Biochimie 112 (2015) 1e9
Contents lists available at ScienceDirect
Biochimie journal homepage: www.elsevier.com/locate/biochi
Research paper
Transcriptional regulation of the apolipoprotein F (ApoF) gene by ETS and C/EBPa in hepatoma cells Xue-Bin Shen a, b, Ling Huang a, b, Shao-Hong Zhang c, De-Ping Wang a, d, Yun-Li Wu a, Wan-Nan Chen a, Shang-Hua Xu b, **, Xu Lin a, * a
Key Laboratory of Ministry of Education for Gastrointestinal Cancer, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China Department of Cardiology, Affiliated Nanping First Hospital, Fujian Medical University, Nanping, China Department of Medical Laboratory, Affiliated Nanping First Hospital, Fujian Medical University, Nanping, China d Department of Endocrinology and Metabolism, Hongqi Hospital of MuDanJiang Medical College, Mudanjiang, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 October 2014 Accepted 17 February 2015 Available online 26 February 2015
Apolipoprotein F (ApoF) inhibits cholesteryl ester transfer protein (CETP) activity and plays an important role in lipid metabolism. In the present study, the full-length human ApoF promoter was cloned, and the molecular mechanism of the regulation of ApoF was investigated. The ApoF promoter displayed higher activities in hepatoma cell lines, and the 198 nt to þ79 nt promoter region contained the maximum promoter activity. In the promoter region of 198 nt to 2 nt there were four putative binding sites for transcription factors ETS-1/ETS-2 (named EBS-1 to EBS-4) and one for C/EBP. Mutation of EBS-2, EBS4 and the C/EBP binding site abolished the promoter activity, and ETS-1/ETS-2 and C/EBPa could interact with corresponding binding sites. In addition, overexpression of ETS-1/2 or C/EBPa enhanced, while dominant-negative mutants of ETS-1/2 and knockdown of C/EBPa decreased, ApoF promoter activities. ETS-1 and C/EBPa associated physically, and acted synergistically to activate ApoF transcription. These results demonstrated combined activation of the ApoF promoter by liver-enriched and ubiquitous transcription factors. Direct interactions between C/EBPa and ETS-1 were important for high liverspecific expression of ApoF. te Française de Biochimie et Biologie Mole culaire (SFBBM). All rights © 2015 Elsevier B.V. and Socie reserved.
Keywords: Apolipoprotein F Lipid transfer inhibitor protein Promoter E-twenty-six CCAAT/enhancer binding protein
1. Introduction
Abbreviations: ApoF, apolipoprotein F; LTIP, lipid transfer inhibitor protein; C/ EBPa, CCAAT/enhancer binding protein a; ETS, E-twenty-six; EBS, ETS binding site; DN, dominant-negative; EMSA, electrophoretic mobility shift assay; Co-IP, coimmunoprecipitation; ChIP, chromatin immunoprecipitation; siRNA, small interfering RNA; bZIP, basic region leucine zipper; CVD, cardiovascular disease; DMEM, Dulbecco's modified Eagle medium; FBS, fetal bovin serum; MEM, modified Eagle's medium; RT-PCR, reverse transcription-polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. * Corresponding author. Key Laboratory of Ministry of Education for Gastrointestinal Cancer, School of Basic Medical Sciences, Fujian Medical University, 1 Xueyuan Road, Minhou, Fuzhou 350108, China. Tel.: þ86 591 22862648; fax: þ86 591 83569132. ** Corresponding author. Department of Cardiology, Affiliated Nanping First Hospital, Fujian Medical University, Zhongshan Road, Nanping 353000, China. Tel.: þ86 599 8636186; fax: þ86 599 8622948. E-mail addresses: [email protected] (S.-H. Xu), [email protected] (X. Lin).
Apolipoprotein F (ApoF), also known as lipid transfer inhibitor protein (LTIP) [1], is a 29 kDa sialoglycoprotein with an isoelectric point of 3.7 [2]. ApoF occurs at concentrations of about 83.5 mg/ML in human plasma [3]. Approximately 75% of the circulating ApoF is associated with high-density lipoprotein (HDL), while the remaining 25% resides on low-density lipoprotein (LDL) [2,4e7]. ApoF tailors cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester (CE) and triglyceride (TG) transfer [6,8], and ApoF levels are elevated in hypercholesterolemia [9], and correlated negatively with TG levels, although this correlation appears to occur only in males [10]. Overexpression of ApoF reduces HDL cholesterol levels in mice by increasingclearance of HDL-CE [5]. ApoF knockout female mice had increased liver cholesteryl ester content relative to wild-type controls on a chow diet [11]. Clinical trials also showed that the ApoF concentration in hypertriglyceridemic patients was lower than in
http://dx.doi.org/10.1016/j.biochi.2015.02.013 te Française de Biochimie et Biologie Mole culaire (SFBBM). All rights reserved. 0300-9084/© 2015 Elsevier B.V. and Socie
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X.-B. Shen et al. / Biochimie 112 (2015) 1e9
healthy controls [3], and ApoF levels in HDL were significantly reduced in a small set of subjects with coronary heart disease [12]. Thus, aberrant expression of ApoF may result in abnormality of lipid metabolism, and is closely related to the cardiovascular disease. ApoF mRNA is found only in liver tissue, as detected by northern blotting [1,9]. However, little is known about the gene regulation of ApoF. In the present study, the ApoF promoter was cloned and the factors critical for expression of ApoF in hepatocytes were determined. Our data suggested that interplay between liver-enriched transcription factor C/EBPa and ubiquitous transcription factors of ETS is required for liver-specific expression of ApoF. 2. Materials and methods 2.1. ApoF promoter luciferase reporter constructs A DNeasy Tissue Kit (Qiagen, Hilden, Germany) was used to extract genomic DNA from HepG2 cells, which was used as a template for polymerase chain reaction (PCR) amplification. The plasmid p-1618-Luc with the ApoF promoter driving firefly luciferase was constructed by ligation of the PCR-generated full-length ApoF promoter (nucleotides 1618 to þ79, relative to the transcription start site) into the KpnI and XhoI (New England BioLabs, Beverly, MA, USA) cleaved sites of the luciferase reporter plasmid pGL3-Basic (Promega, Madison, WI, USA). Various ApoF promoter deletion constructs, which included p-972-Luc (nucleotides 972 to þ79), p-478-Luc (nucleotides 478 to þ79), p-252-Luc (nucleotides 252 to þ79), p-198 (nucleotides 198 to þ79) and p-2-Luc (nucleotides 2 to þ79) were also constructed by insertion of the corresponding PCR-generated fragment into the KpnI and XhoI sites of the pGL3-Basic plasmid. The primers used for PCR amplification are shown in Supplementary Table S1. The p-198-Luc plasmid was used as a template to generate ETS binding site (EBS) and C/EBP binding sites mutants. To make the mutants, the Gene tailor site-directed mutagenesis kit (Invitrogen, Carlsbad, CA, USA) was used, following the manufacturer's instructions. The primers used are shown in Supplementary Table S1. The resulting plasmids were pEBS1mut-Luc, pEBS2mut-Luc, pEBS3mut-Luc, pEBS4mut-Luc, pEBS24mut-Luc (which included both EBS2 and EBS4 binding site mutations) and pC/EBPmut-Luc. Taq DNA Polymerase High Fidelity (Invitrogen) was used for PCR amplification, and DNA sequencing was used to confirm the amplified sequences. 2.2. Overexpression and dominant-negative mutant constructs The TRIzol Reagent (Invitrogen) was used to extract total RNA from HepG2 cells, which was used as a template for reverse transcription PCR (RT-PCR). The plasmid pETS-1 or pETS-2 was constructed by inserting the RT-PCR generated ETS-1 (GenBank accession no. NM_005238) or ETS-2 (GenBank accession no. NM_005239) genes into the NheI and KpnI sites of pcDNA3.1/ Hygro(þ) (Invitrogen). The plasmids pC/EBPa or pC/EBPb were constructed by insertion of the RT-PCR generated C/EBPa (GenBank accession no. BC063874) or C/EBPb gene (GenBank accession no. BC021931) into the KpnI and Xhol sites of pcDNA3.1/Hygro(þ) (Invitrogen). The paired primers used for PCR amplification are shown in Supplementary Table S1. pETS-1DN, expressing a dominant negative mutant of ETS-1 (lacking of exons IIIeVI of ETS-1) [13], was constructed by fusion PCR using pETS-1 as a template. pETS-2DN, expressing a dominant negative mutant of ETS-2 (lacking the sequence encoding the Nterminal 328 amino acid residues of ETS-2) [14], was constructed by PCR using pETS-2 as a template. The primers used are shown in Supplementary Table S1.
2.3. Cell culture and transfection Human hepatoblastoma cell line HepG2 (HB-8065, ATCC, VA), hepatoma cell line Huh7 (JCRB0403, Japan), embryonic kidney cell line 293A (R705-07, Invitrogen), gastric adenocarcinoma cell line AGS (CRL-1739, ATCC, VA), pancreatic adenocarcinoma cells BxPC-3 (CRL-1687, ATCC, VA) were maintained in Dulbecco's modified Eagle medium (DMEM, Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS, Invitrogen). The human intestinal carcinoma cell line HIC (China Center for Type Culture Collection, Wuhan, China) was grown in modified Eagle's medium (MEM, HyClone, Logan, UT, USA) supplemented with 10% (v/v) FBS. Cells were plated at a density of 2 105 cells/well. The X-tremeGENE HP DNA transfection reagent (Roche Diagnostics, GmbH, Mannheim, Germany) was used to carry out DNA transfection in 12-well plates, in accordance with the manufacturer's recommendations. 2.4. Dual-luciferase reporter assay Cells were transfected with the ApoF promoter luciferase reporter constructs. The renilla luciferase expression vector pRL-SV40 (Promega) was used for normalization, and the promoterless vector pGL3-Basic served as the negative control. Cells were lysed 48 h after transfection. The Dual-Luciferase Reporter Assay System (Promega) was used to detect intracellular luciferase activity in 20 ml of cell lysates, following the manufacturers' recommendations. Luminescence measurement was carried out on an illuminometer (Orion II Microplate Luminometer, Berthold Detection Systems, Germany). Each transfection was performed in duplicate and the data were expressed as the mean ± SD of three separate experiments. 2.5. Western blotting Cells were lysed using RIPA buffer containing a protease inhibitor cocktail (Roche). The protein concentrations of the cell lysates were determined by the Bradford method (Bio-Rad, Hercules, CA, USA). Protein (30 mg) was subjected to 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and electrophoretic transfer to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA). Protein blots were incubated separately with a panel of specific antibodies that included anti-C/EBPa (sc-61, 1:500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-C/EBPb (sc-150,1:500 dilution, Santa Cruz), anti-ETS-1 (sc-350 1:500 dilution, Santa Cruz), anti-ETS-2 (sc-351 1:500 dilution, Santa Cruz), anti-ApoF (1:1000 dilution, Pierce, Rockford, IL, USA) or antib-actin (1:4000 dilution, Sigma) antibodies. The addition of alkaline phosphatase (AP)-conjugated secondary antibody detected the proteins. The addition of CDP STAR reagents (Roche Diagnostics) permitted visualization of the immunoreactive proteins. The densitometric software Quantity One (Biorad, Hercules, CA, USA) was used to quantify the intensities of the protein signals and the relative intensity to the b-actin internal control was calculated. A western blot showing the overexpression of ETS-1, ETS-2, C/EBPa and C/EBPb with the right size was provided in Supplementary Fig. S1, and a western blot showing the specificity of antibodies against ETS-1, ETS-2, C/EBPa, C/EBPb and b-actin was provided in Supplementary Fig. S2. 2.6. RNA extraction and real-time RT-PCR analysis The TRIzol Reagent (Invitrogen) was used to extract total RNA from HepG2 or Huh7 cells. Agarose gel electrophoresis and ethidium bromide staining assessed the integrity of the extracted total RNA. A spectrophotometer (Eppendorf, Hamburg, Germany)
X.-B. Shen et al. / Biochimie 112 (2015) 1e9
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was used to determine the RNA concentration. The reverse transcription reaction was carried out with 2 mg of RNA in a final volume of 20 ml using the ExScript RT-PCR Kit (TaKaRa, Japan) on the ABI StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), in accordance with the manufacturer's recommendations. The ABI StepOne Real-Time PCR System (Applied Biosystems) and the SYBR Premix Ex Taq Kit (TaKaRa) performed the quantitative real-time PCR. PCR was performed in accordance with the manufacturer's instructions. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene served as an internal control. The primers used for GAPDH and ApoF amplification [15,16] are shown in Supplementary Table 1. Each sample was analyzed in triplicate. The relative expression level of ApoF was calculated by normalization to the endogenous GAPDH mRNA expression before comparative analysis using the 2DDCt method [17].
50 ml of each lysate was removed for analysis of input chromatin DNA. Immunoprecipitation was conducted with 5 mg of specific antibodies (anti-ETS-1, anti-ETS-2 or anti-C/EBPa) followed by 80 ml of pre-blocked protein A/G (Sigma). Normal rabbit IgG or preblocked protein A/G (no antibody control) controls was also performed. Following elution and proteinase K digestion, DNA was recovered by phenol/chloroform extraction and ethanol precipitation in the presence of glycogen, and dissolved in 100 ml distilled sterile water. Bound target DNA fractions were analyzed by PCR with the two pairs of primers, one of which was used to amplify the putative protein-DNA binding sites (187/5), and the other was used to amplify the distal control region (4917/4711) upstream of the promoter region [21,22]. The primers used are shown in Supplementary Table 1. The resulting PCR products were electrophoresed on 2% agarose gels and stained with ethidium bromide.
2.7. Electrophoretic mobility shift assay (EMSA)
2.9. RNA interference assay
The 50 -biotin end-labeled oligonucleotides that corresponded to the EBS2, EBS4 and C/EBPa transcription factor recognition sequences within the ApoF promoter region were synthesized (Invitrogen) and used as probes. Unlabeled wild-type (cold probes) or mutated oligonucleotides (mutated cold probes, which included EBS2mut, EBS4mut and C/EBPamut) were used as competitors. The oligonucleotide sequences are shown in Supplementary Table 1 [18e20]. Double-stranded oligonucleotides were obtained by annealing equal amounts (0.1 mg) of the complementary singlestranded oligonucleotides by heating to 95 C for 5 min and then gradually cooling to room temperature. A Nuclear Extraction Kit (Panomics, Inc., Fremont, CA) was used to prepare nuclear proteins from HepG2 cells, following the manufacturer's instructions. The Bio-Rad DC (detergent compatible) Protein Assay (Bio-Rad, Richmond, CA) was used to determine the protein concentration of the nuclear extracts. An EMSA Gel Shift Kit (Panomics) was used to examine the interaction between ETS-1, ETS-2 or C/EBPa in the nuclear extract with the probe, in accordance with the manufacturer's instructions. Briefly, 7 mg of nuclear extracts were incubated with 1 mg of poly (dI-dC) and 0.02 pmol labeled probe in a final volume of 10 ml. The competition assay was performed using 50 to 100 or 200 fold molar excess of cold probes or cold mutated probes, preincubated with the reaction mixture before addition of biotinlabeled probes. In the supershift assays, 4 mg of the specific antibodies (anti-ETS-1, anti-ETS-2, or anti-C/EBPa) was added to the mixtures of nuclear extracts and DNA probes. The DNA-protein complexes were incubated at 15 C for 30 min, and then subjected to electrophoresis on a 6.0% non-denaturing pre-made polyacrylamide gel (Invitrogen). The complexes were then transferred to a nylon membrane (Amersham Bioscience, Piscataway, NJ, USA) and fixed for 3 min using a UV crosslinker (UPV Inc., Upland, CA, USA). The biotin end-labeled DNA was detected by addition of a streptavidin-horseradish peroxidase conjugate and a chemiluminescent substrate.
Cells were transiently transfected with the small interfering RNA (siRNA) mix against C/EBPa (sc-37047, Santa Cruz) or the negative control that had no homology with known human genes, as assessed using X-tremeGENE (Roche). TRIzol (Invitrogen) was used to isolate total RNA and proteins at 48 h after transfection in accordance with the manufacturer's instructions.
2.8. Chromatin immunoprecipitation assay (ChIP) HepG2 cells (1 107) were cultured and cross-linked by adding formaldehyde to a final concentration of 1% at room temperature for 10 min. The cross-linking was stopped by the addition of 125 mM glycine at room temperature for 5 min. Cells were rinsed twice and collected with ice-cold phosphate-buffered saline (PBS), resuspended in 0.75 ml FA Lysis Buffer (Abcam, Cambridge, MA, USA) containing a protease inhibitor cocktail (Roche) and then incubated on ice for 10 min. Cell lysates were sonicated for 20 min with a Branson model 350 Sonifier (Branson Sonic Power Co., Danbury, Conn.) at 20% duty cycle. Before immunoprecipitation,
2.10. Co-immunoprecipitation reactions (Co-IP) Huh7 cells were washed three times with PBS and lysed using RIPA lysis buffer (Pierce, Rockford, IL, USA) containing a proteinase inhibitor cocktail (Roche Diagnostics). The soluble proteins were pre-cleared with 100 ml of a 50% slurry of protein A agarose (Invitrogen), and then the clear lysates were mixed with 100 ml of a 50% slurry of protein A agarose and 3 mg of each of the rabbit antibody for ETS-1, ETS-2, C/EBPa or non-immune rabbit IgG. The immunoprecipitated complexes were washed with lysis buffer and then analyzed by 10% SDS-PAGE and western blotting, using specific rabbit antibodies, including anti-ETS-1, anti-ETS-2 and anti-C/EBPa. 2.11. Identification of putative transcription factor binding sites A computer-based search for potential transcription factor binding site motifs was carried out on the TRANSFAC 6.0 (transfac. gbf.de/) professional database using TFSEARCH (http://www.cbrc. jp/research/db/TFSEARCH.html) [23,24]and TESS (http://www. cbil.upenn.edu/cgi-bin/tess/tess) programs [25]. 2.12. Statistical analysis The SPSS software (SPSS 13.0 version for windows) (SPSS Inc., Chicago, IL) was used to perform the statistical analyses, such as analysis of variance (ANOVA) and the t-test. All values were expressed as means ± standard deviation (SD) from duplicate experiments. A p-value
Contents lists available at ScienceDirect
Biochimie journal homepage: www.elsevier.com/locate/biochi
Research paper
Transcriptional regulation of the apolipoprotein F (ApoF) gene by ETS and C/EBPa in hepatoma cells Xue-Bin Shen a, b, Ling Huang a, b, Shao-Hong Zhang c, De-Ping Wang a, d, Yun-Li Wu a, Wan-Nan Chen a, Shang-Hua Xu b, **, Xu Lin a, * a
Key Laboratory of Ministry of Education for Gastrointestinal Cancer, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China Department of Cardiology, Affiliated Nanping First Hospital, Fujian Medical University, Nanping, China Department of Medical Laboratory, Affiliated Nanping First Hospital, Fujian Medical University, Nanping, China d Department of Endocrinology and Metabolism, Hongqi Hospital of MuDanJiang Medical College, Mudanjiang, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 October 2014 Accepted 17 February 2015 Available online 26 February 2015
Apolipoprotein F (ApoF) inhibits cholesteryl ester transfer protein (CETP) activity and plays an important role in lipid metabolism. In the present study, the full-length human ApoF promoter was cloned, and the molecular mechanism of the regulation of ApoF was investigated. The ApoF promoter displayed higher activities in hepatoma cell lines, and the 198 nt to þ79 nt promoter region contained the maximum promoter activity. In the promoter region of 198 nt to 2 nt there were four putative binding sites for transcription factors ETS-1/ETS-2 (named EBS-1 to EBS-4) and one for C/EBP. Mutation of EBS-2, EBS4 and the C/EBP binding site abolished the promoter activity, and ETS-1/ETS-2 and C/EBPa could interact with corresponding binding sites. In addition, overexpression of ETS-1/2 or C/EBPa enhanced, while dominant-negative mutants of ETS-1/2 and knockdown of C/EBPa decreased, ApoF promoter activities. ETS-1 and C/EBPa associated physically, and acted synergistically to activate ApoF transcription. These results demonstrated combined activation of the ApoF promoter by liver-enriched and ubiquitous transcription factors. Direct interactions between C/EBPa and ETS-1 were important for high liverspecific expression of ApoF. te Française de Biochimie et Biologie Mole culaire (SFBBM). All rights © 2015 Elsevier B.V. and Socie reserved.
Keywords: Apolipoprotein F Lipid transfer inhibitor protein Promoter E-twenty-six CCAAT/enhancer binding protein
1. Introduction
Abbreviations: ApoF, apolipoprotein F; LTIP, lipid transfer inhibitor protein; C/ EBPa, CCAAT/enhancer binding protein a; ETS, E-twenty-six; EBS, ETS binding site; DN, dominant-negative; EMSA, electrophoretic mobility shift assay; Co-IP, coimmunoprecipitation; ChIP, chromatin immunoprecipitation; siRNA, small interfering RNA; bZIP, basic region leucine zipper; CVD, cardiovascular disease; DMEM, Dulbecco's modified Eagle medium; FBS, fetal bovin serum; MEM, modified Eagle's medium; RT-PCR, reverse transcription-polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. * Corresponding author. Key Laboratory of Ministry of Education for Gastrointestinal Cancer, School of Basic Medical Sciences, Fujian Medical University, 1 Xueyuan Road, Minhou, Fuzhou 350108, China. Tel.: þ86 591 22862648; fax: þ86 591 83569132. ** Corresponding author. Department of Cardiology, Affiliated Nanping First Hospital, Fujian Medical University, Zhongshan Road, Nanping 353000, China. Tel.: þ86 599 8636186; fax: þ86 599 8622948. E-mail addresses: [email protected] (S.-H. Xu), [email protected] (X. Lin).
Apolipoprotein F (ApoF), also known as lipid transfer inhibitor protein (LTIP) [1], is a 29 kDa sialoglycoprotein with an isoelectric point of 3.7 [2]. ApoF occurs at concentrations of about 83.5 mg/ML in human plasma [3]. Approximately 75% of the circulating ApoF is associated with high-density lipoprotein (HDL), while the remaining 25% resides on low-density lipoprotein (LDL) [2,4e7]. ApoF tailors cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester (CE) and triglyceride (TG) transfer [6,8], and ApoF levels are elevated in hypercholesterolemia [9], and correlated negatively with TG levels, although this correlation appears to occur only in males [10]. Overexpression of ApoF reduces HDL cholesterol levels in mice by increasingclearance of HDL-CE [5]. ApoF knockout female mice had increased liver cholesteryl ester content relative to wild-type controls on a chow diet [11]. Clinical trials also showed that the ApoF concentration in hypertriglyceridemic patients was lower than in
http://dx.doi.org/10.1016/j.biochi.2015.02.013 te Française de Biochimie et Biologie Mole culaire (SFBBM). All rights reserved. 0300-9084/© 2015 Elsevier B.V. and Socie
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X.-B. Shen et al. / Biochimie 112 (2015) 1e9
healthy controls [3], and ApoF levels in HDL were significantly reduced in a small set of subjects with coronary heart disease [12]. Thus, aberrant expression of ApoF may result in abnormality of lipid metabolism, and is closely related to the cardiovascular disease. ApoF mRNA is found only in liver tissue, as detected by northern blotting [1,9]. However, little is known about the gene regulation of ApoF. In the present study, the ApoF promoter was cloned and the factors critical for expression of ApoF in hepatocytes were determined. Our data suggested that interplay between liver-enriched transcription factor C/EBPa and ubiquitous transcription factors of ETS is required for liver-specific expression of ApoF. 2. Materials and methods 2.1. ApoF promoter luciferase reporter constructs A DNeasy Tissue Kit (Qiagen, Hilden, Germany) was used to extract genomic DNA from HepG2 cells, which was used as a template for polymerase chain reaction (PCR) amplification. The plasmid p-1618-Luc with the ApoF promoter driving firefly luciferase was constructed by ligation of the PCR-generated full-length ApoF promoter (nucleotides 1618 to þ79, relative to the transcription start site) into the KpnI and XhoI (New England BioLabs, Beverly, MA, USA) cleaved sites of the luciferase reporter plasmid pGL3-Basic (Promega, Madison, WI, USA). Various ApoF promoter deletion constructs, which included p-972-Luc (nucleotides 972 to þ79), p-478-Luc (nucleotides 478 to þ79), p-252-Luc (nucleotides 252 to þ79), p-198 (nucleotides 198 to þ79) and p-2-Luc (nucleotides 2 to þ79) were also constructed by insertion of the corresponding PCR-generated fragment into the KpnI and XhoI sites of the pGL3-Basic plasmid. The primers used for PCR amplification are shown in Supplementary Table S1. The p-198-Luc plasmid was used as a template to generate ETS binding site (EBS) and C/EBP binding sites mutants. To make the mutants, the Gene tailor site-directed mutagenesis kit (Invitrogen, Carlsbad, CA, USA) was used, following the manufacturer's instructions. The primers used are shown in Supplementary Table S1. The resulting plasmids were pEBS1mut-Luc, pEBS2mut-Luc, pEBS3mut-Luc, pEBS4mut-Luc, pEBS24mut-Luc (which included both EBS2 and EBS4 binding site mutations) and pC/EBPmut-Luc. Taq DNA Polymerase High Fidelity (Invitrogen) was used for PCR amplification, and DNA sequencing was used to confirm the amplified sequences. 2.2. Overexpression and dominant-negative mutant constructs The TRIzol Reagent (Invitrogen) was used to extract total RNA from HepG2 cells, which was used as a template for reverse transcription PCR (RT-PCR). The plasmid pETS-1 or pETS-2 was constructed by inserting the RT-PCR generated ETS-1 (GenBank accession no. NM_005238) or ETS-2 (GenBank accession no. NM_005239) genes into the NheI and KpnI sites of pcDNA3.1/ Hygro(þ) (Invitrogen). The plasmids pC/EBPa or pC/EBPb were constructed by insertion of the RT-PCR generated C/EBPa (GenBank accession no. BC063874) or C/EBPb gene (GenBank accession no. BC021931) into the KpnI and Xhol sites of pcDNA3.1/Hygro(þ) (Invitrogen). The paired primers used for PCR amplification are shown in Supplementary Table S1. pETS-1DN, expressing a dominant negative mutant of ETS-1 (lacking of exons IIIeVI of ETS-1) [13], was constructed by fusion PCR using pETS-1 as a template. pETS-2DN, expressing a dominant negative mutant of ETS-2 (lacking the sequence encoding the Nterminal 328 amino acid residues of ETS-2) [14], was constructed by PCR using pETS-2 as a template. The primers used are shown in Supplementary Table S1.
2.3. Cell culture and transfection Human hepatoblastoma cell line HepG2 (HB-8065, ATCC, VA), hepatoma cell line Huh7 (JCRB0403, Japan), embryonic kidney cell line 293A (R705-07, Invitrogen), gastric adenocarcinoma cell line AGS (CRL-1739, ATCC, VA), pancreatic adenocarcinoma cells BxPC-3 (CRL-1687, ATCC, VA) were maintained in Dulbecco's modified Eagle medium (DMEM, Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS, Invitrogen). The human intestinal carcinoma cell line HIC (China Center for Type Culture Collection, Wuhan, China) was grown in modified Eagle's medium (MEM, HyClone, Logan, UT, USA) supplemented with 10% (v/v) FBS. Cells were plated at a density of 2 105 cells/well. The X-tremeGENE HP DNA transfection reagent (Roche Diagnostics, GmbH, Mannheim, Germany) was used to carry out DNA transfection in 12-well plates, in accordance with the manufacturer's recommendations. 2.4. Dual-luciferase reporter assay Cells were transfected with the ApoF promoter luciferase reporter constructs. The renilla luciferase expression vector pRL-SV40 (Promega) was used for normalization, and the promoterless vector pGL3-Basic served as the negative control. Cells were lysed 48 h after transfection. The Dual-Luciferase Reporter Assay System (Promega) was used to detect intracellular luciferase activity in 20 ml of cell lysates, following the manufacturers' recommendations. Luminescence measurement was carried out on an illuminometer (Orion II Microplate Luminometer, Berthold Detection Systems, Germany). Each transfection was performed in duplicate and the data were expressed as the mean ± SD of three separate experiments. 2.5. Western blotting Cells were lysed using RIPA buffer containing a protease inhibitor cocktail (Roche). The protein concentrations of the cell lysates were determined by the Bradford method (Bio-Rad, Hercules, CA, USA). Protein (30 mg) was subjected to 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and electrophoretic transfer to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA). Protein blots were incubated separately with a panel of specific antibodies that included anti-C/EBPa (sc-61, 1:500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-C/EBPb (sc-150,1:500 dilution, Santa Cruz), anti-ETS-1 (sc-350 1:500 dilution, Santa Cruz), anti-ETS-2 (sc-351 1:500 dilution, Santa Cruz), anti-ApoF (1:1000 dilution, Pierce, Rockford, IL, USA) or antib-actin (1:4000 dilution, Sigma) antibodies. The addition of alkaline phosphatase (AP)-conjugated secondary antibody detected the proteins. The addition of CDP STAR reagents (Roche Diagnostics) permitted visualization of the immunoreactive proteins. The densitometric software Quantity One (Biorad, Hercules, CA, USA) was used to quantify the intensities of the protein signals and the relative intensity to the b-actin internal control was calculated. A western blot showing the overexpression of ETS-1, ETS-2, C/EBPa and C/EBPb with the right size was provided in Supplementary Fig. S1, and a western blot showing the specificity of antibodies against ETS-1, ETS-2, C/EBPa, C/EBPb and b-actin was provided in Supplementary Fig. S2. 2.6. RNA extraction and real-time RT-PCR analysis The TRIzol Reagent (Invitrogen) was used to extract total RNA from HepG2 or Huh7 cells. Agarose gel electrophoresis and ethidium bromide staining assessed the integrity of the extracted total RNA. A spectrophotometer (Eppendorf, Hamburg, Germany)
X.-B. Shen et al. / Biochimie 112 (2015) 1e9
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was used to determine the RNA concentration. The reverse transcription reaction was carried out with 2 mg of RNA in a final volume of 20 ml using the ExScript RT-PCR Kit (TaKaRa, Japan) on the ABI StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), in accordance with the manufacturer's recommendations. The ABI StepOne Real-Time PCR System (Applied Biosystems) and the SYBR Premix Ex Taq Kit (TaKaRa) performed the quantitative real-time PCR. PCR was performed in accordance with the manufacturer's instructions. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene served as an internal control. The primers used for GAPDH and ApoF amplification [15,16] are shown in Supplementary Table 1. Each sample was analyzed in triplicate. The relative expression level of ApoF was calculated by normalization to the endogenous GAPDH mRNA expression before comparative analysis using the 2DDCt method [17].
50 ml of each lysate was removed for analysis of input chromatin DNA. Immunoprecipitation was conducted with 5 mg of specific antibodies (anti-ETS-1, anti-ETS-2 or anti-C/EBPa) followed by 80 ml of pre-blocked protein A/G (Sigma). Normal rabbit IgG or preblocked protein A/G (no antibody control) controls was also performed. Following elution and proteinase K digestion, DNA was recovered by phenol/chloroform extraction and ethanol precipitation in the presence of glycogen, and dissolved in 100 ml distilled sterile water. Bound target DNA fractions were analyzed by PCR with the two pairs of primers, one of which was used to amplify the putative protein-DNA binding sites (187/5), and the other was used to amplify the distal control region (4917/4711) upstream of the promoter region [21,22]. The primers used are shown in Supplementary Table 1. The resulting PCR products were electrophoresed on 2% agarose gels and stained with ethidium bromide.
2.7. Electrophoretic mobility shift assay (EMSA)
2.9. RNA interference assay
The 50 -biotin end-labeled oligonucleotides that corresponded to the EBS2, EBS4 and C/EBPa transcription factor recognition sequences within the ApoF promoter region were synthesized (Invitrogen) and used as probes. Unlabeled wild-type (cold probes) or mutated oligonucleotides (mutated cold probes, which included EBS2mut, EBS4mut and C/EBPamut) were used as competitors. The oligonucleotide sequences are shown in Supplementary Table 1 [18e20]. Double-stranded oligonucleotides were obtained by annealing equal amounts (0.1 mg) of the complementary singlestranded oligonucleotides by heating to 95 C for 5 min and then gradually cooling to room temperature. A Nuclear Extraction Kit (Panomics, Inc., Fremont, CA) was used to prepare nuclear proteins from HepG2 cells, following the manufacturer's instructions. The Bio-Rad DC (detergent compatible) Protein Assay (Bio-Rad, Richmond, CA) was used to determine the protein concentration of the nuclear extracts. An EMSA Gel Shift Kit (Panomics) was used to examine the interaction between ETS-1, ETS-2 or C/EBPa in the nuclear extract with the probe, in accordance with the manufacturer's instructions. Briefly, 7 mg of nuclear extracts were incubated with 1 mg of poly (dI-dC) and 0.02 pmol labeled probe in a final volume of 10 ml. The competition assay was performed using 50 to 100 or 200 fold molar excess of cold probes or cold mutated probes, preincubated with the reaction mixture before addition of biotinlabeled probes. In the supershift assays, 4 mg of the specific antibodies (anti-ETS-1, anti-ETS-2, or anti-C/EBPa) was added to the mixtures of nuclear extracts and DNA probes. The DNA-protein complexes were incubated at 15 C for 30 min, and then subjected to electrophoresis on a 6.0% non-denaturing pre-made polyacrylamide gel (Invitrogen). The complexes were then transferred to a nylon membrane (Amersham Bioscience, Piscataway, NJ, USA) and fixed for 3 min using a UV crosslinker (UPV Inc., Upland, CA, USA). The biotin end-labeled DNA was detected by addition of a streptavidin-horseradish peroxidase conjugate and a chemiluminescent substrate.
Cells were transiently transfected with the small interfering RNA (siRNA) mix against C/EBPa (sc-37047, Santa Cruz) or the negative control that had no homology with known human genes, as assessed using X-tremeGENE (Roche). TRIzol (Invitrogen) was used to isolate total RNA and proteins at 48 h after transfection in accordance with the manufacturer's instructions.
2.8. Chromatin immunoprecipitation assay (ChIP) HepG2 cells (1 107) were cultured and cross-linked by adding formaldehyde to a final concentration of 1% at room temperature for 10 min. The cross-linking was stopped by the addition of 125 mM glycine at room temperature for 5 min. Cells were rinsed twice and collected with ice-cold phosphate-buffered saline (PBS), resuspended in 0.75 ml FA Lysis Buffer (Abcam, Cambridge, MA, USA) containing a protease inhibitor cocktail (Roche) and then incubated on ice for 10 min. Cell lysates were sonicated for 20 min with a Branson model 350 Sonifier (Branson Sonic Power Co., Danbury, Conn.) at 20% duty cycle. Before immunoprecipitation,
2.10. Co-immunoprecipitation reactions (Co-IP) Huh7 cells were washed three times with PBS and lysed using RIPA lysis buffer (Pierce, Rockford, IL, USA) containing a proteinase inhibitor cocktail (Roche Diagnostics). The soluble proteins were pre-cleared with 100 ml of a 50% slurry of protein A agarose (Invitrogen), and then the clear lysates were mixed with 100 ml of a 50% slurry of protein A agarose and 3 mg of each of the rabbit antibody for ETS-1, ETS-2, C/EBPa or non-immune rabbit IgG. The immunoprecipitated complexes were washed with lysis buffer and then analyzed by 10% SDS-PAGE and western blotting, using specific rabbit antibodies, including anti-ETS-1, anti-ETS-2 and anti-C/EBPa. 2.11. Identification of putative transcription factor binding sites A computer-based search for potential transcription factor binding site motifs was carried out on the TRANSFAC 6.0 (transfac. gbf.de/) professional database using TFSEARCH (http://www.cbrc. jp/research/db/TFSEARCH.html) [23,24]and TESS (http://www. cbil.upenn.edu/cgi-bin/tess/tess) programs [25]. 2.12. Statistical analysis The SPSS software (SPSS 13.0 version for windows) (SPSS Inc., Chicago, IL) was used to perform the statistical analyses, such as analysis of variance (ANOVA) and the t-test. All values were expressed as means ± standard deviation (SD) from duplicate experiments. A p-value
