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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 (RNA-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 this issue, Cyp27b1 null mice on either a normal or a high calcium/phosphate-containing rescue diet were treated with either vehicle or 1,25(OH)2D3 and evaluated 6h later. RNA samples from duodena were then subjected to RNA-seq analysis and the data analyzed bioinformatically. 1,25(OH)2D3 altered expression of large collections of genes raised under either dietary condition. 45 genes were found common to both 1,25(OH)2D3-treated groups, and were comprised of genes previously linked to intestinal calcium uptake including S100g, Trpv6, Atp2b1 and Cldn2 as well as others. An additional distinct network of 56 genes was regulated exclusively by diet. This study revealed a global network of genes in the intestine that both represent direct targets of vitamin D action in mice and are involved in calcium absorption.

ORGANISM(S): Mus musculus

PROVIDER: GSE69170 | GEO | 2015/06/03

SECONDARY ACCESSION(S): PRJNA284754

REPOSITORIES: GEO

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Project description:Human kidney was obtained from the University of Wisconsin Madison Organ Procurement Program from a deceased 69 year old female patient and the cortex was dissected from 3 different cortex locations. Associated publication abstract: Targeted genomic deletions identify diverse enhancer functions and generate a kidney-specific, endocrine-deficient Cyp27b1 pseudo-null mouse Vitamin D3 is terminally bioactivated in the kidney to 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) via cytochrome P450 family 27 subfamily B member 1 (CYP27B1), whose gene is regulated by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and 1,25(OH)2D3. Our recent genomic studies in the mouse have revealed a complex kidney-specific enhancer module within the introns of adjacent methyltransferase like 1 (Mettl1) and Mettl21b that mediate basal and PTH induced expression of Cyp27b1 and FGF23- and 1,25(OH)2D3-mediated repression. Gross deletion of these segments in mice has severe consequences on Cyp27b1 regulation and skeletal phenotype, but does not affect Cyp27b1 expression in non-renal target cells (NRTCs). Here, we report a bimodal activity in the Mettl1 intronic enhancer with components responsible for PTH-mediated Cyp27b1 induction and 1,25(OH)2D3-mediated repression and additional activities, including FGF23 repression, within the Mettl21b enhancers. Deletion of both submodules eliminated basal Cyp27b1 expression and regulation in the kidney, leading to systemic and skeletal phenotypes similar to those of Cyp27b1-null mice. However, basal expression and lipopolysaccharide-induced regulation of Cyp27b1 in NRTCs was unperturbed. Importantly, dietary normalization of calcium, phosphate, PTH, and FGF23 rescued the skeletal phenotype of this mutant mouse, creating an ideal in vivo model to study non-renal 1,25(OH)2D3 production in health and disease. Finally, we confirmed a conserved chromatin landscape in human kidney that is similar to that in mouse. These findings define a finely balanced homeostatic mechanism involving PTH and FGF23 together with protection from 1,25(OH)2D3 toxicity that is responsible for both adaptive vitamin D metabolism and mineral regulation.

Project description:Heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2 plays a pivotal role in vitamin D receptor (VDR) signaling by acting as a vitamin D response element (VDRE)-binding protein (VDRE-BP). Transcriptional regulation by active 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) involves occupancy of VDRE by VDRE-BP or 1,25(OH)2D3 bound-VDR. This relationship is disrupted by over-expression of VDRE-BP and can cause a form of human hereditary vitamin D-resistant rickets (HVDRR). DNA array analyses using B-cells from an HVDRR patient and matched control defined a sub-cluster of genes where 1,25(OH)2D3-regulated transcription was abrogated by over-expression of VDRE-BP. Amongst these, the DNA-damage-inducible transcript 4 (DDIT4), an inhibitor of mammalian target of rapamycin (mTOR) signaling, was also induced by 1,25(OH)2D3 in human osteoblasts. Chromatin immunoprecipitation using 1,25(OH)2D3-treated osteoblasts confirmed that liganded VDR and VDRE-BP compete for binding to the proximal promoter of the DDIT4 gene in a similar fashion to other known 1,25(OH)2D3-target genes. Treatment of osteoblasts with 1,25(OH)2D3 induced DDIT4 expression and suppressed phosphorylated S6K1T389 protein (a downstream target of mTOR). The functional importance of this for 1,25(OH)2D3 responses in osteoblasts was underlined by the fact that siRNA knockdown of DDIT4 expression suppressed antiproliferative and cell growth responses to 1,25(OH)2D3. These data confirm that VDRE-BP is required for normal 1,25(OH)2D3-mediated transcription and cell function in osteoblasts. Conversely over-expression of VDRE-BP exerts a dominant-negative effect on transcription of 1,25(OH)2D3-target genes. Characterization of VDRE-BP action in 1,25(OH)2D3-treated osteoblasts highlights an entirely novel role for vitamin D as a regulator of mTOR – a known ‘master regulator’ of cell function. We performed gene expression microarray analysis in HVDRR EBV-transformed B-cells and control cells in the presence or absence of vitamin D.

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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 (RNA-seq)

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