Volume 141, Issue 1, Pages 326-337.e3 (July 2011) The Promoter of Human Telomerase Reverse Transcriptase Is Activated During Liver Regeneration and Hepatocyte Proliferation  Hüseyin Sirma, Mukesh Kumar, Jitendra K. Meena, Britta Witt, Julia M. Weise, Andre Lechel, Satyanarayana Ande, Vadim Sakk, Christiane Guguen–Guillouzo, Lars Zender, Karl–Lenhard Rudolph, Cagatay Günes  Gastroenterology  Volume 141, Issue 1, Pages 326-337.e3 (July 2011) DOI: 10.1053/j.gastro.2011.03.047 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Up-regulation of hTERT-promoter activity in regenerating mouse liver. (A) Representative radioactive RT-PCR for lacZ, mTert, and Gapdh. (B) Real-time RT-PCR for lacZ and (C) determination of β-Gal enzymatic activity. (D) Real-time RT-PCR for mTert. A-D: n = 4 samples per time point, from at least 3 independent repeat experiments per sample. (E) Telomerase activity measured by the Telomeric Repeat Amplification Protocol (TRAP) assay; the ladder of extension product indicates telomerase activity (n = 3 mice per time point). (F) Representative picture showing the immunohistologic detection of β-Gal protein in regenerating liver. Immunohistochemistry was performed on 4-μm–thick paraffin sections from livers of wild-type and hTERTp-lacZ transgenic mice at the indicated time points (n = 2 mice per group, 2 repeat staining per sample). Histology samples were visualized using a Zeiss Axiophot microscope, and images were captured at 400× magnification using AxioCam MRc camera and AxioVision Version 4.7 imaging software (Carl Zeiss, Oberkochen, Germany). Black arrows indicate hepatocytes without and red arrows indicate hepatocytes with hTERT-promoter activation (β-Gal protein). Gastroenterology 2011 141, 326-337.e3DOI: (10.1053/j.gastro.2011.03.047) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Characterization of HepaRG differentiation. (A) Determination of HepaRG proliferation. Cells were seeded at a density of 1 × 105, and cell numbers were counted at the indicated days postplating in duplicate, in 3 independent experiments. P values were in comparison with the previous day (n.s., not significant). (B) Determination of mRNA levels of liver-specific markers pre-albumin and APOA1 (Apolipoprotein A1) by real-time RT-PCR. Experiments were performed in triplicate from at least 2 independent approaches. (C) Detection of liver-specific protein pre-albumin by immunoblot. (D) Morphology of HepaRG cells. Phase contrast micrographs of HepaRG cells under proliferating (2 days postplating) or differentiated (maintained for 14 days without dimethyl sulfoxide then treated with 2% dimethyl sulfoxide for 14 days [d28 pp]) state. Cells were visualized using Olympus Mikroskop (Olympus, Tokyo, Japan), and images were captured at 100× magnification using Till Vision Software (Till Photonics GmbH, Gräfelfing, Germany). Hepatocyte-like cells and epithelium-like cells are indicated, respectively, by “h” and “e.” Gastroenterology 2011 141, 326-337.e3DOI: (10.1053/j.gastro.2011.03.047) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Telomerase activity is required for HepaRG cell proliferation. (A) Radioactive Telomeric Repeat Amplification Protocol (TRAP) assay. Two hundred nanograms HepaRG cellular extract and an equivalent of 1000 cells from HeLa control cell extract were used for the TRAP assay. HeLaΔ, Heat-treated extracts; CHAPS, no extracts added; TSR8, control reaction. Representative results from 3 independent experiments. (B) hTERT mRNA level is down-regulated during differentiation. Endogenous hTERT mRNA levels were determined by real-time RT-PCR in triplicate. Representative result from at least 5 independent experiments. (C and D) Suppression of telomerase activity and reduction in hTERT mRNA levels in HepaRG cells. Cells were transfected with a vector encoding for a DN-hTERT protein or for a shRNA sequence directed against hTERT mRNA, and arising clones were used for TRAP assay (C) or hTERT mRNA determination (D). (E) Colony formation assay. HepaRG cells were transfected with empty vector (pBABE), wild-type hTERT (pBABE-wt-hTERT), pBABE-shTERT, and pBABE-DN-hTERT, and numbers of colonies were determined. Experiment was performed in triplicate. Gastroenterology 2011 141, 326-337.e3DOI: (10.1053/j.gastro.2011.03.047) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Core hTERT-promoter activity is sufficient for hTERT-promoter down-regulation during HepaRG differentiation. (A) Schematic drawing of 8-kbp and 0.3-kbp hTERT-promoter luciferase-reporter constructs. (B) Down-regulation of hTERTp-Luc reporter constructs in stably transfected HepaRG cells was determined by luciferase assay. Relative luciferase activity was calculated per milligram protein extract. Representative results from at least 3 independent experiments performed in duplicate. (C) Identification of cis-regulatory elements. Schematic drawing of 0.3-kbp hTERT-promoter constructs with mutations (top); hm1–hm14 designate the single mutations.13 (D) Activity of mutant hTERT-promoter constructs in proliferating or nonproliferating HepaRG cells. Cells were transfected in triplicate with reporter plasmids 1-day postplating, and luciferase activity was determined 2 days after transfection. Gastroenterology 2011 141, 326-337.e3DOI: (10.1053/j.gastro.2011.03.047) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Determination of endogenous E2F1, E2F2, E2F7, and E2F8 mRNA levels by real-time RT-PCR (A) during differentiation of HepaRG cells (B) or in regenerating mouse liver. Determination of endogenous E2F2 and E2F7 protein levels by immunoblot (C) during differentiation of HepaRG cells (D) or in regenerating mouse liver. Real-time RT-PCR experiments were performed from at least 2 independent wild-type and transgenic liver samples in duplicates. Representative immunoblots (from at least 4 independent experiments) indicated E2F2 and E2F7 down-regulation during HepaRG differentiation and up-regulation in liver after partial hepatectomy. Gastroenterology 2011 141, 326-337.e3DOI: (10.1053/j.gastro.2011.03.047) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 E2F2 and E2F7 regulate hTERT-promoter in HepaRG cells and in regenerating mouse liver. (A) HepaRG-297 cells stably containing the core hTERT-promoter luciferase reporter construct were transfected with siRNAs, and luciferase activity was determined. Cells were transfected at 1-day postplating and collected 2–3 days after transfection at ∼30%–50% confluency. Each experiment was performed in triplicate in 24-well plates. (B) Endogenous hTERT messenger RNA (mRNA) levels in HepaRG-297 cells transfected with E2F- or CTCF-siRNAs. The values are given in percentage mRNA levels compared with hTERT mRNA levels of untransfected control HepaRG cells (set to 100%). (C–E) Analysis of in vivo regulation of hTERT-promoter. (C) Wild-type but not E2F-site mutant (wild type, hm1 and hm4: see Figure 4C) hTERTp-Luc reporter is induced during liver regeneration in wild-type mice (n = 5). Luciferase activity was calculated relative to the activity of quiescent liver (0 hours, set to 100%). (D and E) siRNA mediated down-regulation of E2F2, or E2F7 impairs induction of the hTERT-promoter activity during liver regeneration in hTERTp-lacZ transgenic mice as determined by lacZ mRNA levels (D) or β-Gal enyzmatic activity (E). siRNAs or luciferase reporter plasmids were injected by hydrodynamic tail vein injection. Partial hepatectomy (PH) (5 mice each group) was performed 48 hours after plasmid DNA injections or 24 hours after siRNA injections. Resected liver samples served as day 0 controls. Mice were killed at day 4 (plasmid injections) or at day 3.5 (siRNA injections) after PH for RNA and protein isolations. Gastroenterology 2011 141, 326-337.e3DOI: (10.1053/j.gastro.2011.03.047) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 In vivo binding of E2F-Rb factors to hTERT gene promoter. Binding of endogenous E2F2, E2F7, or Rb factors on endogenous hTERT-promoter was analyzed by ChIP assay in HepaRG cells (A) and in liver (B). E2F2 and E2F7 bind hTERT-promoter in proliferating HepaRG cells (A) and in proliferating liver samples from hTERTp-lacZ mice (d3.5 after PH, n = 5 mice). (B) In contrast, Rb is associated with hTERT-promoter in differentiated (nonproliferating) HepaRG cells (d28 pp) and in quiescent livers (0 hours). ChIP experiments with HepaRG cells were performed from 3 independent approaches, and at least 2 independent ChIP experiments were done with pooled liver samples from 3–5 animals (from d0 and d3.5, respectively). At least 3 independent PCR reactions were performed for each sample. Gastroenterology 2011 141, 326-337.e3DOI: (10.1053/j.gastro.2011.03.047) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Control for transfection and knock-down efficiency of siRNA. siRNAs were transfected in proliferating HepaRG cells, cells were lysed 2–3 days thereafter, and total RNA was prepared. Quantitative reverse-transcription PCR (A–F) was performed as described in Materials and Methods section. (G) Detection of AllStars Negative AlexaFlour488-siRNA in HepaRG 2 days after transfection. Gastroenterology 2011 141, 326-337.e3DOI: (10.1053/j.gastro.2011.03.047) Copyright © 2011 AGA Institute Terms and Conditions

Supplementry Figure 2 Impairment of HepaRG cell proliferation depleted for E2F2 and E2F7. HepaRG cells were transduced with lentiviruses encoding a (A) control (scrambled) shRNA or shRNAs directed to (B) E2F2 or (C) E2F7, respectively. After selection with puromycin, cells were either harvested to determine the depletion efficiency by quantitative PCR (Supplementary Figure 3) or further cultured for 2 weeks. Cells were visualized using Olympus Mikroskop (Tokyo, Japan), and phase contrast images were captured at 100× magnification using Till Vision Software (Till Photonics GmbH, Gräfelfing, Germany). Gastroenterology 2011 141, 326-337.e3DOI: (10.1053/j.gastro.2011.03.047) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 3 Control for knock-down efficiency of shRNA. The HepaRG cells were harvested after selection with puromycin at low density. Total RNA was prepared, and knock-down efficiency was determined by quantitative reverse-transcription PCR. Control shRNA was kindly provided by Roger Everett, Glasgow. A set of 4 shRNAs for E2F2 (No. RHS4531-NM_004091) and 7 shRNAs for E2F7 were obtained from Open Biosystems (Thermo Fisher Scientific, Huntsville, AL) and tested for efficiency. For E2F2, clones 3 and 4 (V2LHS_172943, Sequence 5′-CAGTGTCTTTCTGAGGTAA and V2LHS_287030, Sequence 5′-GCATCTATGACATCACCAA, respectively) resulted in ∼50% down-regulation of E2F2 messenger RNA levels (A). For E2F7, clone 8 (V2LHS_252159, Sequence: 5′-TTAAATTTACTATGGTCTG) proved to be functional, resulting in ∼85%–90% down-regulation of E2F7 messenger RNA levels (B). Experiments were performed in triplicates from 2 independent approaches. Gastroenterology 2011 141, 326-337.e3DOI: (10.1053/j.gastro.2011.03.047) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 4 Control for knock-down efficiency of mouse E2F2 and E2F7 siRNA. siRNAs were transfected into mouse hepatocellular cancer cell line AL1698, cells were lysed 3 days thereafter, and total RNA was prepared. Quantitative reverse-transcription PCR was performed as described in Materials and Methods section, and E2F2 (A) or E2F7 (B) messenger RNA levels were determined. Relative knock-down efficiencies were calculated in comparison to untransfected cells (100%). Gastroenterology 2011 141, 326-337.e3DOI: (10.1053/j.gastro.2011.03.047) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 5 Control for knock-down efficiencies of (A) E2F2 and (B) E2F7 siRNAs in mouse liver samples. Total RNA was isolated from mouse liver samples at the time point of partial hepatectomy (d0) or at d3.5 after partial hepatectomy, and mRNA levels were determined. RNA levels were plotted as fold change to messenger RNA levels of control siRNA treated mouse liver (d0). Gastroenterology 2011 141, 326-337.e3DOI: (10.1053/j.gastro.2011.03.047) Copyright © 2011 AGA Institute Terms and Conditions