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Patterns of genome-wide VDR locations

ABSTRACT: 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 (ChIP) coupled with massive parallel 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 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 DR3-type binding site screening using HOMER of the six VDR ChIP-seq datasets suggest that DR3 sites are strongly associated with the ligand-responsiveness of VDR occupation. 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE53041 | GEO | 2014/11/14

SECONDARY ACCESSION(S): PRJNA230725

REPOSITORIES: GEO

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Project description:The genome-wide analysis of the binding sites of the transcription factor vitamin D receptor (VDR) is essential for a global appreciation the physiological impact of the nuclear hormone 1M-NM-1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Genome-wide analysis of lipopolysaccharide (LPS)-polarized THP-1 human monocytic leukemia cells via chromatin immunoprecipitation (ChIP) coupled with massive parallel 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 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 DR3-type binding site screening using HOMER of the six VDR ChIP-seq datasets suggest that DR3 sites are strongly associated with the ligand-responsiveness of VDR occupation. 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. Systematic reanalysis of 5 published VDR ChIP-seq datasets together with a new dataset from 24 h LPS-treated THP-1 cells at the unstimulated state and after 80 min ligand (10 nM 1M-NM-1,25(OH)2D3 (1,25D, calcitriol)) treatment. See GSM1280896 and GSM1280896 Sample records for data processing information. GSE53041_README.txt has additional details.

Project description:The biological effects of 1?,25-dihydroxyvitamin D3 (1,25(OH)2D3) on osteoblast differentiation and function differ significantly depending upon the cellular state of maturation. To explore this phenomenon mechanistically, we examined the impact of 1,25(OH)2D3 on the transcriptomes of both pre-osteoblastic (POBs) and differentiated osteoblastic (OBs) MC3T3-E1 cells, and assessed localization of the vitamin D receptor (VDR) at sites of action on a genome-scale using ChIP-seq analysis. We observed that the 1,25(OH)2D3-induced transcriptomes of POBs and OBs were quantitatively and qualitatively different, supporting not only the altered biology observed but the potential for a change in VDR interaction at the genome as well. This idea was confirmed through discovery that VDR cistromes in POBs and OBs were also strikingly different. Depletion of VDR binding sites in OBs, due in part to reduced VDR expression, was the likely cause of the loss of VDR-target gene interaction. Continued novel regulation by 1,25(OH)2D3, however, suggested that factors in addition to the VDR might also be involved. Accordingly, we show that transcriptomic modifications are also accompanied by changes in genome binding of the master osteoblast regulator RUNX2 and the chromatin remodeler C/EBP?. Importantly, genome occupancy was also highlighted by the presence of epigenetic enhancer signatures which were selectively changed in response to both differentiation and 1,25(OH)2D3. The impact of VDR, RUNX2, and C/EBP? on osteoblast differentiation is exemplified by their actions at the Runx2 and Sp7 gene loci. We conclude that each of these mechanisms may contribute to the diverse actions of 1,25(OH)2D3 on differentiating osteoblasts. 4 transcription factors and 5 histone modifications were examined in undifferentiated MC3T3-E1 cells as well as post 15 day osteogenic differentiation MC3T3-E1 cells, which were treated for 3 hours prior to ChIP assay with ethanol vehicle or with 10-7M 1,25(OH)2D3. For the vehicle matched samples for RUNX2, CEBP beta and histones, please refer to study GSE41955. The samples were completed in biological replicate and examined separately.

Project description:The biological effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) on osteoblast differentiation and function differ significantly depending upon the cellular state of maturation. To explore this phenomenon mechanistically, we examined the impact of 1,25(OH)2D3 on the transcriptomes of both pre-osteoblastic (POBs) and differentiated osteoblastic (OBs) MC3T3-E1 cells, and assessed localization of the vitamin D receptor (VDR) at sites of action on a genome-scale using ChIP-seq analysis. We observed that the 1,25(OH)2D3-induced transcriptomes of POBs and OBs were quantitatively and qualitatively different, supporting not only the altered biology observed but the potential for a change in VDR interaction at the genome as well. This idea was confirmed through discovery that VDR cistromes in POBs and OBs were also strikingly different. Depletion of VDR binding sites in OBs, due in part to reduced VDR expression, was the likely cause of the loss of VDR-target gene interaction. Continued novel regulation by 1,25(OH)2D3, however, suggested that factors in addition to the VDR might also be involved. Accordingly, we show that transcriptomic modifications are also accompanied by changes in genome binding of the master osteoblast regulator RUNX2 and the chromatin remodeler C/EBPβ. Importantly, genome occupancy was also highlighted by the presence of epigenetic enhancer signatures which were selectively changed in response to both differentiation and 1,25(OH)2D3. The impact of VDR, RUNX2, and C/EBPβ on osteoblast differentiation is exemplified by their actions at the Runx2 and Sp7 gene loci. We conclude that each of these mechanisms may contribute to the diverse actions of 1,25(OH)2D3 on differentiating osteoblasts. RNA was isolated and applied to gene expression microarrays in undifferentiated MC3T3-E1 cells as well as post 15 day osteogenic differentiation MC3T3-E1 cells, which were treated for 24 hours with 10-7M 1,25(OH)2D3. For the vehicle matched samples, please refer to study GSE41955. The samples were completed in biological triplicate.

Project description:Osteocytes are derived from osteoblast lineage cells that become progressively embedded in mineralized bone. Development of the osteocytogenic cell line IDG-SW3 has enabled a temporal and mechanistic investigation of this process. Through RNA-seq analyses, we show that while substantial changes in gene expression occur during the osteoblast to osteocyte transition, the majority of the transcriptome remains qualitatively osteoblast-like. Genes either up-regulated or expressed uniquely in the osteocyte include local and systemic factors such as Sost and Fgf23 as well as genes implicated in neuronal, muscle, vascular, or regulatory function. As assessed by ChIP-seq, numerous changes in epigenetic histone modifications also occur during osteocytogenesis; these are largely qualitative rather than quantitative. Specific epigenetic changes correlate with altered gene expression patterns that are observed during the transition. These genomic changes likely influence the highly restricted transcriptomic response to 1,25(OH)2D3 that occurs during differentiation. VDR binding in osteocytes revealed an extensive cistrome co-occupied by RXR and located predominantly at sites distal to regulated genes. Although sites of VDR binding were apparent near many 1,25(OH)2D3-regulated genes, the expression of others adjacent to VDR binding sites were unaffected; lack of VDR binding was particularly prevalent at down-regulated genes. Interestingly, 1,25(OH)2D3 was found to induce the Boc and Cdon co-receptors that are active in hedgehog signaling in osteocytes. We conclude that osteocytogenesis is accompanied by changes in gene expression that may be driven by both genetic and epigenetic components. These changes are likely responsible for the osteocyte phenotype and may contribute to reduced sensitivity to 1,25(OH)2D3. ChIP-seq was performed on differentiated IDGSW3 cells at day 35 for VDR and RXR following 3 hour vehicle or 1,25(OH)2d3 treatment in biological replicate. ChIP-seq was also performed for H3K4me1, H3K4me2, H3K4me3, H3K27Ac, H4K5Ac, H3K9Ac, H4K20me1, H3K36me3, or H3K9me3 at day 3 and day 35 of differentiation in the basal condition.

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Patterns of genome-wide VDR locations

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