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

Figures

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  Fig 1. Distribution of glucocorticoid receptor and CTCF in human ANGPTL4 gene locus. (A) Enrichment of the glucocorticoid receptor (GR), CTCF, and modified histone H3 in the ANGPTL4 locus of HepG2 cells. KANK3, ANGPTL4, RAB11B-AS, and RAB11B 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 [15]. (B) Induction of ANGPTL4 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. (C) ANGPTL4 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.…

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  Fig 2. Enrichment of GR, CTCF, acetyl-H3K27, and RNA polymerase II at the ANGPTL4 gene locus in cells treated with Dex. (A) Enrichment of glucocorticoid receptor (GR) in cells treated with Dex. As shown in Fig 1B, 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. *P < 0.05, **P < 0.01, ***P < 0.005.

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  Fig 3. Long-term dexamethasone treatment inhibits the induction of ANGPTL4 transcription, together with down-regulation of the GR. (A) Protocol for Long-Term Dexamethasone Treatment (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 ANGPTL4 induction in LTDT cells. (C) 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 S6 Fig. Asterisks indicate statistically significance between control and LTDT…

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  Fig 4. The role of CTCF in regulating ANGPLT4 transcription. (A) qRT-PCR analysis of HepG2 cells transfected with the siRNAs (siCont, siCTCF, and siRAB11B AS) for 48 h and then treated with Dex (100 nM). Expression levels were normalized to those of 36B4 transcripts. (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. (C) qRT-PCR analysis of ANGPTL4 mRNA expression in siRNA-transfected HepG2 cells treated with Dex (see Fig 4A). (D) qRT-PCR analysis of ANGPTL4 mRNA expression in siRNA-transfected LTDT cells treated with Dex. Expression levels were normalized to those of 36B4 transcripts. (E) 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. *P < 0.05, **P < 0.01, ***P < 0.005.

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  Fig 5. Specific changes in higher-order chromatin conformation of the ANGPTL4 locus in cells treated with dexamethasone. (A) Chromosome conformation capture (3C) assays were performed using of DpnII-digested fragments containing each AC/AG site and the ANGPTL4 promoter. AC3/AG2 sites reside in the same fragment, and AG2 is the ANGPTL4 enhancer [15]. 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 ANGPTL4 promoter (indicated with red) with other DpnII fragments in Dex-treated cells. PCR amplification using internal primers derived from the ANGPTL4 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…

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  Fig 6. Model of ANGPTL4 gene regulation and higher-order chromatin in cells treated with Dex. In its basal state, ANGPTL4 is not activated in the absence of Dex-bound GR within the preexisting higher-order chromatin structure. When ANGPTL4 is transcriptionally active (Dex 3 h), liganded GR binds to AG sites and enhances interactions between enhancer, promoter, and CTCF sites to selectively induce ANGPTL4 but not the neighboring KANK3, RAB11B-AS, and RAB11B genes. In the silenced state (Dex 24 h), the positions of enhancer, promoter, and CTCF sites are restored, leading to down-regulation of ANGPTL4 transcription. CTCF is required for proper induction and subsequent silencing of the ANGPTL4 transcription.