Population-level transcription cycles derive from stochastic timing of single-cell transcription.

Eukaryotic transcription is a dynamic process relying on a large number of proteins. By measuring the cycling expression of the pyruvate dehydrogenase kinase 4 gene in human cells, we constructed a detailed stochastic model for single-gene transcription at the molecular level using realistic kinetics for diffusion and protein complex dynamics. We observed that gene induction caused an approximate 60 min periodicity of several transcription related processes: first, the covalent histone modifications and presence of many regulatory proteins at the transcription start site; second, RNA polymerase II activity; third, chromatin loop formation; and fourth, mRNA accumulation. Our model can predict the precise timing of single-gene activity leading to transcriptional cycling on the cell population level when we take into account the sequential and irreversible multistep nature of transcriptional initiation. We propose that the cyclic nature of population gene expression is primarily based on the intrinsic periodicity of the transcription process itself.

An update on vitamin D signaling and cancer.

A low vitamin D status is associated with an increased risk of various cancers, such as of colon, breast, prostate and hematological cells. The biologically most active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) is a high affinity ligand of the transcription factor vitamin D receptor (VDR). 1,25(OH)2D3 induces via VDR changes to the epigenome of healthy and neoplastic cells and in this way influences their transcriptome. Ligand-activated VDR binds to more than 10,000 loci within the human genome and affects the transcription of some 1000 target genes in a large proportion of human tissues and cell types. From the evolutionary perspective, the prime role of vitamin D was probably the control of energy metabolism later shifting to modulate innate and adaptive immunity as well as to regulate calcium and bone homeostasis. Since rapidly growing immune and cancer cells both use the same pathways and genes for controlling their proliferation, differentiation and apoptosis, not surprisingly, vitamin D signaling changes these processes also in neoplastic cells. Thus, anti-cancer effects of vitamin D may derive from managing growth and differentiation in immunity. This review provides an update on the molecular basis of vitamin D signaling, i.e., the effects of 1,25(OH)2D3 on the epigenome and transcriptome, and its relationship to cancer prevention and therapy.

Nutrition and epigenetic programming.

PURPOSE OF REVIEW: The aim of this study is to highlight the epigenomic programming properties of nutritional molecules and their metabolites in human tissues and cell types. RECENT FINDINGS: Chromatin is the physical expression of the epigenome and has a memory function on the level of DNA methylation, histone modification and 3-dimensional (3D) organization. This epigenetic memory does not only affect transient gene expression but also represents long-lasting decisions on cellular fate. The memory is based on an epigenetic programming process, which is directed by extracellular and intracellular signals that are sensed by transcription factors and chromatin modifiers. Many dietary molecules and their intermediary metabolites serve as such signals, that is they contribute to epigenetic programming and memory. In this context, we will discuss about molecules of intermediary energy metabolism affecting chromatin modifier actions, nutrition-triggered epigenetic memory in pre- and postnatal phases of life; and epigenetic programming of immune cells by vitamin D. These mechanisms explain some of the susceptibility for complex diseases, such as the metabolic syndrome, cancer and immune disorders. SUMMARY: The observation that nutritional molecules are able to modulate the epigenome initiated the new nutrigenomic subdiscipline nutritional epigenetics. The concept that epigenetic memory and programming is directed by our diet has numerous implications for the interpretation of disease risk including their prevention.

In Vivo Regulation of Signal Transduction Pathways by Vitamin D Stabilizes Homeostasis of Human Immune Cells and Counteracts Molecular Stress.

Vitamin D3 is a pre-hormone that regulates hundreds of target genes and dozens of physiological functions, including calcium homeostasis and the activity of the immune system, via its metabolite 1,25-dihydroxyvitamin D3, which is a high-affinity ligand for the transcription factor vitamin D receptor. In this study, we took advantage of data from the VitDHiD vitamin D3 intervention trial (25 healthy individuals) indicating that 442 protein-coding genes were significantly (false discovery rate < 0.05) up- or downregulated in peripheral blood mononuclear cells one day after taking a vitamin D3 bolus. Since more than half of the encoded proteins had "signaling" assigned as a primary biological function, we evaluated their involvement in signal transduction cascades included in the KEGG (Kyoto Encyclopedia of Genes and Genomes) database and found 88 of the vitamin D targets contributing to 16 different pathways. Eight of the pathways show an approximately even contribution of up- and downregulated genes, suggesting that the actions of vitamin D stabilize homeostasis of the physiological processes driven by the respective signaling cascades. Interestingly, vitamin D target genes involved in the signaling pathways of hypoxia-inducible factor 1 (HIF1), tumor necrosis factor (TNF), mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NFκB) are primarily downregulated. This supports the observation that the physiological role of vitamin D in healthy individuals is to tone down certain processes rather than activate them. In conclusion, under in vivo conditions, vitamin D either alleviates the homeostasis of immune cells in healthy individuals or counteracts molecular responses to oxygen deprivation (HIF1), microbe infection (TNF), growth stimulation (MAPKs) and inflammation (NFκB).

Time-Resolved Gene Expression Analysis Monitors the Regulation of Inflammatory Mediators and Attenuation of Adaptive Immune Response by Vitamin D.

Peripheral blood mononuclear cells (PBMCs) belong to the innate and adaptive immune system and are highly sensitive and responsive to changes in their systemic environment. In this study, we focused on the time course of transcriptional changes in freshly isolated human PBMCs 4, 8, 24 and 48 h after onset of stimulation with the active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). Taking all four time points together, 662 target genes were identified and segregated either by time of differential gene expression into 179 primary and 483 secondary targets or by driver of expression change into 293 direct and 369 indirect targets. The latter classification revealed that more than 50% of target genes were primarily driven by the cells' response to ex vivo exposure than by the nuclear hormone and largely explained its down-regulatory effect. Functional analysis indicated vitamin D's role in the suppression of the inflammatory and adaptive immune response by down-regulating ten major histocompatibility complex class II genes, five alarmins of the S100 calcium binding protein A family and by affecting six chemokines of the C-X-C motif ligand family. Taken together, studying time-resolved responses allows to better contextualize the effects of vitamin D on the immune system.

A Single Oral Vitamin D3 Bolus Reduces Inflammatory Markers in Healthy Saudi Males.

Vitamin D deficiency has increased in the general population and is a public health issue. Vitamin D plays an important role in regulating the immune system, e.g., by modulating the production of inflammatory cytokines. In most countries, the recommended maximal daily dose of vitamin D3 is 4000 IU (100 µg) per day. In this study, we investigated whether a single vitamin D3 bolus can reduce the levels of the inflammatory markers interleukin (IL) 6, IL8 and tumor necrosis factor (TNF) within one month. Fifty healthy Saudi males were recruited from the local community in Jeddah city and were orally supplemented with a single dose of 80,000 IU vitamin D3. Serum samples were collected at time points 0, 1 and 30 days, and serum levels of IL6, IL8 and TNF, parathyroid hormone (PTH), 25-hydroxyvitamin D3 (25(OH)D3), triglycerides, cholesterol, calcium (Ca2+) and phosphate (PO4-) were determined. On average, the vitamin D3 bolus resulted in a significant increase in vitamin D status as well as in a significant decrease in the levels of inflammatory cytokines even one month after supplementation without changing serum Ca2+, PO4- or lipid levels. In conclusion, single high-dose vitamin D3 supplementation is safe for reducing inflammation markers and may lead to an update of current recommendations for vitamin D intake, in order to prevent critical health problems.

Skin colour and vitamin D: An update.

Homo sapiens evolved in East Africa and had dark skin, hair, and eyes, in order to protect against deleterious consequences of intensive UV radiation at equatorial latitudes. Intensive skin pigmentation was thought to bear the risk of inefficient vitamin D3 synthesis in the skin. This initiated the hypothesis that within the past 75 000 years, in which humans migrated to higher latitudes in Asia and Europe, the need for vitamin D3 synthesis served as an evolutionary driver for skin lightening. In this review, we summarize the recent archeogenomic reconstruction of population admixture in Europe and demonstrate that skin lightening happened as late as 5000 years ago through immigration of lighter pigmented populations from western Anatolia and the Russian steppe but not primarily via evolutionary pressure for vitamin D3 synthesis. We show that variations in genes encoding for proteins being responsible for the transport, metabolism and signalling of vitamin D provide alternative mechanisms of adaptation to a life in northern latitudes without suffering from consequences of vitamin D deficiency. This includes hypotheses explaining differences in the vitamin D status and response index of European populations.

Vitamin D receptor(s): In the nucleus but also at membranes?

The genomic actions of the vitamin D are mediated via its biologically most potent metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ) and the transcription factor vitamin D receptor (VDR). Activation of VDR by 1,25(OH)2 D3 leads to change in the expression of more 1000 genes in various human tissues. Based on (epi)genome, transcriptome and crystal structure data the molecular details of this nuclear vitamin D signalling pathway are well understood. Vitamin D is known for its role on calcium homeostasis and bone formation, but it also modulates energy metabolism, innate and adaptive immunity as well as cellular growth, differentiation and apoptosis. The observation of rapid, non-genomic effects of 1,25(OH)2 D3 at cellular membranes and in the cytosol initiated the question, whether there are alternative vitamin D-binding proteins in these cellular compartments. So far, the best candidate is the enzyme PDIA3 (protein disulphide isomerase family A member 3), which is found at various subcellular locations. Furthermore, also VDR seems to play a role in membrane-based responses to vitamin D. In this viewpoint, we will dispute whether these rapid, non-genomic pathways are a meaningful addition to the genome-wide effects of vitamin D.

Randomized controlled trial on the effectiveness of web-based Genomics Nursing Education Intervention for undergraduate nursing students: a study protocol.

AIM: To describe a randomized controlled trial protocol that will evaluate the effectiveness of two web-based genomic nursing education interventions. BACKGROUND: Preparing future nurses to be competent in genetic and genomic concepts is fundamental to ensure appropriate clinical application. However, genetics-genomics concepts are still new in the field of nursing. Little is known about what type and kind of web-based nursing education is effective in improving the knowledge of nursing students. To address these knowledge gaps, a web-based 'Genomic Nursing Education Intervention' will be developed and compared with an existing online education programme. DESIGN: A randomized controlled trial of two groups with pre-test and repeated posttesting. METHODS: The Genomic Nursing Concept Inventory, a validated tool, will be used to assess the genetics-genomics knowledge of nursing students. Participants will be randomly allocated to either a control or an intervention group. The control group will receive the standard web-based nursing education, while the intervention group will receive a newly developed web-based education intervention. Outcome measures include the students' knowledge level of nursing genetics-genomics concepts. Participants will be retested at 3 and 6 months. CONCLUSION: Current evidence shows that ensuring nurses have adequate education in genetic-genomic concepts is challenging. This study will demonstrate which of two web-based nursing education methods is more effective in teaching genetic-genomic concepts. This research project will better prepare the nursing profession in their careers for the emerging advance technologies in genetics-genomics and personalized health care. IMPACT: Current evidence shows major challenges in ensuring that nurses have adequate education in genetics-genomics concepts. Less is known about what approaches to web-based education are effective to improve the knowledge gaps of nursing students in genetics-genomics concepts. This study will determine which type of web-based nursing education is effective in improving the genetics-genomics knowledge of nursing students. This research project will help better prepare nurses in dealing with advances in genetics-genomics in their careers. TRIAL REGISTRATION:  This study is registered in ClinicalTrials.gov (ID number NCT03963687) https://clinicaltrials.gov/show/NCT03963687.

Epigenome-wide effects of vitamin D and their impact on the transcriptome of human monocytes involve CTCF.

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.

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.

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

BACKGROUND: The 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. RESULTS: To 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 (r 2 > 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. CONCLUSIONS: These 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.

Tracing the molecular basis of transcriptional dynamics in noisy data by using an experiment-based mathematical model.

Changes in transcription factor levels, epigenetic status, splicing kinetics and mRNA degradation can each contribute to changes in the mRNA dynamics of a gene. We present a novel method to identify which of these processes is changed in cells in response to external signals or as a result of a diseased state. The method employs a mathematical model, for which the kinetics of gene regulation, splicing, elongation and mRNA degradation were estimated from experimental data of transcriptional dynamics. The time-dependent dynamics of several species of adipose differentiation-related protein (ADRP) mRNA were measured in response to ligand activation of the transcription factor peroxisome proliferator-activated receptor δ (PPARδ). We validated the method by monitoring the mRNA dynamics upon gene activation in the presence of a splicing inhibitor. Our mathematical model correctly identifies splicing as the inhibitor target, despite the noise in the data.

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 24 h 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.

Multiplex Eukaryotic Transcription (In)activation: Timing, Bursting and Cycling of a Ratchet Clock Mechanism.

Activation of eukaryotic transcription is an intricate process that relies on a multitude of regulatory proteins forming complexes on chromatin. Chromatin modifications appear to play a guiding role in protein-complex assembly on chromatin. Together, these processes give rise to stochastic, often bursting, transcriptional activity. Here we present a model of eukaryotic transcription that aims to integrate those mechanisms. We use stochastic and ordinary-differential-equation modeling frameworks to examine various possible mechanisms of gene regulation by multiple transcription factors. We find that the assembly of large transcription factor complexes on chromatin via equilibrium-binding mechanisms is highly inefficient and insensitive to concentration changes of single regulatory proteins. An alternative model that lacks these limitations is a cyclic ratchet mechanism. In this mechanism, small protein complexes assemble sequentially on the promoter. Chromatin modifications mark the completion of a protein complex assembly, and sensitize the local chromatin for the assembly of the next protein complex. In this manner, a strict order of protein complex assemblies is attained. Even though the individual assembly steps are highly stochastic in duration, a sequence of them gives rise to a remarkable precision of the transcription cycle duration. This mechanism explains how transcription activation cycles, lasting for tens of minutes, derive from regulatory proteins residing on chromatin for only tens of seconds. Transcriptional bursts are an inherent feature of such transcription activation cycles. Bursting transcription can cause individual cells to remain in synchrony transiently, offering an explanation of transcriptional cycling as observed in cell populations, both on promoter chromatin status and mRNA levels.

Vitamin D receptor signaling and its therapeutic implications: Genome-wide and structural view.

Vitamin D3 is one of the few natural compounds that has, via its metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and the transcription factor vitamin D receptor (VDR), a direct effect on gene regulation. For efficiently applying the therapeutic and disease-preventing potential of 1,25(OH)2D3 and its synthetic analogs, the key steps in vitamin D signaling need to be understood. These are the different types of molecular interactions with the VDR, such as (i) the complex formation of VDR with genomic DNA, (ii) the interaction of VDR with its partner transcription factors, (iii) the binding of 1,25(OH)2D3 or its synthetic analogs within the ligand-binding pocket of the VDR, and (iv) the resulting conformational change on the surface of the VDR leading to a change of the protein-protein interaction profile of the receptor with other proteins. This review will present the latest genome-wide insight into vitamin D signaling, and will discuss its therapeutic implications.

Chromatin acetylation at transcription start sites and vitamin D receptor binding regions relates to effects of 1α,25-dihydroxyvitamin D3 and histone deacetylase inhibitors on gene expression.

The nuclear hormone 1α,25-dihydroxyvitamin D(3) (1α,25(OH)(2)D(3) or 1,25D) regulates its target genes via activation of the transcription factor vitamin D receptor (VDR) far more specifically than the chromatin modifier trichostatin A (TsA) via its inhibitory action on histone deacetylases. We selected the thrombomodulin gene locus with its complex pattern of five VDR binding sites and multiple histone acetylation and open chromatin regions as an example to investigate together with a number of reference genes, the primary transcriptional responses to 1α,25(OH)(2)D(3) and TsA. Transcriptome-wide, 18.4% of all expressed genes are either up-or down-regulated already after a 90 min TsA treatment; their response pattern to 1α,25(OH)(2)D(3) and TsA sorts them into at least six classes. TsA stimulates a far higher number of genes than 1α,25(OH)(2)D(3) and dominates the outcome of combined treatments. However, 200 TsA target genes can be modulated by 1α,25(OH)(2)D(3) and more than 1000 genes respond only when treated with both compounds. The genomic view on the genes suggests that the degree of acetylation at transcription start sites and VDR binding regions may determine the effect of TsA on mRNA expression and its interference with 1α,25(OH)(2)D(3). Our findings hold true also for other HDAC inhibitors and may have implications on dual therapies using chromatin modifiers and nuclear receptor ligands.

Dynamics of 1α,25-dihydroxyvitamin D3-dependent chromatin accessibility of early vitamin D receptor target genes.

The signaling cascade of the transcription factor vitamin D receptor (VDR) is triggered by its specific ligand 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). In this study we demonstrate that in THP-1 human monocytic leukemia cells 87.4% of the 1034 most prominent genome-wide VDR binding sites co-localize with loci of open chromatin. At 165 of them 1α,25(OH)2D3 strongly increases chromatin accessibility and has at further 217 sites weaker effects. Interestingly, VDR binding sites in 1α,25(OH)2D3-responsive chromatin regions are far more often composed of direct repeats with 3 intervening nucleotides (DR3s) than those in ligand insensitive regions. DR3-containing VDR sites are enriched in the neighborhood of genes that are involved in controling cellular growth, while non-DR3 VDR binding is often found close to genes related to immunity. At the example of six early VDR target genes we show that the slope of their 1α,25(OH)2D3-induced transcription correlates with the basal chromatin accessibility of their major VDR binding regions. However, the chromatin loci controlling these genes are indistinguishable in their VDR association kinetics. Taken together, ligand responsive chromatin loci represent dynamically regulated contact points of VDR with the genome, from where it controls early 1α,25(OH)2D3 target genes.

Effects of a healthy Nordic diet on plasma 25-hydroxyvitamin D concentration in subjects with metabolic syndrome: a randomized, [corrected] controlled trial (SYSDIET).

PURPOSE: At northern latitudes, vitamin D is not synthesized endogenously during winter, causing low plasma 25-hydroxyvitamin D (25(OH)D) concentrations. Therefore, we evaluated the effects of a healthy Nordic diet based on Nordic nutrition recommendations (NNR) on plasma 25(OH)D and explored its dietary predictors. METHODS: In a Nordic multi-centre trial, subjects (n = 213) with metabolic syndrome were randomized to a control or a healthy Nordic diet favouring fish (≥300 g/week, including ≥200 g/week fatty fish), whole-grain products, berries, fruits, vegetables, rapeseed oil and low-fat dairy products. Plasma 25(OH)D and parathyroid hormone were analysed before and after 18- to 24-week intervention. RESULTS: At baseline, 45 % had vitamin D inadequacy (<50 nmol/l), whereas 8 % had deficiency (<25 nmol/l). Dietary vitamin D intake was increased by the healthy Nordic diet (P < 0.001). The healthy Nordic and the control diet reduced the prevalence of vitamin D inadequacy by 42 % (P < 0.001) and 19 % (P = 0.002), respectively, without between-group difference (P = 0.142). Compared with control, plasma 25(OH)D (P = 0.208) and parathyroid hormone (P = 0.207) were not altered by the healthy Nordic diet. Predictors for 25(OH)D were intake of vitamin D, eicosapentaenoic acids (EPA), docosahexaenoic acids (DHA), vitamin D supplement, plasma EPA and plasma DHA. Nevertheless, only vitamin D intake and season predicted the 25(OH)D changes. CONCLUSION: Consuming a healthy Nordic diet based on NNR increased vitamin D intake but not plasma 25(OH)D concentration. The reason why fish consumption did not improve vitamin D status might be that many fish are farmed and might contain little vitamin D or that frying fish may result in vitamin D extraction. Additional ways to improve vitamin D status in Nordic countries may be needed.

Dataset integration identifies transcriptional regulation of microRNA genes by PPARγ in differentiating mouse 3T3-L1 adipocytes.

Peroxisome proliferator-activated receptor γ (PPARγ) is a key transcription factor in mammalian adipogenesis. Genome-wide approaches have identified thousands of PPARγ binding sites in mouse adipocytes and PPARγ upregulates hundreds of protein-coding genes during adipogenesis. However, no microRNA (miRNA) genes have been identified as primary PPARγ-targets. By integration of four separate datasets of genome-wide PPARγ binding sites in 3T3-L1 adipocytes we identified 98 miRNA clusters with PPARγ binding within 50 kb from miRNA transcription start sites. Nineteen mature miRNAs were upregulated ≥2-fold during adipogenesis and for six of these miRNA loci the PPARγ binding sites were confirmed by at least three datasets. The upregulation of five miRNA genes miR-103-1 (host gene Pank3), miR-148b (Copz1), miR-182/96/183, miR-205 and miR-378 (Ppargc1b) followed that of Pparg. The PPARγ-dependence of four of these miRNA loci was demonstrated by PPARγ knock-down and the loci of miR-103-1 (Pank3), miR-205 and miR-378 (Ppargc1b) were also responsive to the PPARγ ligand rosiglitazone. Finally, chromatin immunoprecipitation analysis validated in silico predicted PPARγ binding sites at all three loci and H3K27 acetylation was analyzed to confirm the activity of these enhancers. In conclusion, we identified 22 putative PPARγ target miRNA genes, showed the PPARγ dependence of four of these genes and demonstrated three as direct PPARγ target genes in mouse adipogenesis.