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The myeloid master regulator CCAAT enhancer-binding protein alpha (CEBPA) is known as a pioneer factor. In this study, we report the CEBPA cistrome of THP-1 human monocytes after stimulation with the vitamin D receptor (VDR) ligand 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) for 2, 8 and 24 h. About a third of the genomic VDR binding sites co-located with those of CEBPA. In parallel, the binding strength of 5% of the CEBPA cistrome, i.e. some 1500 sites, is significantly (p < 0.001) affected by 1,25(OH) 2 D 3 . Transcriptome-wide analysis after CEBPA silencing indicated that the pioneer factor enhances both the basal expression and ligand inducibility of 70 vitamin D target genes largely involved in lipid signaling and metabolism. In contrast, CEBPA suppresses 82 vitamin D target genes many of which are related to the modulation of T cell activity by monocytes. The inducibility of the promoter-specific histone marker H3K4me3 distinguishes the former class of genes from the latter. Moreover, prominent occupancy of the myeloid pioneer factor PU.1 on 1,25(OH) 2 D 3 -sensitive CEBPA enhancers mechanistically explains the dichotomy of vitamin D target genes. In conclusion, CEBPA supports vitamin D signaling concerning actions of the innate immune system, but uses the antagonism with PU.1 for suppressing possible overreactions of adaptive immunity., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The micronutrient vitamin D significantly modulates the human epigenome via enhancing genome-wide the rate of accessible chromatin and vitamin D receptor (VDR) binding. This study focuses on histone marks of active chromatin at promoter and enhancer regions and investigates, whether these genomic loci are sensitive to vitamin D. The epigenome of THP-1 human monocytes contains nearly 23,000 sites with H3K4me3 histone modifications, 550 of which sites are significantly (p < 0.05) modulated by stimulation with the VDR ligand 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ). H3K27ac histone modifications mark active chromatin and 2473 of 45,500 sites are vitamin D sensitive. The two types of ligand-dependent histone marks allow to distinguish promoter and enhancer regulation by vitamin D, respectively. Transcription start site overlap is the prime attribute of ligand-dependent H3K4me3 marks, while VDR co-location is the top ranking parameter describing 1,25(OH) 2 D 3 -sensitive H3K27ac marks at enhancers. A categorization of 1,25(OH) 2 D 3 -sensitive histone marks by machine learning algorithms - using the attributes overall peak strength and ligand inducibility - highlights 260 and 287 regions with H3K4me3 and H3K27ac modifications, respectively. These loci are found at the promoter regions of 59 vitamin D target genes and their associated enhancers. In this way, ligand-dependent histone marks provide a link of the effects of 1,25(OH) 2 D 3 on the epigenome with previously reported mRNA expression changes of vitamin D target genes. In conclusion, the human epigenome responds also on the level of histone modifications to 1,25(OH) 2 D 3 stimulation. This allows a more detailed understanding of vitamin D target gene regulation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The transcription factor vitamin D receptor (VDR) is the exclusive nuclear target of the biologically active form of vitamin D (1,25(OH) 2 D 3 ). In THP-1 human monocytes we obtained a highly accurate VDR cistrome after 2 and 24h ligand stimulation comprising >11,600 genomic loci, 78% of which were detected exclusively after 24h. In contrast, a group of 510 persistent VDR sites occurred at all conditions and some 2100 VDR loci were only transiently occupied. Machine learning and statistical analysis as well as a comparison with the re-analyzed B cell VDR cistrome indicated a subgroup of 339 highly conserved persistent VDR sites that were suited best for describing vitamin D-triggered gene regulatory scenarios. The 1,25(OH) 2 D 3 -dependent transcriptome of THP-1 cells comprised 587 genes, 311 of which were primary targets with main functions in the immune system. More than 97% of the latter genes were located within 1,25(OH) 2 D 3 -modulated topologically associated domains (TADs). The number of persistent and transient VDR sites was found to be the main discriminator for sorting these TADs into five classes carrying vitamin D target genes involved in distinct biological processes. In conclusion, specific regulation of biological processes by vitamin D depends on differences in time-dependent VDR binding., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The ETS-domain transcription factor PU.1 acts as a pioneer factor for other transcription factors including nuclear receptors. In this study, we report that in THP-1 human monocytes the PU.1 cistrome comprises 122,319 genomic sites. Interestingly, at 6498 (5.3%) of these loci PU.1 binding was significantly modulated by the vitamin D receptor (VDR) ligand 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ). In most cases 1,25(OH) 2 D 3 increased PU.1 association, which correlated strongly with VDR co-location and overlap ratios for canonical DR3-type VDR binding sites. Genome-wide 6488 sites associating both with PU.1 and VDR as well as 5649 non-VDR overlapping, 1,25(OH) 2 D 3 -sensitive PU.1 loci represent the PU.1-VDR crosstalk and can be described by four gene regulatory scenarios, each. Chromatin accessibility was the major discriminator between these models. The location of the PU.1 binding loci in open chromatin coincided with a significantly smaller mean distance to the closest 1,25(OH) 2 D 3 target gene. PU.1 knockdown indicated that the pioneer factor is relevant for the transcriptional activation of 1,25(OH) 2 D 3 target genes but its impact differed in magnitude and orientation. In conclusion, PU.1 is an important modulator of VDR signaling in monocytes, including but also exceeding its role as a pioneer factor, but we found no evidence for a direct interaction of both proteins., (Copyright © 2017 Elsevier B.V. All rights reserved.)

5-Lipoxygenase (5-LO) gene expression is strongly upregulated during induction of myeloid cell differentiation by 1alpha,25-dihydroxyvitamin D(3) (calcitriol) and transforming growth factor-beta (TGFbeta) 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 TGFbeta 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 TGFbeta 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.

5-Lipoxygenase (5-LO) expression is strongly induced by transforming growth factor-beta (TGFbeta) and 1alpha,25-dihydroxyvitamin D(3) in Mono Mac 6 cells. Since Smads have been described as downstream effectors of TGFbeta, we have investigated the role of the TGFbeta/Smad signalling system in the regulation of 5-LO gene expression. The rapid induction of 5-LO mRNA, determined with real-time quantitative RT-PCR, suggests that 5-LO is a primary TGFbeta target gene. In reporter gene assays with plasmids containing the 5-LO promoter plus different parts of the gene, Smads3/4 mediate a prominent upregulation of reporter activity that strongly depends on the coding sequence and to a lesser extent on the 3'-UTR and introns J-M. Deletion studies revealed the most profound decrease of inducibility by Smads3/4 when exons 10-14 are deleted. Sequence analysis and deletion studies indicate the existence of up to four Smad binding elements and at least one TGFbeta responsive element far downstream of the transcriptional start site.