR knockout mouse model (VDRΔIEC) and cultured cell lines, influences of VDR status on the expression of Axin1 was evaluated by Western blots and real-time PCR. Loss- and gain-of-function assays were used to investigate the regulation of VDR on Axin1 at the transcriptional and translational levels. Cells were treated with cycloheximide or actinomycin for molecular mechanistic studies. Candidate genomic VDR binding sites for Axin1 were tested by chromatin immunoprecipitation (ChIP) assay. Physical interactions among VDR, Axin1, and β-catenin were tested by immunoprecipitation. Cellular localization of Axin1 with different VDR status was determined by fractionation and immunohistochemistry. ResultsWe found that VDR deletion led to lower protein and mRNA levels of Axin1, whereas knockdown of Axin1 did not change the expression level of VDR protein. Immunoprecipitation data did not support physical interaction between VDR and Axin1. The VDR regulation of Axin1 was through a VDR genomic binding site for Axin1 gene on the regulatory region. Fractionation data showed that cytosolic Axin1 was significantly reduced due to VDR deletion, leaving the nuclear fraction unchanged. In ileum, Axin1 was distributed in the cytosol of apical epithelium and crypts. ConclusionVDR is important for the maintenance of physiological level of Axin1. The discovery of Axin1 as a VDR target gene provides novel and fundamental insights into the interactions between the VDR and β-catenin signaling pathways.

Vitamin D-dependent chromatin association of CTCF in human monocytes

CCCTC-binding factor (CTCF) is a transcription factor being involved in 3D chromatin organization and displays a highly conserved genome-wide binding pattern. In this study, we report the cistrome of CTCF in THP-1 human monocytes and confirm that from the 40,078 CTCF binding sites nearly 85% are identical with those found in K562 monocytes. Quadruplicate chromatin immunoprecipitation sequencing (ChIP-seq) demonstrated that at 2130 loci the association strenght of CTCF with genomic DNA was significantly (p<0.05) modulated by stimulation with the natural vitamin D receptor (VDR) ligand 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). Some 55% of these CTCF sites contribute to DNA looping and mark the anchors of 587 putative topologically associating domains (TADs) containing at least one VDR binding site and one 1,25(OH)2D3 target gene. These TADs can explain the regulatory scenarios of up to 70% of all 1,25(OH)2D3 target genes. A self-organizing map approach subdivided the vitamin D-sensitive CTCF sites into seven classes that can be distinguished by participation in DNA loop formation, binding to open chromatin, carrying binding motifs for CTCF or its relative BORIS, overlap with transcription start site (TSS) regions and binding of VDR. These variant molecular profiles suggest different mechanisms of the 1,25(OH)2D3-dependent action of CTCF. The co-location of VDR and 1,25(OH)2D3-dependent CTCF sites increases in the context of accessible chromatin and TSS regions but does not show any significant correlation with classical DNA binding mechanisms of CTCF. In conclusion, vitamin D-sensitive CTCF sites provide further mechanistic details to the epigenome-wide understanding of 1,25(OH)2D3-mediated gene regulation.

EBNA2 Binds to Genomic Intervals Associated with Multiple Sclerosis and Overlaps with Vitamin D Receptor Occupancy

Epstein-Barr virus (EBV) is a non-heritable factor that associates with multiple sclerosis (MS). However its causal relationship with the disease is still unclear. The virus establishes a complex co-existence with the host that includes regulatory influences on gene expression. Hence, if EBV contributes to the pathogenesis of MS it may do so by interacting with disease predisposing genes. To verify this hypothesis we evaluated EBV nuclear antigen 2 (EBNA2, a protein that recent works by our and other groups have implicated in disease development) binding inside MS associated genomic intervals. We found that EBNA2 binding occurs within MS susceptibility sites more than expected by chance (factor of observed vs expected overlap [O/E] = 5.392-fold, p < 2.0e-05). This remains significant after controlling for multiple genomic confounders. We then asked whether this observation is significant per se or should also be viewed in the context of other disease relevant gene-environment interactions, such as those attributable to vitamin D. We therefore verified the overlap between EBNA2 genomic occupancy and vitamin D receptor (VDR) binding sites. EBNA2 shows a striking overlap with VDR binding sites (O/E = 96.16-fold, p < 2.0e-05), even after controlling for the chromatin accessibility state of shared regions (p <0.001). Furthermore, MS susceptibility regions are preferentially targeted by both EBNA2 and VDR than by EBNA2 alone (enrichment difference = 1.722-fold, p = 0.0267). Taken together, these findings demonstrate that EBV participates in the gene-environment interactions that predispose to MS.

The transcriptional regulator BCL6 participates in the secondary gene regulatory response to vitamin D

The vitamin D metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is the high affinity ligand of the transcription factor vitamin D receptor (VDR) and therefore a direct regulator of transcription. Transcriptome-wide analysis of THP-1 human monocytes had indicated more than 600 genes to be significantly (p<0.05) stimulated after 4h incubation with 1,25(OH)2D3, but only 67 of them where more than 1.5-fold up-regulated. These include the genes encoding for the transcription factors BCL6, NFE2, POU4F2 and ELF4, which are controlled by one or two VDR binding sites within their chromosomal domains. The latter are defined via DNA loop formation mediated by the transcription factor CTCF that is highly conserved in its genome-wide loci. We found BCL6 being most responsive to 1,25(OH)2D3 and selected it for further analysis. An incubation of THP-1 cells with 1,25(OH)2D3 for 24h resulted in a significant (p<0.001) change in the mRNA expression of more than 1600 genes, of which 132 were at least 2-fold up-regulated. About half of the latter genes are secondary 1,25(OH)2D3 targets, since they do not carry any VDR binding site within their chromosomal domain. Chromatin immunoprecipitation sequencing datasets indicated that the majority of these domains contain a BCL6 binding site. We followed the secondary transcriptional response to 1,25(OH)2D3 for eight representative gene examples and confirmed the binding of CTCF and BCL6 to their respective chromosomal domains. In conclusion, our study indicated that in monocytes most of the physiological responses to 1,25(OH)2D3 involve the action of the transcription factor BCL6.

Dissecting high from low responders in a vitamin D3 intervention study

Vitamin D3 is a pleiotropic signaling molecule that has via activation of the transcription factor vitamin D receptor (VDR) a direct effect on the expression of more than 100 genes. The aim of this study was to find transcriptomic and clinical biomarkers that are most suited to identify vitamin D3 responders within 71 pre-diabetic subjects during a 5-month intervention study (VitDmet). In hematopoietic cells, the genes ASAP2, CAMP, CD14, CD97, DUSP10, G0S2, IL8, LRRC8A, NINJ1, NRIP1, SLC37A2 and THBD are known as primary vitamin D targets. We demonstrate that each of these 12 genes carries a conserved VDR binding site within its genomic region and is expressed in human peripheral blood mononuclear cells (PBMCs). The changes in the expression of these genes in human PBMCs at the start and the end of the vitamin D-intervention were systematically correlated with the alteration in the circulating form of vitamin D3, 25-hydroxyvitamin D3 (25(OH)D3). Only 39–44 (55–62%) of the study subjects showed a highly significant response to vitamin D3, i.e., we considered them as “responders”. In comparison, we found for 37–53 (52–75%) of the participants that only 12 biochemical and clinical parameters, such as concentrations of parathyroid hormone (PTH) and insulin, or computed values, such as homeostatic model assessment and insulin sensitivity index, show a correlation with serum 25(OH)D3 levels that is as high as that of the selected VDR target genes. All 24 parameters together described the pleiotropic vitamin D response of the VitDmet study subjects. Interestingly, they demonstrated a number of additional correlations that define a network, in which PTH plays the central role. In conclusion, vitamin D3-induced changes in human PBMCs can be described by transcriptomic and serum biomarkers and allow a segregation into high and low responders.This article is part of a Special Issue entitled ‘17th Vitamin D Workshop’ .

Changes in vitamin D target gene expression in adipose tissue monitor the vitamin D response of human individuals.

Vitamin D₃, its biologically most active metabolite 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), and the vitamin D receptor (VDR) are important for adipose tissue biology. We extrapolated genomic VDR association loci in adipocytes from 55 conserved genome-wide VDR-binding sites in nonfat tissues. Taking the genes DUSP10, TRAK1, NRIP1, and THBD as examples, we confirmed the predicted VDR binding sites upstream of their transcription start sites and showed rapid mRNA up-regulation of all four genes in SGBS human pre-adipocytes. Using adipose tissue biopsy samples from 47 participants of a 5-month vitamin D₃ intervention study, we demonstrated that all four primary VDR target genes can serve as biomarkers for the vitamin D₃ responsiveness of human individuals. Changes in DUSP10 gene expression appear to be the most comprehensive marker, while THBD mRNA changes characterized a rather different group of study participants. We present a new approach to predict vitamin D target genes based on conserved genomic VDR-binding sites. Using human adipocytes as examples, we show that such ubiquitous VDR target genes can be used as markers for the individual's response to a supplementation with vitamin D₃.

Patterns of genome-wide VDR locations.

The genome-wide analysis of the binding sites of the transcription factor vitamin D receptor (VDR) is essential for a global appreciation the physiological impact of the nuclear hormone 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). Genome-wide analysis of lipopolysaccharide (LPS)-polarized THP-1 human monocytic leukemia cells via chromatin immunoprecipitation sequencing (ChIP-seq) resulted in 1,318 high-confidence VDR binding sites, of which 789 and 364 occurred uniquely with and without 1,25(OH)2D3 stimulation, while only 165 were common. We re-analyzed five public VDR ChIP-seq datasets with identical peak calling settings (MACS, version 2) and found, using a novel consensus summit identification strategy, in total 23,409 non-overlapping VDR binding sites, 75% of which are unique within the six analyzed cellular models. LPS-differentiated THP-1 cells have 22% more genomic VDR locations than undifferentiated cells and both cell types display more overlap in their VDR locations than the other investigated cell types. In general, the intersection of VDR binding profiles of ligand-stimulated cells is higher than those of unstimulated cells. De novo binding site searches and HOMER screening for binding motifs formed by direct repeats spaced by three nucleotides (DR3) suggest for all six VDR ChIP-seq datasets that these sequences are found preferentially at highly ligand responsive VDR loci. Importantly, all VDR ChIP-seq datasets display the same relationship between the VDR occupancy and the percentage of DR3-type sequences below the peak summits. The comparative analysis of six VDR ChIP-seq datasets demonstrated that the mechanistic basis for the action of the VDR is independent of the cell type. Only the minority of genome-wide VDR binding sites contains a DR3-type sequence. Moreover, the total number of identified VDR binding sites in each ligand-stimulated cell line inversely correlates with the percentage of peak summits with DR3 sites.