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• W2040994916 abstract "Hepatocyte nuclear factor 4α (HNF4α) plays critical roles during liver development and in the transcriptional regulation of many hepatic genes in adult liver. Here we have demonstrated that in human hepatoma HepG2 cells, HNF4α is expressed at levels as high as in human liver but its activity on target genes is very low or absent. We have discovered that the low expression of key coactivators (PGC1α, SRC1, SRC2, and PCAF) might account for the lack of function of HNF4α in HepG2 cells. Among them, PGC1α and SRC1 are the two most important HNF4α coactivators as revealed by reporter assays with an Apo-CIII promoter construct. Moreover, the expression of these two coactivators was found to be down-regulated in all human hepatomas investigated. Overexpression of SRC1 and PGC1α by recombinant adenoviruses led to a significant up-regulation of well characterized HNF4α-dependent genes (ApoCIII, ApoAV, PEPCK, AldoB, OTC, and CYP7A1) and forced HepG2 cells toward a more differentiated phenotype as demonstrated by increased ureogenic rate. The positive effect of PGC1α was seen to be dependent on HNF4α. Finally, insulin treatment of human hepatocytes and HepG2 cells caused repression of PGC1α and a concomitant down-regulation of ApoCIII, PEPCK, AldoB, and OTC. Altogether, our results suggest that SRC1, and notably PGC1α, are key coactivators for the proper function of HNF4α in human liver and for an integrative control of multiple hepatic genes involved in metabolism and homeostasis. The down-regulation of key HNF4α coactivators could be a determinant factor for the dedifferentiation of human hepatomas. Hepatocyte nuclear factor 4α (HNF4α) plays critical roles during liver development and in the transcriptional regulation of many hepatic genes in adult liver. Here we have demonstrated that in human hepatoma HepG2 cells, HNF4α is expressed at levels as high as in human liver but its activity on target genes is very low or absent. We have discovered that the low expression of key coactivators (PGC1α, SRC1, SRC2, and PCAF) might account for the lack of function of HNF4α in HepG2 cells. Among them, PGC1α and SRC1 are the two most important HNF4α coactivators as revealed by reporter assays with an Apo-CIII promoter construct. Moreover, the expression of these two coactivators was found to be down-regulated in all human hepatomas investigated. Overexpression of SRC1 and PGC1α by recombinant adenoviruses led to a significant up-regulation of well characterized HNF4α-dependent genes (ApoCIII, ApoAV, PEPCK, AldoB, OTC, and CYP7A1) and forced HepG2 cells toward a more differentiated phenotype as demonstrated by increased ureogenic rate. The positive effect of PGC1α was seen to be dependent on HNF4α. Finally, insulin treatment of human hepatocytes and HepG2 cells caused repression of PGC1α and a concomitant down-regulation of ApoCIII, PEPCK, AldoB, and OTC. Altogether, our results suggest that SRC1, and notably PGC1α, are key coactivators for the proper function of HNF4α in human liver and for an integrative control of multiple hepatic genes involved in metabolism and homeostasis. The down-regulation of key HNF4α coactivators could be a determinant factor for the dedifferentiation of human hepatomas. Hepatoma cell lines and hepatocellular carcinomas (HCC) 3The abbreviations used are: HCC, hepatocellular carcinoma; HNF, hepatocyte nuclear factor; PGC1α, peroxisomal proliferator-activated -γ coactivator-1-α; SRC, steroid coactivator; Apo, apolipoprotein; AldoB, aldolase-B; OTC, ornithine transcarbamylase; PEPCK, phosphoenolpyruvate carboxykinase; CBP, cAMP-response element-binding protein-binding protein; L-FABP, liver fatty acid-binding protein; PCAF, P300/CBP-associated factor; m.o.i., multiplicity of infection; Ad, recombinant adenovirus; RT-PCR, reverse transcription PCR. undergo phenotypic dedifferentiation leading to the loss or low expression of typical hepatic functions such as plasma protein synthesis or xenobiotic detoxification (1Dong X.Y. Pang X.W. Yu S.T. Su Y.R. Wang H.C. Yin Y.H. Wang Y.D. Chen W.F. Int. J. Cancer. 2004; 112: 239-248Crossref PubMed Scopus (54) Google Scholar, 2Xu X.R. Huang J. Xu Z.G. Qian B.Z. Zhu Z.D. Yan Q. Cai T. Zhang X. Xiao H.S. Qu J. Liu F. Huang Q.H. Cheng Z.H. Li N.G. Du J.J. Hu W. Shen K.T. Lu G. Fu G. Zhong M. Xu S.H. Gu W.Y. Huang W. Zhao X.T. Hu G.X. Gu J.R. Chen Z. Han Z.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15089-15094Crossref PubMed Scopus (326) Google Scholar, 3Rodriguez-Antona C. Donato M.T. Boobis A. Edwards R.J. Watts P.S. Castell J.V. Gomez-Lechon M.J. Xenobiotica. 2002; 32: 505-520Crossref PubMed Scopus (320) Google Scholar). Dedifferentiation is a key early event in the pathogenesis of HCC that has been associated with an altered expression of liver-enriched transcription factors (4Xu L. Hui L. Wang S. Gong J. Jin Y. Wang Y. Ji Y. Wu X. Han Z. Hu G. Cancer Res. 2001; 61: 3176-3181PubMed Google Scholar, 5Lazarevich N.L. Cheremnova O.A. Varga E.V. Ovchinnikov D.A. Kudrjavtseva E.I. Morozova O.V. Fleishman D.I. Engelhardt N.V. Duncan S.A. Hepatology. 2004; 39: 1038-1047Crossref PubMed Scopus (181) Google Scholar). Similarly, studies in hepatoma cell lines have revealed that the maintenance of a differentiated hepatic phenotype is dependent on the expression of liver-enriched transcription factor (6Gomez-Lechon M.J. Donato T. Jover R. Rodriguez C. Ponsoda X. Glaise D. Castell J.V. Guguen-Guillouzo C. Eur. J. Biochem. 2001; 268: 1448-1459Crossref PubMed Scopus (69) Google Scholar, 7Griffo G. Hamon-Benais C. Angrand P.O. Fox M. West L. Lecoq O. Povey S. Cassio D. Weiss M. J. Cell Biol. 1993; 121: 887-898Crossref PubMed Scopus (63) Google Scholar). Current research supports the notion that hepatocyte nuclear factor 4α (HNF4α) is one of the most important liver-enriched transcription factors for hepatocyte differentiation. HNF4α is a highly conserved member of the nuclear superfamily that was initially identified as a factor required for liver-specific gene expression (8Sladek F.M. Zhong W.M. Lai E. Darnell Jr., J.E. Genes Dev. 1990; 4: 2353-2365Crossref PubMed Scopus (861) Google Scholar). HNF4α plays critical roles not only in the specification of the hepatic phenotype during liver development but also in the transcriptional regulation of genes involved in glucose, cholesterol, fatty acids, and xenobiotic metabolism and in the synthesis of blood coagulation factors (9Sladek F.M. Receptor. 1993; 3: 223-232PubMed Google Scholar, 10Li J. Ning G. Duncan S.A. Genes Dev. 2000; 14: 464-474PubMed Google Scholar, 11Hayhurst G.P. Lee Y.H. Lambert G. Ward J.M. Gonzalez F.J. Mol. Cell. Biol. 2001; 21: 1393-1403Crossref PubMed Scopus (868) Google Scholar, 12Jover R. Bort R. Gomez-Lechon M.J. Castell J.V. Hepatology. 2001; 33: 668-675Crossref PubMed Scopus (201) Google Scholar). Disruption of HNF4α leads to an early embryonic lethal phenotype associated with a failure of differentiation of visceral endoderm (13Chen W.S. Manova K. Weinstein D.C. Duncan S.A. Plump A.S. Prezioso V.R. Bachvarova R.F. Darnell Jr., J.E. Genes Dev. 1994; 8: 2466-2477Crossref PubMed Scopus (485) Google Scholar). Genome-scale location analysis revealed surprising results for HNF4α in hepatocytes. The number of genes that exhibit a binding of HNF4α to their regulatory regions (>1500 genes) was much larger than that observed with other typical liver-specific regulators. Notably, from the genes occupied by RNA polymerase II, 42% were also bound by HNF4α in hepatocytes (14Odom D.T. Zizlsperger N. Gordon D.B. Bell G.W. Rinaldi N.J. Murray H.L. Volkert T.L. Schreiber J. Rolfe P.A. Gifford D.K. Fraenkel E. Bell G.I. Young R.A. Science. 2004; 303: 1378-1381Crossref PubMed Scopus (1093) Google Scholar). Therefore, HNF4α emerges as a widely acting transactivator in the liver, consistent with the observation that the expression of this constitutively active transcription factor overcomes repression of the hepatic phenotype in dedifferentiated hepatoma cells (15Spath G.F. Weiss M.C. Mol. Cell. Biol. 1997; 17: 1913-1922Crossref PubMed Scopus (104) Google Scholar). However, the significance of a correlation between the expressions of both HNF4α and hepatic functions is challenged by several studies showing that hepatic functions could be silent despite HNF4α being expressed. Hepatic functions were found uncoupled or dissociated from HNF4α in hepatoma cell lines, intertypic cell hybrids, and immortalized hepatocytes (3Rodriguez-Antona C. Donato M.T. Boobis A. Edwards R.J. Watts P.S. Castell J.V. Gomez-Lechon M.J. Xenobiotica. 2002; 32: 505-520Crossref PubMed Scopus (320) Google Scholar, 16Chaya D. Fougere-Deschatrette C. Weiss M.C. Mol. Cell. Biol. 1997; 17: 6311-6320Crossref PubMed Scopus (20) Google Scholar, 17Bulla G.A. Nucleic Acids Res. 1999; 27: 1190-1197Crossref PubMed Scopus (9) Google Scholar, 18Bulla G.A. Kraus D.M. Biosci. Rep. 2004; 24: 595-608Crossref PubMed Scopus (4) Google Scholar, 19Butura A. Johansson I. Nilsson K. Warngard L. Ingelman-Sundberg M. Schuppe-Koistinen I. Biochem. Pharmacol. 2004; 67: 1249-1258Crossref PubMed Scopus (21) Google Scholar, 20Amicone L. Spagnoli F.M. Spath G. Giordano S. Tommasini C. Bernardini S. De Luca V. Della Rocca C. Weiss M.C. Comoglio P.M. Tripodi M. EMBO J. 1997; 16: 495-503Crossref PubMed Scopus (160) Google Scholar). Additional evidence of dissociation between the expression of HNF4α and selected hepatic functions was obtained by HNF4α transfection in rat hepatoma cells lacking this factor (15Spath G.F. Weiss M.C. Mol. Cell. Biol. 1997; 17: 1913-1922Crossref PubMed Scopus (104) Google Scholar, 21Bulla G.A. Fournier R.E. Mol. Cell. Biol. 1994; 14: 7086-7094Crossref PubMed Scopus (36) Google Scholar, 22Bailly A. Spath G. Bender V. Weiss M.C. J. Cell Sci. 1998; 111: 2411-2421PubMed Google Scholar). Moreover, indirect evidence suggests that this could also be the case in certain human HCC where HNF4α is well preserved despite significant dedifferentiation (4Xu L. Hui L. Wang S. Gong J. Jin Y. Wang Y. Ji Y. Wu X. Han Z. Hu G. Cancer Res. 2001; 61: 3176-3181PubMed Google Scholar). The existence of such dissociations suggests that in some instances HNF4α could be highly expressed but not fully active. A key element for a correct nuclear function is a balanced, physiologic level of coregulators. It is well known that HNF4α interacts with coactivators and corepressors through its activation function domains. Full activity is achieved through the interaction of HNF4α homodimers with DNA and coactivators. Various studies have shown that HNF4α interacts strongly with the p160 family coactivators (SRC1, 2, and 3) (23Iordanidou P. Aggelidou E. Demetriades C. Hadzopoulou-Cladaras M. J. Biol. Chem. 2005; 280: 21810-21819Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 24Sladek F.M. Ruse Jr., M.D. Nepomuceno L. Huang S.M. Stallcup M.R. Mol. Cell. Biol. 1999; 19: 6509-6522Crossref PubMed Google Scholar, 25Wang J.C. Stafford J.M. Granner D.K. J. Biol. Chem. 1998; 273: 30847-30850Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) and that HNF4α activity can be enhanced by the action of CBP/P300 (26Dell H. Hadzopoulou-Cladaras M. J. Biol. Chem. 1999; 274: 9013-9021Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 27Eeckhoute J. Formstecher P. Laine B. Mol. Endocrinol. 2001; 15: 1200-1210PubMed Google Scholar). In addition, HNF4α has been linked to nutrient metabolism in the liver through interactions with the coactivator PGC1α (28Rhee J. Inoue Y. Yoon J.C. Puigserver P. Fan M. Gonzalez F.J. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4012-4017Crossref PubMed Scopus (476) Google Scholar, 29Yoon J.C. Puigserver P. Chen G. Donovan J. Wu Z. Rhee J. Adelmant G. Stafford J. Kahn C.R. Granner D.K. Newgard C.B. Spiegelman B.M. Nature. 2001; 413: 131-138Crossref PubMed Scopus (1528) Google Scholar). In the present study, we have demonstrated that HNF4α is highly expressed but not fully active in the human hepatoma HepG2. This lack of function can be accounted for by the low levels of the coactivators SRC1 and, notably, PGC1α, which after re-expression caused a marked improvement of the HNF4α function and its target genes and enhanced the hepatic phenotype significantly. Expression analysis in several human hepatomas also suggests that the down-regulation of PGC1α and SRC1 could be an important mechanism involved in hepatocyte dedifferentiation and progression of HCC. Cell Culture—Human hepatoma cells (HepG2, Hep3B, Mz-Hep-1, and Chang Liver) were plated in Ham's F-12/Leibovitz L-15 (1/1, v/v) supplemented with 6% fetal calf serum and cultured to 70–80% confluence. Human hepatoma BC2 cells were cultured in a mixture of 75% minimal essential medium and 25% Medium 199, supplemented with 10% fetal bovine serum, 1 mg/ml bovine serum albumin, 0.7 μm insulin and hydrocortisone hemisuccinate, and maintained at confluence for 3 weeks. HeLa (human cervix carcinoma) and 293 cells (AdE1A-transformed human embryonic kidney) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and maintained as monolayer cultures. Culture medium for 293 cells was also supplemented with 3.5 g/liter of glucose. Human hepatocytes were isolated from liver biopsies (1–3 g) of patients undergoing liver surgery after informed consent. None of the patients habitually consumed alcohol or other drugs. A total of five liver biopsies (two male and three female of ages ranging from 26 to 65 years) were used. Hepatocytes were isolated using a two-step perfusion technique (30Gomez-Lechon M. Castell J. Berry M.N. Edwards A.M. The Hepatocyte Review. Kluwer Academic Publishers, London2000: 11-17Google Scholar) and seeded on plates coated with fibronectin (3.6 μg/cm2) at a density of 8 × 104 cells/cm2. The culture medium was Ham's F-12/Leibovitz L-15 (1/1, v/v) supplemented with 2% newborn calf serum, 5 mm glucose, 0.2% bovine serum albumin, and 10–8 m insulin. The medium was changed 1 h later to remove unattached hepatocytes. After 24 h, the culture medium was changed to serum-free medium containing 10–8 m dexamethasone. Cultures were routinely supplemented with 50 units/ml penicillin and 50 μg/ml streptomycin. Development of Adenoviral Vectors—A recombinant adenovirus was prepared for the expression of human HNF4α as follows: HNF4α2 cDNA was released from the expression vector pMT2-HNF4B (Dr. Talianidis) by EcoRI digestion and subcloned into the EcoRI site of the adenoviral shuttle vector pAC/CMVpLpA. This plasmid was cotransfected with pJM17, containing the full-length adenovirus-5 genome (dl309), into 293 cells by calcium phosphate/DNA coprecipitation. Homologous recombination between adenovirus sequences in the shuttle vector pAC/CMVpLpA and in the pJM17 plasmid generates a genome of a packable size in which most of the adenovirus early region 1 is lacking, thus rendering the recombinant virus replication defective (12Jover R. Bort R. Gomez-Lechon M.J. Castell J.V. Hepatology. 2001; 33: 668-675Crossref PubMed Scopus (201) Google Scholar). The resulting virus (named Ad-HNF4α) was plaque purified, expanded into a high concentration stock, and titrated by plaque assay as previously described (31Castell J.V. Hernandez D. Gomez-Foix A.M. Guillen I. Donato T. Gomez-Lechon M.J. Gene Ther. 1997; 4: 455-464Crossref PubMed Scopus (46) Google Scholar). A recombinant adenovirus for the coactivator SRC1 was prepared by using the AdEasy™ adenoviral vector system (Stratagene). SRC1 cDNA was released from pCR3.1-SRC1a (Dr. O'Malley) by ApaI digestion, subcloned into the pSPORT vector (Invitrogen), and ligated into the BglII and KpnI sites of the adenoviral pShuttle-CMV vector (Stratagene). To generate recombinant adenovirus, the linearized plasmid (PmeI digestion) was transferred into BJ 5183 cells that contained the pretransferred Ad-Easy-1 vector. Colonies containing the correct recombinant adenovirus were identified using restriction enzymes and PCR with insert-specific primers. The recombinant adenovirus DNA was then linearized by PacI and transfected into human embryonic kidney 293 cells by the calcium phosphate precipitation method. After several days of culture, infected 293 cells were collected and subjected to three cycles of freezing/thawing. The generation of a high titer adenovirus stock was performed as described (31Castell J.V. Hernandez D. Gomez-Foix A.M. Guillen I. Donato T. Gomez-Lechon M.J. Gene Ther. 1997; 4: 455-464Crossref PubMed Scopus (46) Google Scholar). The adenoviral vector for the expression of PGC1α was a kind gift from Dr. Puigserver (32Lehman J.J. Barger P.M. Kovacs A. Saffitz J.E. Medeiros D.M. Kelly D.P. J. Clin. Investig. 2000; 106: 847-856Crossref PubMed Scopus (1026) Google Scholar). The Ad-PGC-1 vector contains, in tandem, the green fluorescent protein gene and the PGC1α cDNA (containing FLAG and HA epitope tags) downstream of separate cytomegalovirus promoters. Cell lines and primary hepatocytes were infected with recombinant adenoviruses for 120 min at a m.o.i. (multiplicity of infection) ranging from 1 to 40 plaque-forming units/cell. Thereafter, cells were washed and fresh medium added. 48 h post-transfection, cells were analyzed or directly frozen in liquid N2. Transfection and Reporter Gene Assays—The chimeric luciferase reporter construct containing three in-tandem copies of a HNF4α response element for human ApoCIII in front of a TK promoter (pGL3-B-3xApoCIII-TK-LUC) (33Ktistaki E. Talianidis I. Science. 1997; 277: 109-112Crossref PubMed Scopus (109) Google Scholar) and its control reporter vector (pGL3-B-TK-LUC) were kindly provided by Dr. Talianidis. The expression vectors for transcription factors and coactivators were the following: pMT2-HNF4B (Dr. Talianidis), pcDNA3-HA-hPGC1 (Dr. Kralli), pCR3.1-SRC1a (Dr. O'Malley), pCMX-FLAG-PCAF (Dr. Talianidis), pSG5-GRIPI (Dr. Stallcup), pSG5-TIF-II (Dr. Gronemeyer), pCMX-ACTR (Dr. Evans). Plasmid DNAs were purified with Qiagen Maxiprep kit columns (Qiagen) and quantified by A260 and fluorescence using PicoGreen® (Molecular Probes). The day before transfection, cells were plated in 35-mm dishes with 1.5 ml of Dulbecco's modified Eagle's medium/Nut F12 (Invitrogen) supplemented with 6% newborn calf serum, 50 units/ml penicillin, and 50 μg/ml streptomycin. Firefly luciferase expression constructs (pGL3-B-3xApoCIII-TK-LUC and pGL3-B-TK-LUC) (0.5 μg) were transfected with varying amounts of expression plasmids (0.2–3.0 μg) by the calcium phosphate precipitation method as indicated in the figures. The total amount of expression vector was kept constant by adding empty expression vector. In parallel, 0.08 μg of pRL-CMV (a plasmid expressing Renilla reniformis luciferase under the cytomegalovirus immediate early enhancer/promoter) was cotransfected to correct variations in transfection efficiency. Calcium phosphate/DNA coprecipitates were added directly to each culture, and cells were incubated for an additional 48 h. Luciferase activities were assayed using the Dual-Luciferase® reporter kit (Promega). Quantification of mRNA Levels—Total cellular RNA was extracted with the RNeasy Total RNA kit (Qiagen), and contaminating genomic DNA was removed by incubation with DNase I Amplification Grade (Invitrogen). RNA (1 μg) was reverse transcribed as described (34Perez G. Tabares B. Jover R. Gomez-Lechon M.J. Castell J.V. Toxicol. In Vitro. 2003; 17: 643-649Crossref PubMed Scopus (39) Google Scholar, 35Rodriguez-Antona C. Jover R. Gomez-Lechon M.J. Castell J.V. Arch. Biochem. Biophys. 2000; 376: 109-116Crossref PubMed Scopus (93) Google Scholar). Diluted cDNA (3 μl) was amplified with a rapid thermal cycler (LightCycler Instrument; Roche Diagnostics) in 15 μl of LightCycler DNA Master SYBR Green I (Roche Applied Science), 5 mm MgCl2, and 0.3 μm of each oligonucleotide. We designed specific primer sets for 18 different cDNAs including liver genes, transcription factors, and coactivators (supplemental Table S1). Whenever possible, primer sequences were chosen to span exon boundaries. In parallel, we always analyzed the mRNA concentration of the human housekeeping porphobilinogen deaminase (hydroxymethylbilane synthase) as an internal control for normalization (supplemental Table S1). A stable expression of the housekeeping porphobilinogen deaminase gene was validated by comparison with TATA box-binding protein expression as a second constitutive control gene (Human TBP Primer Set; Invitrogen). We found that the expression ratio of these two internal control genes was practically constant in the different tissues and cells investigated. Moreover, human porphobilinogen deaminase and TATA box-binding protein do not harbor pseudogenes and show genomic stability in cancer (36Vandesompele J. De Preter K. Pattyn F. Poppe B. Van Roy N. De Paepe A. Speleman F. Genome. Biol. 2002; 3 (RESEARCH0034.1–0034.11)Crossref PubMed Google Scholar). PCR amplicons were confirmed to be specific by size (agarose gel electrophoresis) and melting curve analysis. After denaturing for 30 s at 95 °C, amplification was performed in 40 cycles of 1 s at 94°C, 5 s at 62 °C, and 15–20 s at 72 °C. The real-time monitoring of the PCR reaction and the precise quantification of the products in the exponential phase of the amplification were performed with the LightCycler quantification software according to the manufacturer's recommendations. Reproducibility of the measurements was assessed by conducting triplicate reactions. Extraction of Nuclear Proteins and Immunoblotting—Nuclear extracts from cultured cells were prepared as described (37Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2214) Google Scholar) and electrophoresed in an SDS-polyacrylamide gel (20 μg of protein/lane). Proteins were transferred to polyvinylidene fluoride membranes (Immobilon; Millipore), and sheets were incubated with a goat polyclonal antibody raised against a carboxyl-terminal epitope of human HNF4α (Santa Cruz Biotechnology). After washing, blots were developed with horseradish peroxidase-labeled IgG using an Enhanced Chemiluminescence kit (Amersham Biosciences). Equal loading was verified by Coomassie Blue staining of the membrane blots. Chromatin Immunoprecipitation (ChIP) Assay and RNAPol-ChIP—Cells were treated with 1% formaldehyde in phosphate-buffered saline buffer under gentle agitation for 10 min at room temperature in order to cross-link transcription factors to DNA. Thereafter, cells were collected by centrifugation, washed, resuspended in lysis buffer, and sonicated on ice for 6 × 10-s steps at 75% output in a Branson Sonicator. Cross-linking and sonication of chromatin from human liver tissue (750 mg) was carried out following a partially different protocol (38Sandoval J. Rodriguez J.L. Tur G. Serviddio G. Pereda J. Boukaba A. Sastre J. Torres L. Franco L. Lopez-Rodas G. Nucleic Acids Res. 2004; 32: e88Crossref PubMed Scopus (118) Google Scholar). Sonicated samples were centrifuged to clear supernatants. DNA content was carefully measured by fluorescence with PicoGreen dye (Molecular Probes) and properly diluted to obtain an equivalent amount of DNA in all samples (input DNA). For immunoprecipitation, two different antibodies for HNF4α (sc-6556 and sc-8987; Santa Cruz Biotechnology) and a specific antibody against the RPB1 subunit of RNApol II (sc-899) were used. The immunofractionation of protein-DNA complexes was performed by the addition of 10 μg/ml of specific antibodies with incubation at 4 °C overnight on a 360° rotator (antibody-bound DNA fraction). For each cell preparation, an additional mock immunoprecipitation with rabbit preimmune IgG (sc-2027; Santa Cruz Biotechnology) was performed (background DNA fraction). The immunocomplexes were affinity absorbed with 10 mg of protein A/G-Sepharose (prewashed with lysis buffer for 4 h at 4°C under gentle rotation) and collected by centrifugation (6500 × g, 1 min). The antibody-bound and background DNA fractions were washed as described (38Sandoval J. Rodriguez J.L. Tur G. Serviddio G. Pereda J. Boukaba A. Sastre J. Torres L. Franco L. Lopez-Rodas G. Nucleic Acids Res. 2004; 32: e88Crossref PubMed Scopus (118) Google Scholar). The cross-links were reversed by heating the samples at 65 °C overnight. The DNA from bound, background, and input fractions was purified, diluted (1/10 bound and background fractions, 1/400 input fraction), and subjected to quantitative real-time PCR with a LightCycler instrument. Amplification was real-time monitored and allowed to proceed in the exponential phase until fluorescent signal from input samples reached a significant value. Amplified DNA was then analyzed by agarose gel electrophoresis. Amplification and quantification of ApoCIII gene sequences (–740 and –80-bp 5′-flanking regions, and exons 3 and 4) among the pull of DNA was performed with specific primers flanking these regions (supplemental Table S2). The detection of RNA polymerase II within the coding region of the ApoCIII gene allows the quantification of the actual transcriptional rate (38Sandoval J. Rodriguez J.L. Tur G. Serviddio G. Pereda J. Boukaba A. Sastre J. Torres L. Franco L. Lopez-Rodas G. Nucleic Acids Res. 2004; 32: e88Crossref PubMed Scopus (118) Google Scholar). To ensure reproducibility, immunoprecipitation and PCR analysis were performed in duplicate from different liver tissue samples, cultured hepatocytes, and cell lines. Ureagenesis—The ureogenic rate was assessed in HepG2 cells incubated with 3 mm ammonium chloride by measuring the appearance of urea in the culture medium. Urea concentration was determined by the diacetylmonoxime method (30Gomez-Lechon M. Castell J. Berry M.N. Edwards A.M. The Hepatocyte Review. Kluwer Academic Publishers, London2000: 11-17Google Scholar). HNF4α Levels in Different Hepatic and Non-hepatic Cell Models and in Human Liver—HNF4α expression levels in cultured human hepatocytes and hepatoma cell lines (HepG2, Hep3B, and BC2) were similar to those of liver tissue as assessed by RT-PCR (Fig. 1A) and immunoblotting analysis (Fig. 1B). Among the several hepatomas analyzed, the highest HNF4α expression level was found in the widely used cell line HepG2. The expression of HNF4α was, however, very low or absent in non-hepatic cell lines (i.e. 293 and HeLa) and in the more dedifferentiated human hepatoma Mz-Hep-1 (Fig. 1). HNF4α Function Is Impaired in Human Hepatoma HepG2 Cells—The expression of many hepatic genes has an absolute dependence on HNF4α. Data obtained from HNF4α null mice demonstrated that this transcription factor is indispensable for the constitutive expression of key hepatic genes such as apolipoproteins (A, B, and C families), L-FABP, PEPCK, AldoB, OTC, and CYP7A1 (10Li J. Ning G. Duncan S.A. Genes Dev. 2000; 14: 464-474PubMed Google Scholar, 11Hayhurst G.P. Lee Y.H. Lambert G. Ward J.M. Gonzalez F.J. Mol. Cell. Biol. 2001; 21: 1393-1403Crossref PubMed Scopus (868) Google Scholar, 39Inoue Y. Hayhurst G.P. Inoue J. Mori M. Gonzalez F.J. J. Biol. Chem. 2002; 277: 25257-25265Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 40Stoffel M. Duncan S.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13209-13214Crossref PubMed Scopus (341) Google Scholar, 41Parviz F. Matullo C. Garrison W.D. Savatski L. Adamson J.W. Ning G. Kaestner K.H. Rossi J.M. Zaret K.S. Duncan S.A. Nat. Genet. 2003; 34: 292-296Crossref PubMed Scopus (468) Google Scholar). A comparative analysis of eight well characterized HNF4α target genes in different cell types revealed high expression levels in cultured human hepatocytes and null or very low levels in HepG2 cells (supplemental Fig. S1). Among the eight mRNA measured, we specifically found that ApoCIII, AldoB, PEPCK, and OTC were essentially not expressed in HepG2 cells, whereas ApoAII, ApoAV, CYP7A1, and L-FABP showed levels of ∼20% of human liver (supplemental Fig. S1). The expression profile in HepG2 cells was closer to that of non-hepatic cell lines (293 and HeLa). To gain a better understanding of the discrepancy between high HNF4α levels and the low or null expression of target genes in HepG2, we performed chromatin immunoprecipitation assays and analyzed the occupancy of two different binding sites in the human ApoCIII gene by HNF4α (Fig. 2A). In parallel, we performed RNAPol-chromatin immunoprecipitation to measure the binding of RNApol-II to the promoter and the transcription through ApoCIII coding regions (exons 3 and 4) (Fig. 2B). We confirmed an appropriate binding of HNF4α to the –740 and –80-bp elements in human liver samples, as well as RNApol-II binding to the promoter and active transcription through exons 3 and 4. Similar results were found in cultured human hepatocytes (data not shown). However, in the human hepatoma HepG2, binding of HNF4α to the –80-bp element was substantially decreased (Fig. 2A) and RNApol-II occupancy at the promoter and coding regions was almost undetectable (Fig. 2B). As a negative control, we also analyzed non-hepatic HeLa cells where HNF4α and ApoCIII are not expressed. Our results suggest that binding and transactivation by HNF4α is impaired in HepG2 cells. Possible Mechanisms Underlying the Dysfunction of HNF4α in Hepatoma Cells—HNF4α exists in several isoforms, all of which are capable of binding to the same regulatory elements but with different transactivating properties. An imbalanced expression of HNF4α isoforms could explain why HNF4α is non-operative in HepG2. However, this apparently is not the case; the major HNF4α splicing variants (α1, α2, α3, and α7) had similar expression levels in HepG2 and in human hepatocytes (data not shown). Another mechanism causing HNF4α dysfunction in hepatoma cells could be an increase of negative factors that block HNF4α activity. The small heterodimeric partner, which lacks a DNA binding domain and exhibits inhibiting interactions with HNF4α, might be involved. Similarly, the" @default.
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• W2040994916 title "Underexpressed Coactivators PGC1α AND SRC1 Impair Hepatocyte Nuclear Factor 4α Function and Promote Dedifferentiation in Human Hepatoma Cells" @default.
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