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Abstract Vitamin D modulates innate and adaptive immunity, the molecular mechanisms of which we aim to understand under human in vivo conditions. Therefore, we designed the study VitDHiD (NCT03537027) as a human investigation, in which 25 healthy individuals were supplemented with a single vitamin D3 bolus (80,000 IU). Transcriptome-wide differential gene expression analysis of peripheral blood mononuclear cells (PBMCs), which were isolated directly before and 24 h after supplementation, identified 452 genes significantly (FDR

Anti-Müllerian hormone (AMH) is a peptide belonging to the transforming growth factor beta superfamily and acts exclusively through its receptor type 2 (AMHR2). From the 8th week of pregnancy, AMH is produced by Sertoli cells, and from the 23rd week of gestation, it is produced by granulosa cells of the ovary. AMH plays a critical role in regulating gonadotropin secretion, ovarian tissue responsiveness to pituitary hormones, and the pathogenesis of polycystic ovarian syndrome. It inhibits the transition from primordial to primary follicles and is considered the best marker of ovarian reserve. Therefore, measuring AMH concentration of the hormone is valuable in managing assisted reproductive technologies. AMH was initially discovered through its role in the degeneration of Müllerian ducts in male fetuses. However, due to its ability to inhibit the cell cycle and induce apoptosis, it has also garnered interest in oncology. For example, antibodies targeting AMHR2 are being investigated for their potential in diagnosing and treating various cancers. Additionally, AMH is present in motor neurons and functions as a protective and growth factor. Consequently, it is involved in learning and memory processes and may support the treatment of Alzheimer’s disease. This review aims to provide a comprehensive overview of the biology of AMH and its role in both endocrinology and oncology.

The concept of the vitamin D response index was developed based on vitamin D intervention studies conducted with Finnish cohorts. In this study, we challenged the concept by performing a single vitamin D3 bolus (80,000 IU) intervention with a cohort of 100 native Saudis. The change of serum levels of the proinflammatory cytokines interleukin 6, interleukin 8 and tumor necrosis factor measured directly before intervention in comparison to samples taken one and thirty days after vitamin D3 supplementation were used as biomarkers for distinguishing low, mid and high responders. Interestingly, we identified 39 % of the study participants as low responders. In contrast, when we used in a subset of 37 study participants whole blood expression changes of seven well-known vitamin D target genes one and thirty days after supplementation as alternative biomarkers, only 9 persons (24 %) were identified as low responders. In conclusion, in Saudi Arabia the rate of low vitamin D responders is equal or even higher than that in Finland. Therefore, similar to Nordic countries also in Saudi Arabia appropriate vitamin D3 supplementation is essential, in order to fulfill the needs of low responders.

Fanconi anemia, a rare disorder with an incidence of 1 in 300,000, is caused by mutations in FANC genes, which affect the repair of DNA interstrand crosslinks. The disease is characterized by congenital malformations, bone marrow failure within the first decade of life, and recurrent squamous cell carcinomas of the oral cavity, esophagus, and anogenital regions starting around age 20. In this review, we propose that Fanconi anemia should be considered a premature-aging syndrome. Interestingly, the onset and severity of the life-limiting clinical features of Fanconi anemia can be influenced by lifestyle choices, such as a healthy diet and physical activity. These factors shape the epigenetic status of at-risk cell types and enhance the competence of the immune system through nutritional signaling. Fanconi anemia may serve as a model for understanding the aging process in the general population, addressing research gaps in its clinical presentation and suggesting prevention strategies. Additionally, we will discuss how the balance of genetic and environmental risk factors—affecting both cancer onset and the speed of aging—is interlinked with signal transduction by dietary molecules. The underlying nutrigenomic principles will offer guidance for healthy aging in individuals with Fanconi anemia as well as for the general population.

During our whole lifespan, from conception to death, the epigenomes of all tissues and cell types of our body integrate signals from the environment. This includes signals derived from our diet and the uptake of macro- and micronutrients. In most cases, this leads only to transient changes, but some effects of this epigenome programming process are persistent and can even be transferred to the next generation. Both epigenetic programming and redox processes are affected by the individual choice of diet and other lifestyle decisions like physical activity. The nutrient-gene communication pathways have adapted during human evolution and are essential for maintaining health. However, when they are maladaptive, such as in long-term obesity, they significantly contribute to diseases like type 2 diabetes and cancer. The field of nutrigenomics investigates nutrition-related signal transduction pathways and their effect on gene expression involving interactions both with the genome and the epigenomes. Several of these diet-(epi)genome interactions and the involved signal transduction cascades are redox-regulated. Examples include the effects of the NAD+/NADH ratio, vitamin C levels and secondary metabolites of dietary molecules from plants on the acetylation and methylation state of the epigenome as well as on gene expression through redox-sensitive pathways via the transcription factors NFE2L2 and FOXO. In this review, we summarize and extend on these topics as well as those discussed in the articles of this Special Issue and take them into the context of redox biology.

Fanconi anemia (FA) is a rare disease (incidence of 1:300,000) primarily based on the inheritance of pathogenic variants in genes of the FA/BRCA (breast cancer) pathway. These variants ultimately reduce the functionality of different proteins involved in the repair of DNA interstrand crosslinks and DNA double-strand breaks. At birth, individuals with FA might present with typical malformations, particularly radial axis and renal malformations, as well as other physical abnormalities like skin pigmentation anomalies. During the first decade of life, FA mostly causes bone marrow failure due to reduced capacity and loss of the hematopoietic stem and progenitor cells. This often makes hematopoietic stem cell transplantation necessary, but this therapy increases the already intrinsic risk of developing squamous cell carcinoma (SCC) in early adult age. Due to the underlying genetic defect in FA, classical chemo-radiation-based treatment protocols cannot be applied. Therefore, detecting and treating the multi-step tumorigenesis process of SCC in an early stage, or even its progenitors, is the best option for prolonging the life of adult FA individuals. However, the small number of FA individuals makes classical evidence-based medicine approaches based on results from randomized clinical trials impossible. As an alternative, we introduce here the concept of multi-level dynamical modelling using large, longitudinally collected genome, proteome- and transcriptome-wide data sets from a small number of FA individuals. This mechanistic modelling approach is based on the “hallmarks of cancer in FA”, which we derive from our unique database of the clinical history of over 750 FA individuals. Multi-omic data from healthy and diseased tissue samples of FA individuals are to be used for training constituent models of a multi-level tumorigenesis model, which will then be used to make experimentally testable predictions. In this way, mechanistic models facilitate not only a descriptive but also a functional understanding of SCC in FA. This approach will provide the basis for detecting signatures of SCCs at early stages and their precursors so they can be efficiently treated or even prevented, leading to a better prognosis and quality of life for the FA individual.

Nutrigenomics attempts to characterize and integrate the relation between dietary molecules and gene expression on a genome-wide level. One of the biologically active nutritional compounds is vitamin D3, which activates via its metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) the nuclear receptor VDR (vitamin D receptor). Vitamin D3 can be synthesized endogenously in our skin, but since we spend long times indoors and often live at higher latitudes where for many winter months UV-B radiation is too low, it became a true vitamin. The ligand-inducible transcription factor VDR is expressed in the majority of human tissues and cell types, where it modulates the epigenome at thousands of genomic sites. In a tissue-specific fashion this results in the up- and downregulation of primary vitamin D target genes, some of which are involved in attenuating oxidative stress. Vitamin D affects a wide range of physiological functions including the control of metabolism, bone formation and immunity. In this review, we will discuss how the epigenome- and transcriptome-wide effects of 1,25(OH)2D3 and its receptor VDR serve as a master example in nutrigenomics. In this context, we will outline the basis of a mechanistic understanding for personalized nutrition with vitamin D3.

Nutrigenomics describes the interaction between nutrients and our genome. Since the origin of our species most of these nutrient-gene communication pathways have not changed. However, our genome experienced over the past 50,000 years a number of evolutionary pressures, which are based on the migration to new environments concerning geography and climate, the transition from hunter-gatherers to farmers including the zoonotic transfer of many pathogenic microbes and the rather recent change of societies to a preferentially sedentary lifestyle and the dominance of Western diet. Human populations responded to these challenges not only by specific anthropometric adaptations, such as skin color and body stature, but also through diversity in dietary intake and different resistance to complex diseases like the metabolic syndrome, cancer and immune disorders. The genetic basis of this adaptation process has been investigated by whole genome genotyping and sequencing including that of DNA extracted from ancient bones. In addition to genomic changes, also the programming of epigenomes in pre- and postnatal phases of life has an important contribution to the response to environmental changes. Thus, insight into the variation of our (epi)genome in the context of our individual's risk for developing complex diseases, helps to understand the evolutionary basis how and why we become ill. This review will discuss the relation of diet, modern environment and our (epi)genome including aspects of redox biology. This has numerous implications for the interpretation of the risks for disease and their prevention.

The pro-hormone vitamin D3 is an important modulator of both innate and adaptive immunity since its biologically active metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) regulates via the transcription factor VDR (vitamin D receptor) the epigenome and transcriptome of human immune cells and controls in this way the expression of hundreds of vitamin D target genes. Since the myeloid linage of hematopoiesis is epigenetically programmed by VDR in concert with the pioneer factors PU.1 (purine-rich box 1) and CEBPα (CCAAT/enhancer binding protein α), monocytes, macrophages, and dendritic cells are the most vitamin D-sensitive immune cell types. The central role of the immune system in various aging-related diseases suggests that immunocompetence describes not only the ability of an individual to resist pathogens and parasites but also to contest non-communicative diseases and the process of aging itself. In this review, we argue that the individual-specific responsiveness to vitamin D relates to a person’s immunocompetence via the epigenetic programming function of VDR and its ligand 1,25(OH)2D3 during hematopoiesis as well as in the periphery. This may provide a mechanism explaining how vitamin D protects against major common diseases and, in parallel, promotes healthy aging.

Environmental triggers often work via signal transduction cascades that modulate the epigenome and transcriptome of cell types involved in the disease process. Multiple sclerosis (MS) is an autoimmune disease affecting the central nervous system being characterized by a combination of recurring inflammation, demyelination and progressive loss of axons. The mechanisms of MS onset are not fully understood and genetic variants may explain only some 20% of the disease susceptibility. From the environmental factors being involved in disease development low vitamin D levels have been shown to significantly contribute to MS susceptibility. The pro-hormone vitamin D3 acts via its metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) as a high affinity ligand to the transcription factor VDR (vitamin D receptor) and is a potent modulator of the epigenome at thousands of genomic regions and the transcriptome of hundreds of genes. A major target tissue of the effects of 1,25(OH)2D3 and VDR are cells of innate and adaptive immunity, such as monocytes, dendritic cells as well as B and T cells. Vitamin D induces immunological tolerance in T cells and reduces inflammatory reactions of various types of immune cells, all of which are implicated in MS pathogenesis. The immunomodulatory effects of 1,25(OH)2D3 contribute to the prevention of MS. However, the strength of the responses to vitamin D3 supplementation is highly variegated between individuals. This review will relate mechanisms of individual’s vitamin D responsiveness to MS susceptibility and discuss the prospect of vitamin D3 supplementation as a way to extinguish the autoimmunity in MS.

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).

Vitamin D intervention studies are designed to evaluate the impact of the micronutrient vitamin D3 on health and disease. The appropriate design of studies is essential for their quality, successful execution, and interpretation. Randomized controlled trials (RCTs) are considered the “gold standard” for intervention studies. However, the most recent large-scale (up to 25,000 participants), long-term RCTs involving vitamin D3 did not provide any statistically significant primary results. This may be because they are designed similarly to RCTs of a therapeutic drug but not of a nutritional compound and that only a limited set of parameters per individual were determined. We propose an alternative concept using the segregation of study participants into different groups of responsiveness to vitamin D3 supplementation and in parallel measuring a larger set of genome-wide parameters over multiple time points. This is in accordance with recently developed mechanistic modeling approaches that do not require a large number of study participants, as in the case of statistical modeling of the results of a RCT. Our experience is based on the vitamin D intervention trials VitDmet, VitDbol, and VitDHiD, which allowed us to distinguish the study participants into high, mid, and low vitamin D responders. In particular, investigating the vulnerable group of low vitamin D responders will provide future studies with more conclusive results both on the clinical and molecular benefits of vitamin D3 supplementation. In conclusion, our approach suggests a paradigm shift towards detailed investigations of transcriptome and epigenome-wide parameters of a limited set of individuals, who, due to a longitudinal design, can act as their own controls.

Human peripheral blood mononuclear cells (PBMCs) represent a highly responsive primary tissue that is composed of innate and adaptive immune cells. In this study, we compared modulation of the transcriptome of PBMCs by the vitamin D metabolites 25-hydroxyvitamin D3 (25(OH)D3), 25(OH)D2 and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). Saturating concentrations of 1,25(OH)2D3, 25(OH)D3 and 25(OH)D2 resulted after 24 h stimulation in a comparable number and identity of target genes, but below 250 nM 25(OH)D3 and 25(OH)D2 were largely insufficient to affect the transcriptome. The average EC50 values of 206 common target genes were 322 nM for 25(OH)D3 and 295 nM for 25(OH)D2 being some 600-fold higher than 0.48 nM for 1,25(OH)2D3. The type of target gene, such as primary/secondary, direct/indirect or up-/down-regulated, had no significant effect on vitamin D metabolite sensitivity, but individual genes could be classified into high, mid and lower responders. Since the 1α-hydroxylase CYP27B1 is very low expressed in PBMCs and early (4 and 8 h) transcriptome responses to 25(OH)D3 and 25(OH)D2 were as prominent as to 1,25(OH)2D3, both vitamin D metabolites may directly control gene expression. In conclusion, at supra-physiological concentrations 25(OH)D3 and 25(OH)D2 are equally potent in modulating the transcriptome of PBMCs possibly by directly activating the vitamin D receptor.

Abstract The transcription factor vitamin D receptor (VDR) is the high affinity nuclear target of the biologically active form of vitamin D3 (1,25(OH)2D3). In order to identify pure genomic transcriptional effects of 1,25(OH)2D3, we used VDR cistrome, transcriptome and open chromatin data, obtained from the human monocytic cell line THP-1, for a novel hierarchical analysis applying three bioinformatics approaches. We predicted 75.6% of all early 1,25(OH)2D3-responding (2.5 or 4 h) and 57.4% of the late differentially expressed genes (24 h) to be primary VDR target genes. VDR knockout led to a complete loss of 1,25(OH)2D3–induced genome-wide gene regulation. Thus, there was no indication of any VDR-independent non-genomic actions of 1,25(OH)2D3 modulating its transcriptional response. Among the predicted primary VDR target genes, 47 were coding for transcription factors and thus may mediate secondary 1,25(OH)2D3 responses. CEBPA and ETS1 ChIP-seq data and RNA-seq following CEBPA knockdown were used to validate the predicted regulation of secondary vitamin D target genes by both transcription factors. In conclusion, a directional network containing 47 partly novel primary VDR target transcription factors describes secondary responses in a highly complex vitamin D signaling cascade. The central transcription factor VDR is indispensable for all transcriptome-wide effects of the nuclear hormone.

Introduction: All epidemiological studies suggest that vitamin D deficiency is prevalent among the Polish general population. Since vitamin D deficiency was shown to be among the risk factors for many diseases and for all-cause mortality, concern about this problem led us to update the previous Polish recommendations. Methods: After reviewing the epidemiological evidence, case-control studies and randomized control trials (RCTs), a Polish multidisciplinary group formulated questions on the recommendations for prophylaxis and treatment of vitamin D deficiency both for the general population and for the risk groups of patients. The scientific evidence of pleiotropic effects of vitamin D as well as the results of panelists’ voting were reviewed and discussed. Thirty-four authors representing different areas of expertise prepared position statements. The consensus group, representing eight Polish/international medical societies and eight national specialist consultants, prepared the final Polish recommendations. Results: Based on networking discussions, the ranges of total serum 25-hydroxyvitamin D concentration indicating vitamin D deficiency [3) as the first choice for prophylaxis and treatment of vitamin D deficiency were developed. Calcifediol dosing as the second choice for preventing and treating vitamin D deficiency was introduced. Conclusions: Improving the vitamin D status of the general population and treatment of risk groups of patients must be again announced as healthcare policy to reduce a risk of spectrum of diseases. This paper offers consensus statements on prophylaxis and treatment strategies for vitamin D deficiency in Poland.

Microbe-associated molecular patterns, such as lipopolysaccharide (LPS) and β-glucan (BG), are surrogates of immune challenges like bacterial and fungal infections, respectively. The biologically active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), supports the immune system in its fight against infections. This study investigated significant and prominent changes of the transcriptome of human peripheral blood mononuclear cells that immediately after isolation are exposed to 1,25(OH)2D3-modulated immune challenges over a time frame of 24-48 h. In this in vitro study design, most LPS and BG responsive genes are downregulated and their counts are drastically reduced when cells are treated 24 h after, 24 h before or in parallel with 1,25(OH)2D3. Interestingly, only a 1,25(OH)2D3 pre-treatment of the LPS challenge results in a majority of upregulated genes. Based on transcriptome-wide data both immune challenges display characteristic differences in responsive genes and their associated pathways, to which the actions of 1,25(OH)2D3 often oppose. The joined BG/1,25(OH)2D3 response is less sensitive to treatment sequence than that of LPS/1,25(OH)2D3. In conclusion, the functional consequences of immune challenges are significantly modulated by 1,25(OH)2D3 but largely depend on treatment sequence. This may suggest that a sufficient vitamin D status before an infection is more important than vitamin D supplementation afterwards.

This year we are celebrating 100 years of the naming of vitamin D, but the molecule is, in fact, more than one billion years old [...]

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.

Vitamin D deficiency is a global health problem that not only leads to metabolic bone disease but also to many other illnesses, most of which are associated with chronic inflammation. Thus, our aim was to investigate the safety and effectiveness of a single high dose of vitamin D3 (80,000 IU) on vitamin D status and proinflammatory cytokines such as interleukin (IL)6, IL8 and tumor necrosis factor (TNF) in healthy Saudi females. Fifty healthy females were recruited and orally supplemented with a single vitamin D3 bolus (80,000 IU). All participants donated fasting blood samples at baseline, one day and thirty days after supplementation. Serum 25-hydroxyvitamin D3 (25(OH)D3), IL6, IL8, TNF, calcium, phosphate, parathyroid hormone (PTH) and blood lipid levels were determined. Serum 25(OH)D3 significantly increased one and thirty days after supplementation when compared with baseline without causing elevation in calcium or phosphate or a decrease in PTH to abnormal levels. In contrast, the concentrations of the three representative proinflammatory cytokines decreased gradually until the end of the study period. In conclusion, a single high dose (80,000 IU) is effective in improving serum vitamin D status and reducing the concentration of the proinflammatory cytokines in a rapid and safe way in healthy females.

For at least 1.2 billion years, eukaryotes have been able to synthesize sterols and, therefore, can produce vitamin D when exposed to UV-B. Vitamin D endocrinology was established some 550 million years ago in animals, when the high-affinity nuclear receptor VDR (vitamin D receptor), transport proteins and enzymes for vitamin D metabolism evolved. This enabled vitamin D to regulate, via its target genes, physiological process, the first of which were detoxification and energy metabolism. In this way, vitamin D was enabled to modulate the energy-consuming processes of the innate immune system in its fight against microbes. In the evolving adaptive immune system, vitamin D started to act as a negative regulator of growth, which prevents overboarding reactions of T cells in the context of autoimmune diseases. When, some 400 million years ago, species left the ocean and were exposed to gravitation, vitamin D endocrinology took over the additional role as a major regulator of calcium homeostasis, being important for a stable skeleton. Homo sapiens evolved approximately 300,000 years ago in East Africa and had adapted vitamin D endocrinology to the intensive exposure of the equatorial sun. However, when some 75,000 years ago, when anatomically modern humans started to populate all continents, they also reached regions with seasonally low or no UV-B, i.e., and under these conditions vitamin D became a vitamin.

The vitamin D metabolite 1α,25-dihydroxyvitamin D3 is the natural, high-affinity ligand of the transcription factor vitamin D receptor (VDR). In many tissues and cell types, VDR binds in a ligand-dependent fashion to thousands of genomic loci and modulates, via local chromatin changes, the expression of hundreds of primary target genes. Thus, the epigenome and transcriptome of VDR-expressing cells is directly affected by vitamin D. Vitamin D target genes encode for proteins with a large variety of physiological functions, ranging from the control of calcium homeostasis, innate and adaptive immunity, to cellular differentiation. This review will discuss VDR’s binding to genomic DNA, as well as its genome-wide locations and interaction with partner proteins, in the context of chromatin. This information will be integrated into a model of vitamin D signaling, explaining the regulation of vitamin D target genes.

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.

The default supply of vitamin D3 to humans is its endogenous production in UV-B-exposed skin [...]

Vitamin D3 is an essential micronutrient mediating pleiotropic effects in multiple tissues and cell types via its metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), which activates the transcription factor vitamin D receptor. In this study, we used peripheral blood mononuclear cells (PBMCs) obtained from five healthy adults and investigated transcriptome-wide, whether the precursor of 1,25(OH)2D3, 25-hydroxyvitamin D3 (25(OH)D3), has gene regulatory potential on its own. Applying thresholds of >2 in fold change of gene expression and 3 (250 nM (nmol/L)) none of the study participants had a significant effect on their PBMC transcriptome. In contrast, 1000 and 10,000 nM 25(OH)D3 regulated 398 and 477 genes, respectively, which is comparable to the 625 genes responding to 10 nM 1,25(OH)2D3. The majority of these genes displayed specificity to the tested individuals, but not to the vitamin D metabolite. Interestingly, the genes MYLIP (myosin regulatory light chain interacting protein) and ABCG1 (ATP binding cassette subfamily G member 1) showed to be specific targets of 10,000 nM 25(OH)D3. In conclusion, 100- and 1000-fold higher 25(OH)D3 concentrations than the reference 10 nM 1,25(OH)2D3 are able to affect the transcriptome of PBMCs with a profile comparable to that of 1,25(OH)2D3.

The vitamin D3 metabolite 1α,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 α (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.

The molecular basis of vitamin D signaling implies that the metabolite 1α,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.

Fanconi anemia (FA) is a rare disorder with the clinical characteristics of (i) specific malformations at birth, (ii) progressive bone marrow failure already during early childhood and (iii) dramatically increased risk of developing cancer in early age, such as acute myeloid leukemia and squamous cell carcinoma. Patients with FA show DNA fragility due to a defect in the DNA repair machinery based on predominately recessive mutations in 23 genes. Interestingly, patients originating from the same family and sharing an identical mutation, frequently show significant differences in their clinical presentation. This implies that epigenetics plays an important role in the manifestation of the disease. The biologically active form of vitamin D, 1α,25-dihydroxyvitamin D3 controls cellular growth, differentiation and apoptosis via the modulation of the immune system. The nuclear hormone activates the transcription factor vitamin D receptor that affects, via fine-tuning of the epigenome, the transcription of >1000 human genes. In this review, we discuss that changes in the epigenome, in particular in immune cells, may be central for the clinical manifestation of FA. These epigenetic changes can be modulated by vitamin D suggesting that the individual FA patient’s vitamin D status and responsiveness are of critical importance for disease progression.

The biologically active form of vitamin D3, 1α,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.

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 D3 has, via its metabolite 1,25-dihydroxyvitaminD3 (1,25(OH)2D3), a high affinity to the transcription factor vitamin D receptor (VDR), and thereby directly affects the epigenome of its target tissues. Changing the transcriptome results in modulation of physiological function, such as calcium homeostasis and the response of innate and adaptive immunity. Genome-wide datasets on the 1,25(OH)2D3-triggered binding of VDR, its pioneer factors PU.1 and CEPBA, histone markers and chromatin accessibility in THP-1 human monocytes compared to those obtained in peripheral blood mononuclear cells from vitaminD3-supplemented human volunteers (VitDbol study) allow the assessment of the epigenome-wide effects of vitamin D.

Nutrigenomics studies how environmental factors, such as food intake and lifestyle, influence the expression of the genome. Vitamin D3 represents a master example of nutrigenomics, since via its metabolite 1α,25-dihydroxyvitamin D3, which binds with high-affinity to the vitamin D receptor, the secosteroid directly affects the epigenome and transcriptome at thousands of loci within the human genome. Vitamin D is important for both cellular metabolism and immunity, as it controls calcium homeostasis and modulates the response of the innate and adaptive immune system. At sufficient UV-B exposure, humans can synthesize vitamin D3 endogenously in their skin, but today’s lifestyle often makes the molecule a true vitamin and micronutrient that needs to be taken up by diet or supplementation with pills. The individual’s molecular response to vitamin D requires personalized supplementation with vitamin D3, in order to obtain optimized clinical benefits in the prevention of osteoporosis, sarcopenia, autoimmune diseases, and possibly different types of cancer. The importance of endogenous synthesis of vitamin D3 created an evolutionary pressure for reduced skin pigmentation, when, during the past 50,000 years, modern humans migrated from Africa towards Asia and Europe. This review will discuss different aspects of how vitamin D interacts with the human genome, focusing on nutritional epigenomics in context of immune responses. This should lead to a better understanding of the clinical benefits of vitamin D.

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 D3 has transcriptome- and genome-wide effects and activates, via the binding of its metabolite 1α,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.

The vitamin D receptor (VDR) is a transcription factor that mediates the genomic effects of 1α,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.

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.

Vitamin D deficiency has been associated with an increased risk of developing a number of diseases. Here we investigated samples from 71 pre-diabetic individuals of the VitDmet study, a 5-month high dose vitamin D3 intervention trial during Finnish winter, for their changes in serum 25-hydroxyvitamin D3 (25(OH)D3) concentrations and the expression of primary vitamin D target genes in peripheral blood mononuclear cells and adipose tissue. A negative correlation between serum concentrations of parathyroid hormone and 25(OH)D3 suggested an overall normal physiological vitamin D response among the participants. The genes CD14 and thrombomodulin (THBD) are up-regulated primary vitamin D targets and showed to be suitable gene expression markers for vitamin D signaling in both primary tissues. However, in a ranking of the samples concerning their expected response to vitamin D only the top half showed a positive correlation between the changes of CD14 or THBD mRNA and serum 25(OH)D3 concentrations. Interestingly, this categorization allows unmasking a negative correlation between changes in serum concentrations of 25(OH)D3 and the inflammation marker interleukin 6. We propose the genes CD14 and THBD as transcriptomic biomarkers, from which the effects of a vitamin D3 supplementation can be evaluated. These biomarkers allow the classification of subjects into those, who might benefit from a vitamin D3 supplementation, and others who do not.

Genome-wide analysis of vitamin D receptor (VDR) binding sites in THP-1 human monocyte-like cells highlighted the interleukin 8 gene, also known as chemokine CXC motif ligand 8 (CXCL8). CXCL8 is a chemotactic cytokine with important functions during acute inflammation as well as in the context of various cancers. The nine genes of the CXCL cluster and the strong VDR binding site close to the CXCL8 gene are insulated from neighboring genes by CCCTC-binding factor (CTCF) binding sites. Only CXCL8, CXCL6 and CXCL1 are expressed in THP-1 cells, but all three are up-regulated primary 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) target genes. Formaldehyde-assisted isolation of regulatory elements sequencing analysis of the whole CXCL cluster demonstrated 1,25(OH)2D3-dependent chromatin opening exclusively for the VDR binding site. In differentiated THP-1 cells the CXCL8 gene showed a 33-fold higher basal expression, but is together with CXCL6 and CXCL1 still a primary 1,25(OH)2D3 target under the control of the same genomic VDR binding site. In summary, both in undifferentiated and differentiated THP-1 cells the genes CXCL8, CXCL6 and CXCL1 are under the primary control of 1,25(OH)2D3 and its receptor VDR. Our observation provides further evidence for the immune-related functions of vitamin D.

The topology of nuclear receptor (NR) signaling is captured in a systems biological graphical notation. This enables us to identify a number of ‘design’ aspects of the topology of these networks that might appear unnecessarily complex or even functionally paradoxical. In realistic kinetic models of increasing complexity, calculations show how these features correspond to potentially important design principles, e.g.: (i) cytosolic ‘nuclear’ receptor may shuttle signal molecules to the nucleus, (ii) the active export of NRs may ensure that there is sufficient receptor protein to capture ligand at the cytoplasmic membrane, (iii) a three conveyor belts design dissipating GTP‐free energy, greatly aids response, (iv) the active export of importins may prevent sequestration of NRs by importins in the nucleus and (v) the unspecific nature of the nuclear pore may ensure signal‐flux robustness. In addition, the models developed are suitable for implementation in specific cases of NR‐mediated signaling, to predict individual receptor functions and differential sensitivity toward physiological and pharmacological ligands.

BACKGROUND: The differentiation of fibroblast-like pre-adipocytes to lipid-loaded adipocytes is regulated by a network of transcription factors, the most prominent one being the nuclear receptor peroxisome proliferator-activated receptor (PPAR) γ. However, many of the other 47 members of the nuclear receptor superfamily have an impact on adipogenesis, which in human cells has not been investigated in detail. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed by quantitative PCR all human nuclear receptors at multiple time points during differentiation of SGBS pre-adipocytes. The earliest effect was the down-regulation of the genes RARG, PPARD, REV-ERBA, REV-ERBB, VDR and GR followed by the up-regulation of PPARG, LXRA and AR. These observations are supported with data from 3T3-L1 mouse pre-adipocytes and primary human adipocytes. Investigation of the effects of the individual differentiation mix components in short-term treatments and of their omission from the full mix showed that the expression levels of the early-regulated nuclear receptor genes were most affected by the glucocorticoid receptor (GR) ligand cortisol and the phosphodiesterase inhibitor IBMX. Interestingly, the effects of both compounds converged to repress the genes PPARD, REV-ERBA, REV-ERBB, VDR and GR, whereas cortisol and IBMX showed antagonistic interaction for PPARG, LXRA and AR causing a time lag in their up-regulation. We hypothesize that the well-known auto-repression of GR fine-tunes the detected early responses. Consistently, chromatin immunoprecipitation experiments showed that GR association increased on the transcription start sites of the genes RARG, REV-ERBB, VDR and GR. CONCLUSIONS/SIGNIFICANCE: Adipocyte differentiation is a process, in which many members of the nuclear receptor superfamily change their mRNA expression. The actions of cortisol and IBMX converged to repress several nuclear receptors early in differentiation, while up-regulation of other nuclear receptor genes showed a time lag due to antagonisms of the signals. Our results place GR and its ligand cortisol as central regulatory factors controlling early regulatory events in human adipogenesis that precedes the regulation of the later events by PPARG.

Ghrelin may influence the development of obesity through its role in the control of energy balance, food intake, and regulation of body weight. The effects of ghrelin are mediated via the growth hormone secretagogue receptor (GHSR).We genotyped 7 single nucleotide polymorphisms (SNPs) in the GHSR gene and assessed the association between those SNPs and obesity and type 2 diabetes-related phenotypes from 507 middle-aged overweight persons with impaired glucose tolerance participating in the Finnish Diabetes Prevention Study (DPS). Additionally, we performed in silico screening of the 5'-regulatory region of GHSR and evaluated SNPs disrupting putative transcription factor (TF) binding sites in vitro with gelshift assays to determine differences in protein binding between different alleles of SNPs. Rs9819506 in the promoter region of GHSR was associated with body weight (p = 0.036); persons with rs9819506-AA genotype having the lowest body weight. Individuals with rs490683-CC genotype displayed highest weight loss in the whole study population (p = 0.032). The false discovery rate for these results was