The Glucocorticoid Receptor Regulates the <i>ANGPTL4</i> Gene in a CTCF-Mediated Chromatin Context in Human Hepatic Cells

<div><p>Glucocorticoid signaling through the glucocorticoid receptor (GR) plays essential roles in the response to stress and in energy metabolism. This hormonal action is integrated to the transcriptional control of GR-target genes in a cell type-specific and condition-dependent manner. In the present study, we found that the GR regulates the <i>angiopoietin-like 4</i> gene (<i>ANGPTL4</i>) in a CCCTC-binding factor (CTCF)-mediated chromatin context in the human hepatic HepG2 cells. There are at least four CTCF-enriched sites and two GR-binding sites within the <i>ANGPTL4</i> locus. Among them, the major CTCF-enriched site is positioned near the <i>ANGPTL4</i> enhancer that binds GR. We showed that CTCF is required for induction and subsequent silencing of <i>ANGPTL4</i> expression in response to dexamethasone (Dex) and that transcription is diminished after long-term treatment with Dex. Although the <i>ANGPTL4</i> locus maintains a stable higher-order chromatin conformation in the presence and absence of Dex, the Dex-bound GR activated transcription of <i>ANGPTL4</i> but not that of the neighboring three genes through interactions among the <i>ANGPTL4</i> enhancer, promoter, and CTCF sites. These results reveal that liganded GR spatiotemporally controls <i>ANGPTL4</i> transcription in a chromosomal context.</p></div

Long-term dexamethasone treatment inhibits the induction of <i>ANGPTL4</i> transcription, together with down-regulation of the GR.

<p><b>(A)</b> Protocol for <u>L</u>ong-<u>T</u>erm <u>D</u>examethasone <u>T</u>reatment (LTDT). For LTDT, HepG2 cells were initially cultured in DMEM medium supplemented with 10% DCC-treated FBS and 100 nM dexamethasone (Dex) for 14 days. Black arrows show sampling times. (B) Decrease in <i>ANGPTL4</i> induction in LTDT cells. <b>(C)</b> Decreased enrichment of GR after Dex treatment of LTDT cells. ChIP-qPCR analysis was performed using an anti-GR antibody and an anti-rabbit IgG (control), followed by quantitative PCR using specific primers for each AG site and the control (NC). (D) CTCF enrichment in control and LTDT cells. ChIP-qPCR analysis was performed using an anti-CTCF antibody and anti-rabbit IgG (control), followed by quantitative PCR using specific primers for each AC site. (E) Expression of CTCF and GR after Dex treatment of control and LTDT cells. The amount of GR decreased in most LTDT cells. The relative level of GR normalized to that of β-tubulin is shown below. Uncropped image of western blot analysis is shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169225#pone.0169225.s006" target="_blank">S6 Fig</a></b>. Asterisks indicate statistically significance between control and LTDT cells at each time point. **<i>P</i> < 0.01, ***<i>P</i> < 0.005.</p

Enrichment of GR, CTCF, acetyl-H3K27, and RNA polymerase II at the <i>ANGPTL4</i> gene locus in cells treated with Dex.

<p>(A) Enrichment of glucocorticoid receptor (GR) in cells treated with Dex. As shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169225#pone.0169225.g001" target="_blank">Fig 1B</a></b>, HepG2 cells were treated with Dex for 24 h. ChIP-qPCR analysis was performed using an anti-GR antibody and an anti-rabbit IgG (control), followed by quantitative PCR using specific primers for each AG site and the control (NC). (B–D) Enrichment of CTCF, acetyl-H3K27 (H3K27ac), and active RNA polymerase II (Pol2 ser5-P) in cells treated with Dex. ChIP-qPCR analyses were performed using an anti-CTCF antibody and an anti-rabbit IgG (control) (B), anti-H3K27ac (C), and anti-Pol2 ser5-P (D), followed by quantitative PCR using specific primers for each indicated site. Relative enrichment of the control (NC) site was normalized to 1 (D). Asterisks indicate statistically significance between control (Dex 0 h) and Dex-treated cells at each time point. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.005.</p

Specific changes in higher-order chromatin conformation of the <i>ANGPTL4</i> locus in cells treated with dexamethasone.

<p>(A) Chromosome conformation capture (3C) assays were performed using of DpnII-digested fragments containing each AC/AG site and the <i>ANGPTL4</i> promoter. AC3/AG2 sites reside in the same fragment, and AG2 is the <i>ANGPTL4</i> enhancer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169225#pone.0169225.ref015" target="_blank">15</a>]. The relative interaction frequencies of the reference AC3/AG2 fragment (indicated with red) with other DpnII fragments were determined using qPCR analysis of at least three distinct samples from HepG2 cells treated with Dex. Gray arrowheads indicate the orientation of CTCF-binding sites. (B) The relative interaction frequencies of the reference <i>ANGPTL4</i> promoter (indicated with red) with other DpnII fragments in Dex-treated cells. PCR amplification using internal primers derived from the <i>ANGPTL4</i> locus was used as a loading control to normalize the amount of DNA fragments. The efficiencies of DpnII digestions and subsequent ligations were determined at each restriction site. The relative frequencies of interactions between the reference and its closest site in the control state (Dex 0 h) were normalized to 1. Asterisks indicate statistically significance between control (Dex 0 h) and Dex-treated cells (Dex 3h). *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Distribution of glucocorticoid receptor and CTCF in human <i>ANGPTL4</i> gene locus.

<p><b>(A)</b> Enrichment of the glucocorticoid receptor (GR), CTCF, and modified histone H3 in the <i>ANGPTL4</i> locus of HepG2 cells. <i>KANK3</i>, <i>ANGPTL4</i>, <i>RAB11B-AS</i>, and <i>RAB11B</i> are located across an approximately 80-kb region. The arrow at the transcription start site of each gene indicates the direction of transcription. According to publically available data and our ChIP-Seq results, two GR-binding sites (designated AG1 and AG2) and four CTCF-enriched sites (designated AC1–AC4) are indicated in orange and green, respectively. NC, negative control. Modifications of histone H3, such as acetylation and methylation, are shown. AG2/AC3 sites are close to each other, and AG2 is an enhancer that has been demonstrated in rat cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169225#pone.0169225.ref015" target="_blank">15</a>]. <b>(B)</b> Induction of <i>ANGPTL4</i> transcription by dexamethasone (Dex). HepG2 cells were grown in DMEM medium supplemented with 10% dextran-coated charcoal (DCC)-treated FBS and were treated with Dex (100 nM). Black arrows show the sampling times of the assays. <b>(C)</b> <i>ANGPTL4</i> as a direct GR target in HepG2 cells. The GR antagonist mifepristone was added to the medium (100 μM for 1 h) before Dex treatment. The relative expression level is indicated as a value normalized to the level of <i>36B4</i> mRNA. Asterisks indicate statistically significance between control (Dex 0 h) and Dex-treated cells at each time point. ***<i>P</i> < 0.005.</p

The role of CTCF in regulating <i>ANGPLT4</i> transcription.

<p>(A) qRT-PCR analysis of HepG2 cells transfected with the siRNAs (siCont, siCTCF, and si<i>RAB11B AS</i>) for 48 h and then treated with Dex (100 nM). Expression levels were normalized to those of <i>36B4</i> transcripts. <b>(B)</b> Western blot analysis of CTCF and GR expression in siRNA-transfected cells. Asterisks indicate statistically significance among siRNA-transfected cells at each time point. <b>(C)</b> qRT-PCR analysis of <i>ANGPTL4</i> mRNA expression in siRNA-transfected HepG2 cells treated with Dex (see <b>Fig 4A</b>). <b>(D)</b> qRT-PCR analysis of <i>ANGPTL4</i> mRNA expression in siRNA-transfected LTDT cells treated with Dex. Expression levels were normalized to those of <i>36B4</i> transcripts. <b>(E)</b> Enrichment of CTCF at AC3 and GR at AG sites in siRNA-transfected cells. ChIP-qPCR analysis was performed using anti-CTCF, anti-GR, and anti-rabbit IgG (control) antibodies, followed by quantitative PCR using primers specific for each site. Asterisks indicate statistically significance between control and CTCF-knockdown cells at each time point. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.005.</p