Vitamin D receptor ChIP-seq in primary CD4+ cells: relationship to serum 25-hydroxyvitamin D levels and autoimmune disease.
ABSTRACT: Vitamin D insufficiency has been implicated in autoimmunity. ChIP-seq experiments using immune cell lines have shown that vitamin D receptor (VDR) binding sites are enriched near regions of the genome associated with autoimmune diseases. We aimed to investigate VDR binding in primary CD4+ cells from healthy volunteers.We extracted CD4+ cells from nine healthy volunteers. Each sample underwent VDR ChIP-seq. Our results were analyzed in relation to published ChIP-seq and RNA-seq data in the Genomic HyperBrowser. We used MEMEChIP for de novo motif discovery. 25-Hydroxyvitamin D levels were measured using liquid chromatography-tandem mass spectrometry and samples were divided into vitamin D sufficient (25(OH)D ?75 nmol/L) and insufficient/deficient (25(OH)D <75 nmol/L) groups.We found that the amount of VDR binding is correlated with the serum level of 25-hydroxyvitamin D (r = 0.92, P= 0.0005). In vivo VDR binding sites are enriched for autoimmune disease associated loci, especially when 25-hydroxyvitamin D levels (25(OH)D) were sufficient (25(OH)D ?75: 3.13-fold, P<0.0001; 25(OH)D <75: 2.76-fold, P<0.0001; 25(OH)D ?75 enrichment versus 25(OH)D <75 enrichment: P= 0.0002). VDR binding was also enriched near genes associated specifically with T-regulatory and T-helper cells in the 25(OH)D ?75 group. MEME ChIP did not identify any VDR-like motifs underlying our VDR ChIP-seq peaks.Our results show a direct correlation between in vivo 25-hydroxyvitamin D levels and the number of VDR binding sites, although our sample size is relatively small. Our study further implicates VDR binding as important in gene-environment interactions underlying the development of autoimmunity and provides a biological rationale for 25-hydroxyvitamin D sufficiency being based at 75 nmol/L. Our results also suggest that VDR binding in response to physiological levels of vitamin D occurs predominantly in a VDR motif-independent manner.
Project description:The 25-Hydroxyvitamin D (25[OH)D) serum concentration depends on vitamin D intake, endogenous vitamin D production and genetic factors. The latter have been demonstrated in large genome-wide association studies indicating that single nucleotide polymorphisms (SNPs) in genes related to the vitamin D metabolism are as important for serum 25(OH)D levels as the influence of season. The mechanism on how these SNPs influence serum 25(OH)D levels are still unclear. The aim of the present study was to investigate the genetic effects of ten selected SNPs related to vitamin D metabolism on 25-hydroxyvitamin D increase (?25(OH)D) after vitamin D supplementation in three randomized controlled trials. Genotypes of SNPs related to vitamin D metabolism were determined in 411 participants with 25(OH)D concentrations < 75 nmol/l receiving 20,000 IU cholecalciferol per week for 8 or 12 weeks after study inclusion. For the vitamin D receptor (VDR) rs10783219 polymorphism, the minor A-allele was associated with lower ?25(OH)D values in the entire study population (p = 0.022), which was not consistent in all three cohorts when analysed separately. VDR rs10783219 might therefore be a genetic modulator of increasing 25-hydroxyvitamin D concentrations. Considering the wide-spread use of vitamin D supplementation, future large and well-designed randomized controlled trials (RCTs) should investigate the clinical impact of this polymorphism.
Project description:Maternal vitamin D deficiency has been associated with numerous adverse health outcomes, but its association with fetal growth restriction remains uncertain. We sought to elucidate the association between maternal serum 25-hydroxyvitamin D [25(OH)D] concentrations in early pregnancy and the risk of small-for-gestational age birth (SGA) and explore the association between maternal single nucleotide polymorphisms (SNP) in the vitamin D receptor (VDR) gene and the risk of SGA. We conducted a nested case-control study of nulliparous pregnant women with singleton pregnancies who delivered SGA infants (n = 77 white and n = 34 black) or non-SGA infants (n = 196 white and n = 105 black). Women were followed from <16 wk gestation to delivery. Women's banked sera at <22 wk were newly measured for 25(OH)D and DNA extracted for VDR genotyping. SGA was defined as live-born infants that were <10th percentile of birth weight according to nomograms based on gender and gestational age. After confounder adjustment, there was a U-shaped relation between serum 25(OH)D and risk of SGA among white mothers, with the lowest risk from 60 to 80 nmol/L. Compared with serum 25(OH)D 37.5-75 nmol/L, SGA odds ratios (95% CI) for levels <37.5 and >75 nmol/L were 7.5 (1.8, 31.9) and 2.1 (1.2, 3.8), respectively. There was no relation between 25(OH)D and SGA risk among black mothers. One SNP in the VDR gene among white women and 3 SNP in black women were significantly associated with SGA. Our results suggest that vitamin D has a complex relation with fetal growth that may vary by race.
Project description: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 1?,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.
Project description:INTRODUCTION:Low levels of vitamin D in pregnancy have been associated with the risk of a variety of pregnancy outcomes. Few studies have investigated vitamin D concentrations throughout pregnancy in healthy women, and most guidelines recommend high vitamin D levels. In the present study, we investigated 25-hydroxyvitamin D concentrations in healthy Caucasian Danish women in relation to season, gestational age and possible vitamin D-linked complications. MATERIALS AND METHODS:Eight hundred and one healthy Caucasian Danish women with an expected normal pregnancy were recruited among 2147 women attending first trimester screening. Seven blood samplings were planned throughout the pregnancy and delivery period. The 25-hydroxyvitamin D2 (25(OH)D2) and 25-hydroxyvitamin D3 (25(OH)D3) concentrations were measured by LC-MS/MS and total 25-hydroxyvitamin D (25(OH)D) were calculated. RESULTS:A total of 3304 samples from 694 women were available for 25(OH)D measurements. The mean (25th-75th percentiles) concentrations of 25(OH)D, 25(OH)D3, and 25(OH)D2 were 54.6 (38.8-68.6) nmol/L, 52.2 (36.4-66.4) nmol/L, and 2.4 (2.2-2.2) nmol/L, respectively. Season was the strongest predictor of 25(OH)D concentration, with the lowest values observed in winter and spring, where only 42% and 41% of samples, respectively, were above 50 nmol/L. Nearly all women had values below the suggested optimal level of 75 nmol/L, independent of season. 25(OH)D peaked at gestational weeks 21-34. Plasma 25(OH)D2 levels were low in all seasons. Women with complications during pregnancy had higher 25(OH)D (estimated difference 9.8 nmol/L, standard error 2.7, p<0.001) than did women without complications, and women giving birth vaginally had lower 25(OH)D than did those delivering via elective (10.0 nmol/L, standard error 2.1, p<0.001) or emergency cesarean section (6.8 nmol/L, standard error 2.2, p<0.001). CONCLUSION:The 25(OH)D concentrations vary with both season and gestational age. Healthy women had lower 25(OH)D concentrations than recommended, without an association with an increased risk of pregnancy complications. Guidelines for vitamin D in pregnancy may require revision.
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:Genome wide association studies (GWAS) have identified several SNPs associated with colorectal cancer (CRC) susceptibility. Vitamin D is also inversely associated with CRC risk.We examined main and joint effects of previously GWAS identified genetic markers of CRC and plasma 25-hydroxyvitamin D (25(OH)D) on CRC risk in three prospective cohorts: the Nurses' Health Study (NHS), the Health Professionals Follow-up Study (HPFS), and the Physicians' Health Study (PHS). We included 1895 CRC cases and 2806 controls with genomic DNA. We calculated odds ratios and 95% confidence intervals for CRC associated with additive genetic risk scores (GRSs) comprised of all CRC SNPs and subsets of these SNPs based on proximity to regions of increased vitamin D receptor binding to vitamin D response elements (VDREs), based on published ChiP-seq data. Among a subset of subjects with additional prediagnostic 25(OH)D we tested multiplicative interactions between plasma 25(OH)D and GRS's. We used fixed effects models to meta-analyze the three cohorts.The per allele multivariate OR was 1.12 (95% CI, 1.06-1.19) for GRS-proximalVDRE; and 1.10 (95% CI, 1.06-1.14) for GRS-nonproxVDRE. The lowest quartile of plasma 25(OH)D compared with the highest, had a multivariate OR of 0.63 (95% CI, 0.48-0.82) for CRC. We did not observe any significant interactions between any GRSs and plasma 25(OH)D.We did not observe evidence for the modification of genetic susceptibility for CRC according to vitamin D status, or evidence that the effect of common CRC risk alleles differed according to their proximity to putative VDR binding sites.
Project description:OBJECTIVE:To compare vitamin D status in women with PCOS versus fertile women and subsequently evaluate the association between vitamin D status and metabolic disturbances in PCOS women. METHODS:We conducted a cross-sectional comparison study of 639 women with PCOS and 449 fertile women. Serum 25-hydroxyvitamin D (25(OH)D) was stratified into a severe deficient (< 25 nmol/l), insufficient (25-50 nmol/l), moderate (50-75 nmol/l) and adequate (> 75 nmol/l) status. The main outcome measures were the difference in vitamin D status between PCOS and fertile women, and the association between serum 25(OH)D and metabolic disturbances in PCOS women only. RESULTS:Serum 25(OH)D was significantly lower in PCOS women compared to fertile controls (mean 25(OH)D of 49.0 nmol/l versus 64.5 nmol/l). An adjusted significant difference was seen between serum 25(OH)D and homeostasis model assessment (HOMA-IR) (? = 0.76; 95% CI: 0.63-0.91; p < 0.01), HDL-cholesterol (? = 0.20; 95% CI: 0.05-0.60, p < 0.01) and apolipoprotein A1 (? = 26.2; 95% CI: 7.5-45.0, p < 0.01) between the highest vitamin D group compared to the lowest vitamin D group. CONCLUSIONS:This study demonstrates that women with PCOS have a significantly lower serum 25(OH)D compared to fertile controls. A compromised vitamin D status in PCOS women is associated with a higher HOMA-IR and an unfavourable lipid profile. Large randomized controlled trials are necessary to explore the causality of this linkage.
Project description:The vitamin D receptor (VDR) is a transcription factor that mediates the genomic effects of 1?,25-dihydroxyvitamin D3 (1,25(OH)2D3). Genome-wide there are several thousand binding sites and hundreds of primary 1,25(OH)2D3 target genes, but their functional relation is largely elusive. In this study, we used ChIA-PET data of the transcription factor CTCF in combination with VDR ChIP-seq data, in order to map chromatin domains containing VDR binding sites. In total, we found 1,599 such VDR containing chromatin domains and studied in THP-1 human monocytic leukemia cells four representatives of them. Our combined ChIP-seq and FAIRE-seq time course data showed that each of these four domains contained a master VDR binding site, where an increase of VDR binding pairs with 1,25(OH)2D3-promoted chromatin opening and the presence of a highly significant DR3-type sequence below the peak summit. These sites differed in their relative VDR binding but not in their kinetics, while other loci either had a weaker and delayed VDR association or could not be confirmed at all. All studied chromatin domains contained at least one primary 1,25(OH)2D3 target gene demonstrating a characteristic slope of mRNA increase, while neighboring genes responded delayed, if at all. In conclusion, the observation of ligand-inducible VDR binding and chromatin opening combined with a DR3-type sequence highlighted genome-wide 160 VDR loci that have within their chromatin domain a more than 4-fold increased likelihood to identify a primary 1,25(OH)2D3 target gene than in the vicinity of other genomic VDR binding sites.
Project description:A serum vitamin D [25-hydroxyvitamin D, 25(OH)D] concentration of ?75 nmol/L is recommended for optimal health. We investigated the relationship between serum 25(OH)D and metabolic syndrome (MetS), diabetes, cardiometabolic biomarkers, and cardiorespiratory fitness (CRF) in US adults using clinical cut points recommended by health organizations. Data from USA's National Health and Nutrition Examination Surveys were used. Prevalences and likelihood of having MetS and diabetes according to clinical cut points for serum 25(OH)D (<30 nmol/L, 30-<50 nmol/L, 50-<75 nmo/L, and ?75 nmol/L) were determined with multivariate logistic regression. Relations between serum 25(OH)D and various cardiometabolic biomarkers, CRF, MetS, and diabetes were tested using multivariable adjusted regression. Prevalence of MetS and diabetes were significantly lower in individuals with serum 25(OH)D ?75 nmol/L (MetS, 21.6%; diabetes, 4.1%) compared to those with 25(OH)D <30 nmol/L (MetS, 45.5%; diabetes, 11.6%) (p < 0.0001). Individuals with serum 25(OH)D ?75 nmol/L had significantly lower waist circumference (p < 0.0001), C-reactive protein (p = 0.003), glycated hemoglobin (p < 0.0002), fasting triglycerides (p < 0.0001), total homocysteine (p < 0.0001), and insulin resistance (p = 0.0001) and had significantly higher HDL-cholesterol (p < 0.0001) and maximal oxygen uptake (marker for CRF) (p< 0.0009) compared to those with 25(OH)D <30 nmol/L. In conclusion, serum 25(OH)D ?75 nmol/L is associated with positive indicators related to cardiometabolic diseases in US adults.
Project description:Vitamin D deficiency has adverse health effects in young children. Our aims were to determine predictors of vitamin D status and then to use these factors to develop a practical tool to predict low 25(OH)D concentrations in preschool New Zealand children. A cross-sectional sample of 1329 children aged 2 to <5?years were enrolled from throughout New Zealand in late-winter to spring 2012. 25-Hydroxyvitamin D (25(OH)D) was measured on dried blood spot (DBS) samples collected using finger-prick sampling. Caregivers completed a questionnaire. Mean (SD) DBS 25(OH)D concentration was 52(19)nmol/L. 25(OH)D?<?25?nmol/L was present in 86(7%), 25(OH)D?<?50?nmol/L in 642(48%), 25(OH)D 50-?<?75?nmol/L in 541(41%) and 25(OH)D?>?75?nmol/L in 146(11%) of children. Factors independently associated with the risk of 25(OH)D <?25?nmol/L were female gender (OR 1.92,95%CI 1.17-3.14), other non-European ethnicities (not including M?ori or Pacific) (3.51,1.89-6.50), had olive-dark skin colour (4.52,2.22-9.16), did not take vitamin D supplements (2.56,1.06-6.18), had mothers with less than secondary-school qualifications (5.00,2.44-10.21) and lived in more deprived households (1.27,1.06-1.53). Children who drank toddler milk (vitamin D fortified cow's milk formula marketed to young children) had a zero risk of 25(OH)D?<?25?nmol/L. The predictive tool identified children at risk of 25(OH)D?<?25?nmol/L with sensitivity 42%, specificity 97% and ROC area-under-curve 0.76(95%CI 0.67-0.86, p?<?0.001). Predictors of low vitamin D status were consistent with those identified in previous studies of New Zealand children. The tool had insufficient predictive ability for use in clinical situations, and suggests a need to promote safe, inexpensive testing to determine vitamin D status in preschool children.