Cell Culture and Subcellular Fractionation
Primary mouse hepatocytes were isolated from 8–10-week-old male C57BL/6N mice as described previously (Kim et al., 2012). The liver was perfused with Hanks’ Balanced Salt Solution containing 0.05 mM ethylene glycol tetra acetic acid for 3 min, followed by a solution containing 0.05% collagenase type IV (Merck, Darmstadt, Germany). After centrifugation at 500 rpm for 3 min, the cell pellet containing hepatocytes was plated in collagen-coated plates with Medium 199 (HyClone, Logan, UT) supplemented with 10% fetal bovine serum, 23 mM HEPES, and 10 nM dexamethasone and maintained under 5% CO2 and 95% air at 37°C. AML12 cells were obtained from the American Type Culture Collection and cultured in DMEM/F-12 medium supplemented with insulin–transferrin–selenium (ITS-G; Thermo Fisher Scientific, Waltham, MA), dexamethasone (Sigma-Aldrich, St Louis, MO), and antibiotics under 5% CO2 and 95% air at 37°C. Subcellular fractionation was performed as described previously (Miao et al.). Briefly, primary hepatocytes were resuspended in a buffer containing 10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and a protease inhibitor and incubated on ice for 15 min. After adding 0.5% NP-40, the mixture was vortexed vigorously and centrifuged. The supernatant was saved as the cytoplasmic fraction. The precipitated nuclear pellet was resuspended in a 50% glycerol buffer and mixed with nucleus lysis buffer (20 mM HEPES pH 7.9, 7.5 mM MgCl2, 0.2 mM EDTA, 0.3 M NaCl, 1 M urea, 1% NP-40, and 1 mM DTT) and centrifuged. The supernatant was collected as the nucleoplasmic fraction. The remaining chromatin pellet was washed with cold phosphate-buffered saline and saved as the chromatin fraction. Easy-Blue (iNtRON Biotechnology, Seongnam, South Korea) was added to each fraction to extract RNA. 8-(4-Chlorophenylthio) adenosine 3¢,5¢-cyclic monophosphate sodium salt (pCPT-cAMP; C-3912) and RU486 (M-8046) were purchased from Merck.
Plasmids, si-RNAs, ASO, and Adenovirus Infusion
The eukaryotic expression vector encoding GR was described previously (Xie et al., 2009). and that encoding mouse hnRNPL was constructed by cloning cDNA (MR208796, OriGene, Rockville, MD) into p3XFLAG-CMV-10 (Sigma-Aldrich) through conventional gene recombination. To construct eukaryotic expression vector for Ctcflos, the Ctcflos 202-S2 fragments amplified from the mouse liver cDNA was cloned into pCDNA 3.1 plus. si-RNAs targeting mouse GR, hnRNPL, and GFP were synthesized by Bioneer Co. (Daejeon, South Korea) (Supplementary Table). The antisense oligonucleotide (ASO) targeting Ctcflos, ASO-Ctcflos, was synthesized by Qiagen (Hilden, Germany) (Supplementary Table). Transient transfection of si-RNAs and ASO was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), as described previously (Han et al., 2019). The adenovirus encoding Ctcflos, Ad-Ctcflos, was constructed by recombination of pcDNA3.1 encoding Ctcflos with an adenoviral backbone plasmid, pAdEasy-1. The titers of the adenovirus preparations were determined by plaque counts. Ad-Ctcflos was generated and kindly provided by Dr. SH. Koo (Division of Life Sciences, Korea University, Seoul, Korea). Transduction of the adenovirus was conducted as described previously (Kim et al., 2012).
Animal Experiments
Eight-week-old male C57BL/6N mice were obtained from Orient Bio Inc. (Seongnam, South Korea) and housed in an air-conditioned room at 22–24°C and 50%–60% humidity with a 12-h light/dark cycle. To analyze the tissue distribution of Ctcflos, Pck1, and Ctcfl in mice, eight tissues, including the liver and kidney, were dissected from mice and rapidly excised, and portions of tissues were stored for further experiments. To establish the fasting or feeding conditions, mice were fasted for 24 h or fed ad libitum. Dexamethasone dissolved in 5% DMSO and 45% PEG30 was administered at a dose of 1 mg/kg of body weight 1 h prior to sacrifice. To overexpress Ctcflos in the liver, Ad-Ctcflos or Ad-GFP (1.0 × 109 plaque-forming units/mouse) was injected into mice via the tail vein. On the 7th day, the mice were fasted for 24 h and liver tissues were collected. All animal experiments were conducted according to the guidelines of the Seoul National University Institutional Animal Care and Use Committee (SNU-200407-3).
Analysis of Chromatin Immunoprecipitation Sequencing (ChIP-seq) Data and ChIP Analysis
The binding signals of H3K27ac, H3K4me3, H3K4me1, and GR on the mouse Pck1 enhancer region were identified in the Gene Expression Omnibus database (GSE31039, GSE72087, and GSE46047). To visualize ChIP-seq from the public database, we used the Integrative Genomics Viewer Software (Broad Institute, Cambridge, MA). The ChIP assay was conducted using the following antibodies: anti-H3K27ac (ab4729, Abcam, Cambridge, UK), anti-H3K4me3 (ab8580, Abcam), anti-H3K4me1 (ab8895, Abcam), anti-GR (24050-1-AP, Proteintech, Rosemont, IL), or anti-hnRNPL (ab6106, Abcam); or control IgG (SC-2025, Santa Cruz Biotechnology, Santa Cruz, CA, and 2729s, Cell Signaling Technology, Danvers, MA), as described previously (Kim et al., 2017). The immunoprecipitated genome region was amplified using the SYBR Green Master mix (Applied Biosystems, Foster City, CA) with specific primers (Supplementary Table). Data were normalized to the input and analyzed relative to the nonspecific IgG control.
Rapid Amplification of cDNA Ends (RACE)
Total RNA was isolated from the primary mouse hepatocytes using an RNeasy Mini kit according to the manufacturer’s protocol (Qiagen). The GeneRacerTM Kit (Invitrogen) was used to amplify the 5¢ and 3¢ RACE-ready cDNAs according to the manufacturer’s instructions. Briefly, 2.5 mg of total RNA was subjected to GeneRacer Oligo ligation and then the M-MLV reverse transcriptase (Invitrogen) was used to produce RACE-ready cDNAs. The RACE and nested RACE PCRs were performed using Biotechnology i-StarTaqTM DNA Polymerase (iNtRON Biotechnology). RACE-specific primers were designed by Integrated DNA technologies. The resulting RACE PCR products were cloned using the TOPO TA cloning kit (Invitrogen) and sequenced.
PCR and Quantitative Real-time PCR (qPCR)
Total RNA was extracted from primary hepatocytes using easy-BLUETM (iNtRON Biotechnology) according to the manufacturer’s instructions. Total RNA was reverse transcribed with M-MLV reverse transcriptase (Invitrogen) and PCR was performed using specific primers (Supplementary Table). PCR products were analyzed by ethidium-bromide-containing agarose gel electrophoresis. qPCR was performed using an ABI StepOnePlusTM Real-time PCR system (Applied Biosystems). Relative RNA levels were measured using the equation 2–DCt (DCt = Ct of the target gene – Ct of 18S rRNA) and are presented as fold change from the level of the control, which was designated as 1 (Han et al., 2014).
Western Blotting, Co-immunoprecipitation, and in situ Proximity Ligation Assays (PLAs)
Western blotting was basically performed as described previously using specific antibodies against Pck1 (ab28455, Abcam), hnRNPL (ab6106, Abcam), GR (12041s, Cell Signaling), or b-actin (SC-47778, Santa Cruz), or control IgG (SC-2025, Santa Cruz or 2729s, Cell Signaling) (Kim et al., 2017). The coimmunoprecipitation assay was performed using specific antibodies against GR (24050-1-AP, Proteintech) and hnRNPL (ab6106, Abcam), as described previously (Ka et al., 2017). In situ PLAs were performed using the Duolink In Situ Red Starter Kit Mouse/Rabbit (Sigma-Aldrich), as described previously (Ka et al., 2017). Briefly, cells grown in 8-well chamber slides were transfected with 20 pmol ASO-Ctcflos. Cells were fixed and incubated with primary antibodies against GR (24050-1-AP, Proteintech) and hnRNPL (ab6106, Abcam). The slides were incubated with PLA probes and subsequent ligation and rolling-circle amplification were performed. The PLA signals were visualized using a Zeiss LSM 710 confocal microscope.
Reporter Gene Analysis
The enhancer region of Pck1 (chr 2: 172,965,854–172,966,544) was amplified from the mouse genomic DNA (G3091, Promega, Madison, WI, USA) as a template using the Pfu-X polymerase (SolGent, Daejeon, South Korea). The obtained DNA fragment was cloned into the MluI/BglII site upstream of the luciferase coding region of the pGL2-promoter vector (Promega) by conventional gene recombination. For reporter gene assays, AML12 cells were transfected with the Pck1 enhancer-Luc reporter and eukaryotic expression vectors encoding GR and hnRNPL using LipofectamineTM 2000 (Invitrogen). Luciferase activity was normalized to b-galactosidase activity, as described previously (Han et al., 2019).
RNA Pull-down Assay
Ctcflos and its antisense strand were synthesized in vitro using the MEGAscript T7/SP6 Transcription Kit (Invitrogen) and biotinylated using a Biotin RNA Labeling mix (Roche). After purification, RNAs were diluted using an RNA structure buffer (10 mM Tris-Cl pH 7.0, 0.1 M KCL, and 10 mM MgCl2) to induce proper RNA folding. Nuclear pellets were obtained from primary hepatocytes and resuspended in RNA immunoprecipitation buffer (150 mM KCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 100 U/mL RNase inhibitor, and protease inhibitor cocktail). The suspended nuclei were sheared using a Dounce homogenizer. Aliquots of 30 pmol of folded RNAs were added to the pre-cleared nuclear lysates and incubated at 4℃ for 2 h with agitation. Streptavidin-conjugated agarose beads (Millipore, Billerica, MA) were added and incubated at 4℃ for 1 h with agitation. The beads were washed with the RNA immunoprecipitation buffer and boiled in SDS sample buffer. The resulting protein mixture was applied to gel electrophoresis and visualized by silver staining. The target protein band was analyzed by mass spectrometry.
Statistics
GraphPad Prism (https://www.graphpad.com/) (GraphPad Software) was applied to all data obtained in this research. All values are expressed as the mean ± standard deviation (SD) based on three independent experiments, unless indicated otherwise. Statistical analysis was performed using the nonparametric Mann–Whitney U test or unpaired Student’s t-test for simple comparison. P < 0.05 was considered statistically different.