Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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1,25-Dihydroxyvitamin D3 Controls a Cohort of Vitamin D Receptor Target Genes in the Proximal Intestine That Is Enriched for Calcium Regulating Components (ChIP-seq)

ABSTRACT: 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) plays an integral role in calcium homeostasis in higher organisms through its actions in the intestine, kidney and skeleton. Interestingly, while several intestinal genes are known to play a contributory role in calcium homeostasis, the entire caste of key components remains to be identified. To examine the vitamin D receptor (VDR) cistrome in this issue, we conducted a ChIP-seq analysis of binding sites for the VDR across the proximal intestine in vitamin D-sufficient normal mice treated with vehicle or 1,25(OH)2D3. The residual VDR cistrome was comprised of 4617 sites which was increased almost 4-fold following hormone treatment. Interestingly, the majority of the genes regulated by 1,25(OH)2D3 in each diet group as well as those found in common in both groups contained frequent VDR sites that likely regulated their expression. This study revealed a global VDR cistrome regulating a network of genes in the intestine that both represent direct targets of vitamin D action in mice and are involved in calcium absorption. Wildtype mice were fed a standard rodent chow diet (Harlan Teklad, #5008). At 8 weeks of age, 3 mice were treated with 1,25(OH)2D3 (10 ng/g bw) or vehicle control and the proximal half of the small intestine (duodenum and jejunum) was collected 1 h later.

ORGANISM(S): Mus musculus

SUBMITTER: Mark Meyer

PROVIDER: E-GEOD-69179 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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: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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1,25-Dihydroxyvitamin D3 Controls a Cohort of Vitamin D Receptor Target Genes in the Proximal Intestine That Is Enriched for Calcium Regulating Components (ChIP-seq)

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