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Spatial genome organization is tightly controlled by several regulatory mechanisms and is essential for gene expression control. Nuclear receptors are ligand-activated transcription factors that modulate physiological and pathophysiological processes and are primary pharmacological targets. DNA binding of the important loop-forming insulator protein CCCTC-binding factor (CTCF) was modulated by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). We performed CTCF HiChIP assays to produce the first genome-wide dataset of CTCF long-range interactions in 1,25(OH)2D3-treated cells, and to determine whether dynamic changes of spatial chromatin interactions are essential for fine-tuning of nuclear receptor signaling. We detected changes in 3D chromatin organization upon vitamin D receptor (VDR) activation at 3.1% of all observed CTCF interactions. VDR binding was enriched at both differential loop anchors and within differential loops. Differential loops were observed in several putative functional roles including TAD border formation, promoter-enhancer looping, and establishment of VDR-responsive insulated neighborhoods. Vitamin D target genes were enriched in differential loops and at their anchors. Secondary vitamin D effects related to dynamic chromatin domain changes were linked to location of downstream transcription factors in differential loops. CRISPR interference and loop anchor deletion experiments confirmed the functional relevance of nuclear receptor ligand-induced adjustments of the chromatin 3D structure for gene expression regulation., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In vitro cell culture studies showed that the hormonal form of vitamin D 3 , 1α,25-dihydroxyvitamin D 3 , significantly (p < 0.05) affects the human epigenome at thousands of genomic loci. Phase II of the VitDbol vitamin D intervention trial (NCT02063334) involved a proof-of-principle study of one individual, who was exposed three times every 28 days to an oral bolus (2000 μg) of vitamin D 3 . Blood samples were taken directly before each supplementation as well as one and two days after, chromatin was isolated from peripheral blood mononuclear cells without any further in vitro culture and at all nine time points epigenome-wide chromatin accessibility was assessed by applying FAIRE-seq (formaldehyde-assisted isolation of regulatory elements sequencing). The vitamin D 3 bolus resulted in an average raise in 25-hydroxyvitamin D 3 (25(OH)D 3 ) serum concentration of 11.9 and 19.4 nM within one and two days, respectively. Consistently accessible chromatin was detected at 5205 genomic loci, the 853 most prominent of which a self-organizing map algorithm classified into early, delayed and non-responding genomic regions: 70 loci showed already after one day and 361 sites after two days significant (p < 0.0001) chromatin opening or closing. Interestingly, more than half of these genomic regions overlap with transcription start sites, but the change of chromatin accessibility at these sites has no direct effect on the transcriptome. Some of the vitamin D responsive chromatin sites cluster at specific loci within the human genome, the most prominent of which is the human leukocyte antigen region in chromosome 6. In conclusion, this study demonstrates that under in vivo conditions a rather minor rise in 25(OH)D 3 serum levels is sufficient to result in significant changes at hundreds of sites within the epigenome of human leukocytes., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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 D 3 (1,25(OH) 2 D 3 ). 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) 2 D 3 target gene. These TADs can explain the regulatory scenarios of up to 70% of all 1,25(OH) 2 D 3 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) 2 D 3 -dependent action of CTCF. The co-location of VDR and 1,25(OH) 2 D 3 -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) 2 D 3 -mediated gene regulation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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 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., (© 2013.)

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.

A global understanding of the actions of the nuclear hormone 1α,25-dihydroxyvitamin D(3) (1α,25(OH)(2)D(3)) and its vitamin D receptor (VDR) requires a genome-wide analysis of VDR binding sites. In THP-1 human monocytic leukemia cells we identified by ChIP-seq 2340 VDR binding locations, of which 1171 and 520 occurred uniquely with and without 1α,25(OH)(2)D(3) treatment, respectively, while 649 were common. De novo identified direct repeat spaced by 3 nucleotides (DR3)-type response elements (REs) were strongly associated with the ligand-responsiveness of VDR occupation. Only 20% of the VDR peaks diminishing most after ligand treatment have a DR3-type RE, in contrast to 90% for the most growing peaks. Ligand treatment revealed 638 1α,25(OH)(2)D(3) target genes enriched in gene ontology categories associated with immunity and signaling. From the 408 upregulated genes, 72% showed VDR binding within 400 kb of their transcription start sites (TSSs), while this applied only for 43% of the 230 downregulated genes. The VDR loci showed considerable variation in gene regulatory scenarios ranging from a single VDR location near the target gene TSS to very complex clusters of multiple VDR locations and target genes. In conclusion, ligand binding shifts the locations of VDR occupation to DR3-type REs that surround its target genes and occur in a large variety of regulatory constellations.

Ligand-regulated nuclear receptors, such as estrogen receptors, glucocorticoid receptor, vitamin D receptor, and peroxisome proliferator-activated receptors, belong to the most widely studied and best understood transcription factors. Therefore, the dynamic nature of transcriptional regulation was observed first with different members of the nuclear receptor superfamily, but is now also extended to other transcription factors, such as nuclear factor κB. Dynamic and in part cyclical processes were observed on the level of translocation into the nucleus, association with genomic binding sites, exchange of co-regulators and chromatin modifiers, occurrence of chromatin marks, and activities of RNA polymerase II resulting in mRNA synthesis. In this review, we summarize recent findings on the dynamic regulation of nuclear receptor target genes in the chromatin context.

A genome-wide data set on vitamin D receptor (VDR) binding sites in human THP-1 cells (monocytes) led us to the genomic region around the ASAP2 (Arf-GAP with SH3 domain, ankyrin repeat and PH domain 2) gene, whose product is involved in the regulation of vesicular transport, cellular migration and autophagy. Using ENCODE data, we demonstrated that the ASAP2 gene is flanked by conserved binding sites of the insulating transcription factor CTCF. These sites define different chromosomal domains containing the ASAP2 gene, up to six additional genes and three VDR binding sites. In human monocytes (THP-1 cells) the ASAP2 gene is more weakly expressed but more and faster inducible by the biologically active form of vitamin D, 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), than in M2-type macrophages (phorbol ester-differentiated THP-1 cells). Within the investigated genomic region, the basal mRNA expressions of the neighboring genes are comparably high in both monocytes and macrophages, but the ASAP2 gene is the only primary 1,25(OH) 2 D 3 target. The three VDR binding sites located 54, 436 and 574 kb downstream of the ASAP2 transcription start site each carry a sequence formed by a direct repeat with three intervening nucleotides (DR3). Ligand-inducible VDR binding was confirmed to all three genomic sites in monocytes and macrophages. Taken together, the region around the ASAP2 gene is genome-wide highlighted as a special attraction point for the VDR, but the presently sole known functional consequence of the binding of VDR to three sites within this chromosomal region is that ASAP2 is a primary 1,25(OH) 2 D 3 target gene in monocytes and macrophages. This article is part of a Special Issue entitled ‘16th Vitamin D Workshop’. [ABSTRACT FROM AUTHOR]

Vitamin D 3 belongs to the few nutritional compounds that has, via the binding of its metabolite 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) to the transcription factor vitamin D receptor (VDR), a direct effect on gene regulation. The relation of thousands of genomic VDR-binding sites to a few hundred primary 1,25(OH) 2 D 3 target genes is still largely unresolved. We studied chromatin domains containing genes for the adhesion molecules CD97 and LRRC8A, the glucose transporter SLC37A2 and the coactivator NRIP1. These domains vary significantly in size (7.3 to 956 kb) but contain each one major VDR-binding site. In monocytic cells these four sites are associated with open chromatin and occupied by VDR, while in macrophage-like cells only the sites of LRRC8A , SLC37A2 and NRIP1 are accessible and receptor bound. The VDR site of CD97 does, in contrast to the three other loci, not carry any DR3-type binding sequence. CD97 , LRRC8A , SLC37A2 and NRIP1 are early responding 1,25(OH) 2 D 3 target genes in monocytic cells, while in macrophage-like cells they respond less and, in part, delayed. In primary human peripheral blood mononuclear cells from 71 prediabetic subjects of a vitamin D 3 intervention study (VitDmet) CD97 , LRRC8A , SLC37A2 and NRIP1 can be used as transcriptomic biomarkers for classifying human individuals for their possible benefit from vitamin D 3 supplementation. In particular, NRIP1 exceeds the potential of the previously identified marker CD14 by more than 40% and seems to be a well-suited molecular marker for the vitamin D 3 status in the hematopoietic system. [ABSTRACT FROM AUTHOR]

Genome- and transcriptome-wide data has significantly increased the amount of available information about primary 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) target genes in cancer cell models, such as human THP-1 myelomonocytic leukemia cells. In this study, we investigated the genes G0S2, CDKN1A and MYC as master examples of primary vitamin D receptor (VDR) targets being involved in the control of cellular proliferation. The chromosomal domains of G0S2 and CDKN1A are 140-170 kb in size and contain one and three VDR binding sites, respectively. This is rather compact compared to the MYC locus that is 15 times larger and accommodates four VDR binding sites. All eight VDR binding sites were studied by chromatin immunoprecipitation in THP-1 cells. Interestingly, the site closest to the transcription start site of the down-regulated MYC gene showed 1,25(OH)2D3-dependent reduction of VDR binding and is not associated with open chromatin. Four of the other seven VDR binding regions contain a typical DR3-type VDR binding sequence, three of which are also occupied with VDR in macrophage-like cells. In conclusion, the three examples suggest that each VDR target gene has an individual regulatory scenario. However, some general components of these scenarios may be useful for the development of new therapy regimens [ABSTRACT FROM AUTHOR]

Abstract: The basic helix-loop-helix protein BHLHE40 functions as a transcriptional repressor and is involved in the control of cellular growth, development and circadian rhythms. By the use of genome-wide data on vitamin D receptor (VDR) location, open chromatin and histone modification backed-up by gene-specific mRNA expression studies we show that the human BHLHE40 gene is dynamically up-regulated by the VDR ligand 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) and down-regulated by the histone deactylase inhibitor trichostatin A. The VDR binding site is located 1.7kb upstream of the transcription start site of the BHLHE40 gene and the chromatin at this genomic site is significantly opened by treatment with 1α,25(OH)2D3. The stair case style fluctuations in the BHLHE40 mRNA accumulation relate to the short half-life of the gene's mRNA of 0.9h. The identification of the widely expressed BHLHE40 gene as a primary VDR target may explain secondary effects of 1α,25(OH)2D3 on BHLHE40 responding genes. This article is part of a Special Issue entitled ‘Vitamin D Workshop’. [Copyright &y& Elsevier]

Abstract: 5-lipoxygenase (5-LO) is the key enzyme in the biosynthesis of proinflammatory leukotrienes. The 5-LO gene is a primary target of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) and its expression is prominently increased during myeloid cell differentiation. Since no functional vitamin D response element (VDRE) has been reported for this gene so far, we performed in silico screening of the whole 5-LO gene area (84 kb, including 10 kb promoter region) and identified 22 putative VDREs. Both gelshift and reporter gene assays identified four of these candidates as functional VDREs. Their approximate positions are −2,250 (promoter), +21,400 (intron 2), +42,000 (intron 4) and +50,600 (intron 5) in relation to the transcription start site (TSS). Remarkably, the VDRE at position +42,000 is one of the strongest known VDREs of the human genome. Chromatin immunoprecipitation (ChIP) assays demonstrated simultaneous association of vitamin D receptor (VDR), retinoid X receptor (RXR) and RNA polymerase II (Pol II) to the 5-LO gene regions containing two of these four putative VDREs. This indicates DNA looping of the TSS to even very distant gene regions. In summary, we suggest that the upregulation of the primary 1α,25(OH)2D3 target 5-LO is mediated in vivo by a prominent VDRE in intron 4. [Copyright &y& Elsevier]

Abstract: 5-Lipoxygenase (5-LO) gene expression is strongly upregulated during induction of myeloid cell differentiation by 1α,25-dihydroxyvitamin D3 (calcitriol) and transforming growth factor-β (TGFβ) in a promoter-independent manner. In an activity-guided approach using reporter gene assays where the distal part of the 5-LO gene was included in the reporter gene plasmid, we localized vitamin D response elements (VDREs) within exon 10, exon 12, and intron M. We found that these newly identified VDRE sites are bound by vitamin D receptor both in vitro by gel-shift analysis and in vivo by chromatin immunoprecipitation assays. In reporter gene assays, the distal part of the 5-LO gene has promoter-like activity that is inducible by calcitriol in a vitamin D receptor-dependent manner. The vitamin D effects were attenuated when the VDREs in exon 10, exon 12, and intron M were deleted or mutated. When we analyzed the effects of calcitriol plus TGFβ on chromatin modifications at exon 10, exon 12, and intron M of the 5-LO gene in Mono Mac 6 cells by chromatin immunoprecipitation analysis, we found an increase in histone H4 K20 monomethylation and a prominent presence of histone H3 K36 trimethylation. Combined treatment with calcitriol and TGFβ also increased histone H4 acetylation, a marker for open chromatin, and the elongation form of RNA polymerase II at these sites, whereas the transcription initiation marker histone H3 K4 trimethylation was almost undetectable. The data suggest that calcitriol induces chromatin opening and transcript elongation via VDREs located at the 3′-end of the 5-LO gene. [Copyright &y& Elsevier]

Abstract: Interleukin 12 (IL-12) is a heterodimeric, pro-inflammatory cytokine that plays a central role in activation and differentiation of CD4+ T cells into interferon-γ secreting T-helper type 1 cells. IL-12B, a gene encoding the larger subunit of active IL-12, has been reported to be down-regulated by the nuclear hormone 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), but the mechanism of the regulation is unknown. In this study, we have examined the molecular mechanism of transcriptional regulation of the IL-12B gene by 1α,25(OH)2D3 in lipopolysaccharide (LPS)-treated human monocytes (THP-1). Quantitative RT-PCR showed that IL-12B mRNA displays a cyclical expression profile and is down-regulated 2.8-fold during the first 8h and even 12.1-fold 24h after exposure to 1α,25(OH)2D3. Gel shift and quantitative chromatin immunoprecipitation (ChIP) assays demonstrated vitamin D receptor (VDR) binding to genomic regions 480 and 6300bp upstream of the IL-12B transcription start site (TSS). Quantitative ChIP assays also revealed that together with VDR and its partner RXR the above regions recruited the co-repressor NCOR2/SMRT and histone deacetylase 3 leading to a decreased histone 4 acetylation and increased histone 3 trimethylation at the IL-12B promoter and its TSS. We suggest that these repressive epigenetic changes eventually cause down-regulation of IL-12 expression. This article is part of a Special Issue entitled: 11th European Symposium on Calcium. [Copyright &y& Elsevier]

Abstract: The biologically most active vitamin D compound, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), influences the status of inflammation by modulating the expression of several cytokine genes. In this study, we have examined the mechanism of transcriptional regulation of interleukin 10 (IL-10) by 1α,25(OH)2D3 in lipopolysaccharide (LPS)-treated human monocytes (THP-1). Quantitative PCR showed that IL-10 mRNA expression was significantly down-regulated (2.8-fold) during the first 8h of 1α,25(OH)2D3 treatment, while after 48h it was up-regulated (3-fold). Gel shift and quantitative chromatin immunoprecipitation (ChIP) assays showed that the vitamin D receptor (VDR) binds in a cyclical fashion to a promoter region 1500–1700bp upstream of the IL-10 transcription start site (TSS) containing two conserved VDR binding sites. Targeting of VDR binding sites by enhancer specific duplex RNAs revealed that only the more distal element is functional and chromosome conformation capture analysis suggested that this region loops 1α,25(OH)2D3-dependently to the TSS. Quantitative ChIP and micrococcal nuclease assays also revealed 1α,25(OH)2D3-dependent cyclical epigenetic changes and nucleosome remodeling at this promoter region. In conclusion, in LPS-treated THP-1 cells the primary effect of 1α,25(OH)2D3 on IL-10 expression is down-regulation, which is achieved via a cyclical recruitment of VDR to the promoter. [Copyright &y& Elsevier]