Corepressors (NCoR and SMRT) as well as coactivators are recruited to positively regulated 1α,25-dihydroxyvitamin D3-responsive genes

doi:10.1016/j.jsbmb.2012.08.006

The Journal of Steroid Biochemistry and Molecular Biology

Volume 136, July 2013, Pages 120-124

https://doi.org/10.1016/j.jsbmb.2012.08.006 Get rights and content

Abstract

Transcription factors require coactivators and corepressors to modulate transcription in mammalian cells. The vitamin D receptor (VDR) utilizes coactivators and corepressors to gain tight control over the activity of a diverse set of genes that can regulate calcium transport, slow proliferation and promote immune responses. We have recently established the VDR/RXR cistrome in human colon cancer cells and have linked these binding sites to the genes that are regulated by 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃). In additional studies described herein, we demonstrate that the coactivators SRC1, CBP and MED1 are recruited to upregulated genes to facilitate transcription as expected. SRC1 was the most highly correlated to VDR/RXR binding (50%). However, we also found that corepressor molecules such as NCoR and SMRT were present along with SRC1, CBP or MED1 at these 1,25(OH)₂D₃ activated gene enhancers. Interestingly, genome-wide NCoR binding mimicked VDR binding by increasing its association with VDR binding in response to 1,25(OH)₂D₃ treatment. Overall, these data indicate a complex role for corepressor and coactivator complexes in the activation or active repression of 1,25(OH)₂D₃ responsive genes.

This article is part of a Special Issue entitled ‘Vitamin D Workshop’.

Highlights

▸ We performed genome-wide ChIP-seq analysis on coactivators and corepressors during 1,25(OH)₂D₃ treatments. ▸ The coactivator SRC-1 was most highly correlated with VDR/RXR during 1,25(OH)₂D₃ activation. ▸ Corepressors NCoR and SMRT were also recruited to activation complexes near genes stimulated by 1,25(OH)₂D₃. ▸ NCoR showed an increased association with VDR only after 1,25(OH)₂D₃ treatment, as exemplified by the gene c-FOS.

Introduction

The steroid hormone 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) tightly regulates calcium and phosphorus homeostasis [1]. 1,25(OH)₂D₃ acts to regulate genes through its receptor, the vitamin D receptor (VDR), and its heterodimeric partner the retinoid X receptor (RXR). These genes are responsible for retention of calcium in the kidney, absorption of dietary calcium in the intestine and direct remodeling processes across the bone through osteoblasts and bone-resorbing osteoclasts. For the regulation of these processes, 1,25(OH)₂D₃ induces genes such as TRPV6, PMCA1B, and S100G whose products are directly involved in the transepithelial transfer of calcium from the gut lumen and SLC34A1 and SLC34A3 that facilitate phosphate uptake [2], [3] and genes such as CYP3A4, CYP3A7, CYP2B6, and ABCB1 that function to detoxify lithocholic acid and other secondary bile acids or are involved in the transport and metabolism of foreign compounds [4], [5], [6]. It is also known that 1,25(OH)₂D₃ exerts activity and regulation on the VDR gene itself as well as its catabolic enzyme CYP24A1 [7], [8].

Coactivators are essential for transcription initiation and are classically believed to provide linkage from receptor complexes to the basal transcriptional machinery [9]. Indeed it is now known that coactivators have catalytic domains that are responsible for modification of the chromatin environment such as acetylation, methylation, phosphorylation and many others [10]. The modulation of these chromatin marks defines the epigenome that drives cell-type and tissue-type specificity. Currently, there are over 300 transcriptional coregulators that have been described in the literature [11]. Coactivators, those believed to facilitate transcription, and corepressors, those believed to inhibit transcription, have been interchangeably described in the activation and/or repression of genes, making characterization difficult. It has been demonstrated, for example, that for full activation of estrogen responsive genes, the corepressor SMRT is required in the activation complex [12]. The coactivators and corepressors are able to directly interact with nuclear receptors like the VDR through LXXLL protein motifs [13] and SMRT is directly involved with vitamin D-mediated transcription [14] as well as other coregulatory molecules [15]. It is believed that ligand activated receptors can change conformations and preference for coactivators based upon which ligands are receptor bound [16]. Furthermore, post translational modifications of coactivators further diversify the activities transferred during coactivation of genes.

Recent advances in transcription research have revealed an extensive array of instructional epigenetic marks that are inserted across the genome in a cell-type specific manner [17]. Some epigenetic marks are associated specifically with regulatory regions, indicating that cellular phenotype is a direct consequence of the establishment of cell-specific enhancers by early lineage-specific transcription factors [18], [19]. Functional binding sites for the VDR/RXR revealed transcription factor interactions as well as the coregulator recruitment processes which identified at least some of the consequences of these interactions at sites on the genome [20]. In the current studies, we expanded on our recent discovery of the VDR/RXR cistrome by analyzing the coactivators (SRC1, CBP, MED1) as well as corepressors (NCoR, SMRT) involved during 1,25(OH)₂D₃-mediated transcription. We found a high correlation between SRC1 and CBP occupancy and transcriptional activation; however, we also found that repressors such as NCoR and SMRT were present in the activation complexes. These studies highlight the complex nature of transcription and coregulatory molecules.

Section snippets

Reagents

1,25(OH)₂D₃ was obtained from Tetrionics, Inc. (Madison, WI). Antibodies to SRC-1 (M-341, sc-8495), CBP (A-22, sc-3996), MED1 (M-255, sc-8998), NCoR (C-20, sc-1609) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). SMRT (PA1-843) antibody was purchased from Affinity Bioreagents (Thermo Fisher, Rockford, IL).

Cell culture

Human LS180 CRC cells were obtained from ATCC (Manassas, VA). LS180 cells were cultured in minimum Eagle's medium supplemented with 10% non-heat-inactivated fetal bovine

VDR/RXR interact with coactivators to modulate transcription

We have previously quantitated the number of DNA binding sites for the vitamin D receptor (VDR) and its heterodimer partner retinoid X receptor (RXR) across the LS180 (human colon adenocarcinoma) genome using ChIP-seq analysis (FDR < 0.001) [22]. Cells were treated with either ethanol vehicle or 1,25(OH)₂D₃ for 3 h and then subjected to the above analyses using validated antibodies to either VDR or RXR. We determined the number of high confidence peaks for VDR and RXR overlap in the 1,25(OH)₂D₃

Acknowledgements

We thank members of the Pike Lab for their helpful discussions and contributions to this manuscript. We also acknowledge the University of Wisconsin DNA Sequencing Facility supported by Marie Adams, Josh Hyman and Eric Cabot. Linux server was maintained by Mindy Preston and Rebecca Hudson. This work was supported by National Institutes of Health grant DK-073995 to J.W.P.

References (26)

M. Meyer et al.
A downstream intergenic cluster of regulatory enhancers contributes to the induction of CYP24A1 expression by 1alpha,25-dihydroxyvitamin D₃
Journal of Biological Chemistry
(2010)
L. Klein-Hitpass et al.
The progesterone receptor stimulates cell-free transcription by enhancing the formation of a stable preinitiation complex
Cell
(1990)
A.B. Johnson et al.
Hypoxia-induced and stress-specific changes in chromatin structure and function
Mutation Research
(2007)
L.A. Zella et al.
The vitamin D receptor interacts preferentially with DRIP205-like LxxLL motifs
Archives of Biochemistry and Biophysics
(2007)
W. Bourguet et al.
Nuclear receptor ligand-binding domains: three-dimensional structures, molecular interactions and pharmacological implications
Trends in Pharmacological Sciences
(2000)
S. Heinz et al.
Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities
Molecular Cell
(2010)
J.S. Carroll et al.
Chromosome-wide mapping of estrogen receptor binding reveals long-range regulation requiring the forkhead protein FoxA1
Cell
(2005)
L.A. Plum et al.
Vitamin D, disease and therapeutic opportunities
Nature Reviews Drug Discovery
(2010)
R. Bouillon et al.
Intestinal calcium absorption: molecular vitamin D mediated mechanisms
Journal of Cellular Biochemistry
(2003)
T. Nijenhuis et al.
(Patho)physiological implications of the novel epithelial Ca²⁺ channels TRPV5 and TRPV6
Pflugers Archiv
(2003)

P.W. Jurutka et al.

Molecular and functional comparison of 1,25-dihydroxyvitamin D(3) and the novel vitamin D receptor ligand, lithocholic acid, in activating transcription of cytochrome P450 3A4

Journal of Cellular Biochemistry

(2005)

K.E. Thummel et al.

Transcriptional control of intestinal cytochrome P-4503A by 1a,25-dihydroxy vitamin D₃

Molecular Pharmacology

(2001)

S. Tachibana et al.

Involvement of Vitamin D receptor in the intestinal induction of human ABCB1

Drug Metabolism and Disposition: The Biological Fate of Chemicals

(2009)

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Citation Excerpt :
VDR mediates the genomic actions of vitamin D. VDR can either homodimerize or heterodimerize with RXR on DNA [74]. The major active form of vitamin D is 1,25(OH)2D3, and the VDR ligand converts DNA-bound VDR homodimers into VDR-RXR heterodimers [75], which recruit corepressors or coactivators to regulate gene transcription [76]. While best known for its physiological function in skeletal health [77], vitamin D has more recently been implicated in metabolic syndrome and NAFLD [78].
As a result of a sedentary lifestyle and excess food consumption in modern society, non-alcoholic fatty liver disease (NAFLD) characterized by fat accumulation in the liver is becoming a major disease burden. Non-alcoholic steatohepatitis (NASH) is an advanced form of NAFLD characterized by inflammation and fibrosis that can lead to hepatocellular carcinoma and liver failure. Nuclear receptors (NRs) are a family of ligand-regulated transcription factors that closely control multiple aspects of metabolism. Their transcriptional activity is modulated by various ligands, including hormones and lipids. NRs serve as potential pharmacological targets for NAFLD/NASH and other metabolic diseases.
In this review, we provide a comprehensive overview of NRs that have been studied in the context of NAFLD/NASH with a focus on their transcriptional regulation, function in preclinical models, and studies of their clinical utility.
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A chromatin-based mechanism controls differential regulation of the cytochrome P450 gene Cyp24a1 in renal and non-renal tissues
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Cytochrome P450 family 27 subfamily B member 1 (CYP27B1) and CYP24A1 function to maintain physiological levels of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) in the kidney. Renal Cyp27b1 and Cyp24a1 expression levels are transcriptionally regulated in a highly reciprocal manner by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and 1,25(OH)₂D₃. In contrast, Cyp24a1 regulation in nonrenal target cells (NRTCs) is limited to induction by 1,25(OH)₂D₃. Herein, we used ChIP-Seq analyses of mouse tissues to identify regulatory regions within the Cyp24a1 gene locus. We found an extended region downstream of Cyp24a1 containing a cluster of sites, termed C24-DS1, binding PTH-sensitive cAMP-responsive element–binding protein (CREB) and a cluster termed C24-DS2 binding the vitamin D receptor (VDR). VDR-occupied sites were present in both the kidney and NRTCs, but pCREB sites were occupied only in the kidney. We deleted each segment in the mouse and observed that although the overt phenotypes of both cluster deletions were unremarkable, RNA analysis in the C24-DS1–deleted strain revealed a loss of basal renal Cyp24a1 expression, total resistance to FGF23 and PTH regulation, and secondary suppression of renal Cyp27b1; 1,25(OH)₂D₃ induction remained unaffected in all tissues. In contrast, loss of the VDR cluster in the C24-DS2–deleted strain did not affect 1,25(OH)₂D₃ induction of renal Cyp24a1 expression yet reduced but did not eliminate Cyp24a1 responses in NRTCs. We conclude that a chromatin-based mechanism differentially regulates Cyp24a1 in the kidney and NRTCs and is essential for the specific functions of Cyp24a1 in these two tissue types.

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Corepressors (NCoR and SMRT) as well as coactivators are recruited to positively regulated 1α,25-dihydroxyvitamin D3-responsive genes

Abstract

Highlights

Introduction

Section snippets

Reagents

Cell culture

VDR/RXR interact with coactivators to modulate transcription

Acknowledgements

Journal of Biological Chemistry

Cell

Mutation Research

Archives of Biochemistry and Biophysics

Trends in Pharmacological Sciences

Molecular Cell

Cell

Vitamin D, disease and therapeutic opportunities

Nature Reviews Drug Discovery

Intestinal calcium absorption: molecular vitamin D mediated mechanisms

Journal of Cellular Biochemistry

(Patho)physiological implications of the novel epithelial Ca2+ channels TRPV5 and TRPV6

Pflugers Archiv

Molecular and functional comparison of 1,25-dihydroxyvitamin D(3) and the novel vitamin D receptor ligand, lithocholic acid, in activating transcription of cytochrome P450 3A4

Journal of Cellular Biochemistry

Transcriptional control of intestinal cytochrome P-4503A by 1a,25-dihydroxy vitamin D3

Molecular Pharmacology

Involvement of Vitamin D receptor in the intestinal induction of human ABCB1

Drug Metabolism and Disposition: The Biological Fate of Chemicals

Corepressors (NCoR and SMRT) as well as coactivators are recruited to positively regulated 1α,25-dihydroxyvitamin D₃-responsive genes

(Patho)physiological implications of the novel epithelial Ca²⁺ channels TRPV5 and TRPV6

Transcriptional control of intestinal cytochrome P-4503A by 1a,25-dihydroxy vitamin D₃