The Mouse Fetoprotein Transcription Factor (FTF) Gene Promoter Is Regulated by Three GATA Elements with Tandem E Box and Nkx Motifs, and FTF in Turn Activates the Hnf3β, Hnf4α, and Hnf1α Gene Promoters
2001; Elsevier BV; Volume: 276; Issue: 16 Linguagem: Inglês
10.1074/jbc.m010737200
ISSN1083-351X
AutoresJean‐François Paré, Sylvie Roy, Luc Galarneau, Luc Bélanger,
Tópico(s)Pediatric Hepatobiliary Diseases and Treatments
ResumoFetoprotein transcription factor (FTF) is an orphan nuclear receptor that activates the α1-fetoprotein gene during early liver developmental growth. Here we sought to define better the position of FTF in transcriptional cascades leading to hepatic differentiation. The mouse FTF gene was isolated and assigned to chromosome 1 band E4 (one mFTF pseudogene was also found). Exon/intron mapping shows an mFTF gene structure similar to that of its close homologue SF1, with two more N-terminal exons in the mFTF gene; exon mapping also delimits several FTF mRNA 5′- and 3′-splice variants. The mFTF transcription initiation site was located in adult liver at 238 nucleotides from the first translation initiator codon, with six canonical GATA, E box, and Nkx motifs clustered between −50/−140 base pairs (bp) from the cap site; DNA/protein binding assays also pinpointed an HNF4-binding element at +36 bp and an FTF-binding element at −257 bp. Transfection assays and point mutations showed that the mFTF promoter is activated by GATA, HNF4α, FTF, Nkx, and basic helix-loop-helix factors, with marked cooperativity between GATA and HNF4α. A tandem GATA/E box activatory motif in the proximal mFTF promoter is strikingly similar to a composite motif coactivated by differentiation inducers in the hematopoietic lineage; a tandem GATA-Nkx motif in the distal mFTF promoter is also similar to a composite motif transducing differentiation signals from transforming growth factor-β-like receptors in the cardiogenic lineage. Three genes encoding transcription factors critical to early hepatic differentiation,Hnf3β, Hnf4α, andHnf1α, each contain dual FTF-binding elements in their proximal promoters, and all three promoters are activated by FTF in transfection assays. Direct DNA binding action and cooperativity was demonstrated between FTF and HNF3β on the Hnf3βpromoter and between FTF and HNF4α on the Hnf1αpromoter. These combined results suggest that FTF is an early intermediary between endodermal specification signals and downstream genes that establish and amplify the hepatic phenotype. Fetoprotein transcription factor (FTF) is an orphan nuclear receptor that activates the α1-fetoprotein gene during early liver developmental growth. Here we sought to define better the position of FTF in transcriptional cascades leading to hepatic differentiation. The mouse FTF gene was isolated and assigned to chromosome 1 band E4 (one mFTF pseudogene was also found). Exon/intron mapping shows an mFTF gene structure similar to that of its close homologue SF1, with two more N-terminal exons in the mFTF gene; exon mapping also delimits several FTF mRNA 5′- and 3′-splice variants. The mFTF transcription initiation site was located in adult liver at 238 nucleotides from the first translation initiator codon, with six canonical GATA, E box, and Nkx motifs clustered between −50/−140 base pairs (bp) from the cap site; DNA/protein binding assays also pinpointed an HNF4-binding element at +36 bp and an FTF-binding element at −257 bp. Transfection assays and point mutations showed that the mFTF promoter is activated by GATA, HNF4α, FTF, Nkx, and basic helix-loop-helix factors, with marked cooperativity between GATA and HNF4α. A tandem GATA/E box activatory motif in the proximal mFTF promoter is strikingly similar to a composite motif coactivated by differentiation inducers in the hematopoietic lineage; a tandem GATA-Nkx motif in the distal mFTF promoter is also similar to a composite motif transducing differentiation signals from transforming growth factor-β-like receptors in the cardiogenic lineage. Three genes encoding transcription factors critical to early hepatic differentiation,Hnf3β, Hnf4α, andHnf1α, each contain dual FTF-binding elements in their proximal promoters, and all three promoters are activated by FTF in transfection assays. Direct DNA binding action and cooperativity was demonstrated between FTF and HNF3β on the Hnf3βpromoter and between FTF and HNF4α on the Hnf1αpromoter. These combined results suggest that FTF is an early intermediary between endodermal specification signals and downstream genes that establish and amplify the hepatic phenotype. At 8–8.5 days of mouse embryogenesis, endodermal cells of the ventral foregut interact with the cardiac mesoderm and become committed to the hepatic differentiation program; these newly specified cells then migrate and proliferate in the mesenchyme of the septum transversum where liver morphogenesis becomes apparent at ≈E10.5 (1Le Douarin N. Med. Biol. 1975; 53: 427-455PubMed Google Scholar,2Zaret K. Curr. Opin. Genet. & Dev. 1998; 8: 526-531Crossref PubMed Scopus (58) Google Scholar). Initial induction of hepatic functions is driven in part by growth factors of the FGF 1The abbreviations used are:FGFfibroblast growth factorAFPα1-fetoproteinbHLHbasic helix-loop-helixFTFfetoprotein transcription factormFTFmouse FTFHNFhepatocyte nuclear factorSF1steroidogenic factor 1CATchloramphenicol acetyltransferasePCRpolymerase chain reactionbpbase pairkbkilobase pairsntnucleotideoligooligonucleotideTGF-βtransforming growth factor-β family, secreted by cardiac mesodermal cells and acting via transmembrane receptor kinases at the endodermal cell surface (3Jung J. Zheng M. Goldfarb M. Zaret K.S. Science. 1999; 284: 1998-2003Crossref PubMed Scopus (595) Google Scholar). The process also involves potentiating transcription factors among which GATA factors appear essential to endodermal determination across vertebrates as well as invertebrates (Ref. 4Zaret K. Dev. Biol. 1999; 209: 1-10Crossref PubMed Scopus (181) Google Scholar and references therein). Following liver specification, transcriptional activation cascades further develop among early hepatic transcription factors creating interactive regulatory networks that amplify the induction signals and imprint the liver phenotype; the HNF4α gene product, for example, activates theHnf1α gene whose product further enhances theHnf4α gene promoter, and both HNF1α and HNF4α activate a broad spectrum of liver functions (5Kuo C.J. Conley P.B. Chen L. Sladek F.M. Darnell Jr., J.E. Crabtree G.R. Nature. 1992; 355: 457-461Crossref PubMed Scopus (367) Google Scholar, 6Zhong W. Mirkovitch J. Darnell Jr., J.E. Mol. Cell. Biol. 1994; 14: 7276-7284Crossref PubMed Scopus (92) Google Scholar). fibroblast growth factor α1-fetoprotein basic helix-loop-helix fetoprotein transcription factor mouse FTF hepatocyte nuclear factor steroidogenic factor 1 chloramphenicol acetyltransferase polymerase chain reaction base pair kilobase pairs nucleotide oligonucleotide transforming growth factor-β One of the earliest events marking endodermal specification to liver function is the activation of the α1-fetoprotein (AFP) locus, one of four albumin-related genes tandemly organized in the genome but differentially expressed during development (7Bélanger L. Roy S. Allard D. J. Biol. Chem. 1994; 269: 5481-5484Abstract Full Text PDF PubMed Google Scholar). The AFP gene is the first to be activated in the foregut endoderm (8Gualdi R. Bossard P. Zheng M. Hamada Y. Coleman J.R. Zaret K.S. Genes Dev. 1996; 10: 1670-1682Crossref PubMed Scopus (459) Google Scholar), and therefore transcription factors that transduce early cell specification signals to unfold AFP chromatin are likely to exert high ranking liver differentiation functions. One prime candidate in that regard is an orphan nuclear receptor originally pinpointed as a potent, highly specific and mandatory activator of the proximal AFP gene promoter (9Guertin M. LaRue H. Bernier D. Wrange O. Chevrette M. Gingras M.-C. Bélanger L. Mol. Cell. Biol. 1988; 8: 1398-1407Crossref PubMed Scopus (79) Google Scholar, 10Bernier D. Thomassin H. Allard D. Guertin M. Hamel D. Blaquière M. Beauchemin M. LaRue H. Estable-Puig M. Bélanger L. Mol. Cell. Biol. 1993; 13: 1619-1633Crossref PubMed Google Scholar, 11Galarneau L. Paré J.-F. Allard D. Hamel D. Lévesque L. Tugwood J.D. Green S. Bélanger L. Mol. Cell. Biol. 1996; 16: 3853-3865Crossref PubMed Google Scholar). The fetoprotein transcription factor (FTF) (NR5A2) (FTF designation as per Genome Data Base Nomenclature Committee (accession number 9837397); NR5A2 as per Nuclear Receptor Nomenclature Committee (12Nuclear Receptors Nomenclature Committee, Cell, 97, 1999, 161, 163.Google Scholar)) belongs to a subgroup of nuclear receptors related to the archetypal Drosophila segmentation gene Ftz-F1(13Lavorgna G. Ueda H. Clos J. Wu C. Science. 1991; 252: 848-851Crossref PubMed Scopus (243) Google Scholar) and that bind as monomers to the DNA motif PyCAAGGPyCPu (where Py is C or T and Pu is A or G)(11). FTF is widespread and tightly conserved among vertebrates (14Ellinger-Ziegelbauer H. Hihi A.K. Laudet V. Keller H. Wahli W. Dreyer C. Mol. Cell. Biol. 1994; 14: 2786-2797Crossref PubMed Google Scholar, 15Kudo T. Sutou S. Gene ( Amst. ). 1997; 197: 261-268Crossref PubMed Scopus (53) Google Scholar, 16Galarneau L. Drouin R. Bélanger L. Cytogenet. Cell Genet. 1998; 82: 269-270Crossref PubMed Google Scholar, 17Lin W.-W. Wang H.-W. Sum C. Liu D. Hew C.L. Chung B.-C. Biochem. J. 2000; 348: 439-446Crossref PubMed Scopus (36) Google Scholar); its closest mammalian relative is SF1 (18Ikeda Y. Lala D.S. Luo X. Kim E. Moisan M.-P. Parker K.L. Mol. Endocrinol. 1993; 7: 852-860Crossref PubMed Google Scholar) that is mainly expressed in steroidogenic cell lineages, whereas FTF is mainly expressed in gut derivatives. Early developmental patterns of FTF expression in several species (14Ellinger-Ziegelbauer H. Hihi A.K. Laudet V. Keller H. Wahli W. Dreyer C. Mol. Cell. Biol. 1994; 14: 2786-2797Crossref PubMed Google Scholar,17Lin W.-W. Wang H.-W. Sum C. Liu D. Hew C.L. Chung B.-C. Biochem. J. 2000; 348: 439-446Crossref PubMed Scopus (36) Google Scholar, 19Rausa F.M. Galarneau L. Bélanger L. Costa R.H. Mech. Dev. 1999; 89: 185-188Crossref PubMed Scopus (73) Google Scholar) and its activation of the AFP locus in hepatocyte progenitors suggested an important role for FTF in endodermal differentiation pathways. To gain better insight into FTF functions and its position in liver induction pathways, we isolated and functionally characterized the mouse FTF gene promoter in search for upstream FTF gene regulators, and we also searched for new FTF downstream gene targets. The combined results make a compelling case for FTF as a key intermediary between initial signals of liver specification and cascade activations of other transcription factors that enhance hepatic differentiation. cis-activating elements pin-pointed in the mFTF promoter may lead to new important effectors of endodermal differentiation pathways. A mouse 129 SV genomic DNA library in bacteriophage λDASH II (Stratagene) was screened by plaque hybridization using rat FTF cDNA (11Galarneau L. Paré J.-F. Allard D. Hamel D. Lévesque L. Tugwood J.D. Green S. Bélanger L. Mol. Cell. Biol. 1996; 16: 3853-3865Crossref PubMed Google Scholar) as a probe. The PCR method was used to screen a mouse 129 SV P1 bacteriophage library (Genome Systems) with the following primers from mouse FTF gene introns: 5′-AGTTGAATCTCTGCTGCCCGTGTCC-3′ (intron 2) and 5′- TAGCCCGAGAGTGTAAAACCAGGAA-3′ (intron 3). λDASH II DNA from the FTF-positive clones was prepared by the plate lysate method, and the P1 clone DNA was prepared by the alkaline lysis procedure. DNA was digested with various restriction enzymes, and DNA fragments were ligated into pBluescript SK+ (Stratagene) and used to transform Escherichia coli DH5α; plasmid DNA was isolated, and positive subclones were identified by Southern analysis using mouse FTF/LRH-1 (11Galarneau L. Paré J.-F. Allard D. Hamel D. Lévesque L. Tugwood J.D. Green S. Bélanger L. Mol. Cell. Biol. 1996; 16: 3853-3865Crossref PubMed Google Scholar) cDNA fragments. DNA was sequenced on both strands by the dideoxynucleotide method, and exon/intron boundaries were mapped against the mFTF/LRH1 cDNA sequence (11Galarneau L. Paré J.-F. Allard D. Hamel D. Lévesque L. Tugwood J.D. Green S. Bélanger L. Mol. Cell. Biol. 1996; 16: 3853-3865Crossref PubMed Google Scholar); mFTF genomic sequences were deposited in GenBankTM under accession number AF239709(BankIt 322861). The mFTF gene transcription start site was mapped by primer extension of mouse liver RNA using a 25-mer oligonucleotide (FTF-PE1) spanning mouse FTF gene sequence +37 to +61 (underlined in Fig.1B). FTF-PE1 was radiolabeled (106 cpm/ng) with [γ-32P]ATP and T4 polynucleotide kinase (Life Technologies, Inc.) and purified by gel electrophoresis and electroelution; 5 × 105 cpm of the FTF-PE1 primer was annealed to 200 μg of total RNA from adult mouse liver overnight at 50 °C in 32 μl of Superscript II reverse transcriptase buffer (Life Technologies, Inc.); the reaction was cooled to 42 °C and pursued for 2 h in the presence of 10 mmdithiothreitol, 1 mm dNTPs, and 100 units of Superscript II reverse transcriptase. Reverse transcription products were subjected to RNase A digestion, phenol/chloroform extraction, and ethanol/sodium acetate precipitation and analyzed by electrophoresis on denaturing 6% polyacrylamide gels; reference DNA sequences used the FTF-PE1 primer with DNA from FTF gene plasmid 4F-CAT (below). Chromosomal localization of the mouse FTF gene was carried out by fluorescence in situ hybridization (Genome Systems). Mouse FTF genomic DNA (P1 clone) was labeled with digoxigenin dUTP by nick translation, mixed with sheared mouse DNA, and hybridized to metaphase chromosomes from normal mouse embryo fibroblasts in 50% formamide, 10% dextran sulfate, and 2× SSC. Hybridization signals were revealed with fluoresceinated anti-digoxigenin antibodies followed by counterstaining with 4,6-diamidino-2-phenylindole. Of 80 metaphases analyzed, 72 exhibited specific labeling; FTF gene assignment was further confirmed by cohybridization with a telomeric probe specific to chromosome 1. FTF gene promoter activity was analyzed with FTF/CAT reporter constructs spanning 4 kb or 280 bp of contiguous DNA 5′-adjacent to the mouse FTF transcription start site. AHindIII-BamHI fragment from pSV0-CAT (9Guertin M. LaRue H. Bernier D. Wrange O. Chevrette M. Gingras M.-C. Bélanger L. Mol. Cell. Biol. 1988; 8: 1398-1407Crossref PubMed Scopus (79) Google Scholar) was cloned into HindIII-BamHI-digested pBluescript SK+ to generate vector SK-CAT; the EcoRI FTF gene fragment −3.9 kb to +79 bp (leftmost fragment in Fig.1A) was isolated, blunted, and inserted inHindIII-blunted SK-CAT to generate construct 4F-CAT. To obtain pF-CAT, the 4F-CAT vector was digested with PstI to leave only FTF gene segment −184/+79 upstream from CAT, and aPstI −280/−185 FTF segment was inserted at −184 in the correct orientation. Mutations in the FTF gene promoter were derived by PCRs using plasmid 4F-CAT and oligonucleotides listed in Table I; two complementary oligonucleotides (125 ng of each) overlapping the targeted region were mixed with 50 ng of 4F-CAT DNA and amplified withPfu DNA polymerase (Stratagene) for 30 s at 95 °C, 1 min at 55 °C, and 2 min/kb of DNA template at 68 °C (18 cycles), followed by 1 h at 37 °C with 10 units of DpnI (Stratagene). The PCR products were transformed into E. coliDH5α, and mutations were confirmed by sequencing. Mutant 4FmG123 was derived from 4FmG3.Table IOligonucleotides used for mutagenesisMutationSequenceFmH4CATACATGCTGGAAAAAaTaaAaAaTaCAGGAGAACACGGACTGGFmG3CAGGGTCCTCTTagAACCGGCACAGGGTCATGFmG12CTTTGCCATTAggTGCCAAGAGTTCCTTAggTCTTTTCGTGTCACFmE1CGGCACAGGGTCATGcGACCAGTTGAAGCCFmE2GCACAGGGTCATGTGACaAGTTGAAGCCTATTGAAAGCFmE12CGGCACAGGGTCATGcGACaAGTTGAAGCCTATTGAAAGCFmNkCTCTTTTCGTGTCAtgTTATAACAGGGTCCTCH1mF1GCGCACGGATAAATATGAAaagTGGAGAATTTCCCCAGCTCCH1mF2CTCACCCCCATGAGGCCTGCACTTGCAAaaCTGAAGTCCAAAGTTCH3mF2GTTAGTACTTTACTTTTCAGTTAAATCagAtcTGCCCAACGCAT Open table in a new tab Reporter vector pH1-CAT driven by the Hnf1α promoter was obtained by PCR amplification of mouse Hnf1α gene region −174/+10 using mouse genomic DNA and the following primers: 5′-GGCTCGAGTGCTCACTCCCAATTGCAGGCCATGACTCC-3′ and 5′-CCAAGCTTGGCCAGTGAATCAGGGCCCCTGCCTGCTC-3′. The PCR product was digested with XhoI-HindIII and cloned in SK-CAT to generate pH1-CAT. Mutations were introduced in pH1-CAT by PCRs as described above for the FTF constructs, using oligonucleotides H1mF1 and H1mF2 (Table I) with pH1-CAT plasmid DNA or its derivative pH1mF1-CAT. HNF3β-CAT reporters pH3-CAT and H3mF1 are constructs HNF3β −184/+69 and its UF2-H3β mutant, described elsewhere (20Pani L. Qian X. Clevidence D. Costa R.H. Mol. Cell. Biol. 1992; 12: 552-562Crossref PubMed Scopus (95) Google Scholar); vector H3mF12 was derived from H3mF1 using PCR with a pTZ18U internal primer and oligonucleotide H3mF2 (Table I). The PCR product was digested with ScaI and SacI and used to replace the ScaI/SacI segment of H3mF1. HNF4α reporter vector pH4-luc is a luciferase expression vector carrying a −363/+182-bp mouse Hnf4α gene segment (6Zhong W. Mirkovitch J. Darnell Jr., J.E. Mol. Cell. Biol. 1994; 14: 7276-7284Crossref PubMed Scopus (92) Google Scholar). Bandshift assays were conducted as described before (10Bernier D. Thomassin H. Allard D. Guertin M. Hamel D. Blaquière M. Beauchemin M. LaRue H. Estable-Puig M. Bélanger L. Mol. Cell. Biol. 1993; 13: 1619-1633Crossref PubMed Google Scholar, 11Galarneau L. Paré J.-F. Allard D. Hamel D. Lévesque L. Tugwood J.D. Green S. Bélanger L. Mol. Cell. Biol. 1996; 16: 3853-3865Crossref PubMed Google Scholar) with total nuclear proteins from adult rat liver and oligonucleotides listed in Table II, using 0.2 ng of 32P-labeled probe, 3 μg of nuclear proteins, and 5–500-fold molar excess of cold competitor oligonucleotides. For FTF binding assays, probe and competitors were coincubated with the nuclear extract for 30 min at 4 °C; for HNF4α supershift assays, 1 μl of anti-HNF4α serum (Santa Cruz Biotechnology) was preincubated with the nuclear extract 1 h at room temperature, and then the probe and competitors were added for an additional 30 min.Table IIOligonucleotides used in electrophoretic mobility shift assayOligonucleotideSequenceFTFαTGTTCAAGGACAmFTFαCTTATGTTCAATGAAAAAGACCFFACCTCAAGTCCACTAH1FTF-1AAATCTTCCAAGGTTCATCTTTH1FTF-2CTTGCAAGGCTGH3FTF-1TTTCAAGGTTACH3FTF-2GTTAAATCCAAGGTGCCCAAH4FTF-1TCACCAAGGTGGACAH4FTF-2CTTCCAAGGCAGFH4AGTGCAGAGTCCAGGmFH4GGCTAAACGTGAAGGDR1GGGTCAAAGGTCAAT Open table in a new tab Transfection assays were carried out by the calcium phosphate procedure detailed previously (9Guertin M. LaRue H. Bernier D. Wrange O. Chevrette M. Gingras M.-C. Bélanger L. Mol. Cell. Biol. 1988; 8: 1398-1407Crossref PubMed Scopus (79) Google Scholar, 10Bernier D. Thomassin H. Allard D. Guertin M. Hamel D. Blaquière M. Beauchemin M. LaRue H. Estable-Puig M. Bélanger L. Mol. Cell. Biol. 1993; 13: 1619-1633Crossref PubMed Google Scholar), using HepG2, Hep3B, HeLa, and F9 cells maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% fetal bovine serum (Wisent) and 1% penicillin/streptomycin (F9 cultures used gelatin-coated dishes). Cells were plated at 1.5 × 106 cells in 6-cm Petri dishes or 75-cm2 flasks and transfected 24 h later with 2–5 μg of CAT reporter plasmid, 2–10 μg of transcription factor expression vector, and 1.5–2.5 μg of RSV-lacZ to standardize for transfection efficiency. Cells were washed after 16 h and harvested 48 h after transfection. CAT activity was measured by thin layer chromatography and phosphorimaging. Luciferase activity was measured with a EG & G Berthold Lumat LB9507 luminometer. Expression vectors for transcription factors used viral enhancer-promoter elements from cytomegalovirus (FTF, HNF4α, C/EBPα, HNF1α, HNF3α, HNF3β, HNF6, SP1, and Nkx2.5), Rous sarcoma virus (GATA1, GATA2, GATA4, GATA5, and GATA6), and simian virus 40 (COUP-TFII); the FTF vector carried full-length human FTF cDNA (16Galarneau L. Drouin R. Bélanger L. Cytogenet. Cell Genet. 1998; 82: 269-270Crossref PubMed Google Scholar, 21Gilbert S. Galarneau L. Lamontagne A. Roy S. Bélanger L. J. Virol. 2000; 74: 5032-5039Crossref PubMed Scopus (44) Google Scholar). We retrieved three independent clones (ZF2, ZF6, and ZF25) from the mouse genomic library screened with a rat FTF cDNA probe; clones ZF2 and ZF25 gave stronger signals on Southern blots and were further characterized. Clone ZF2 contained a contiguous sequence closely matching the mouse FTF/LRH-1 cDNA sequence (11Galarneau L. Paré J.-F. Allard D. Hamel D. Lévesque L. Tugwood J.D. Green S. Bélanger L. Mol. Cell. Biol. 1996; 16: 3853-3865Crossref PubMed Google Scholar) but with many base changes, deletions, insertions, and no introns, all indicative of a retro-pseudogene. Clone ZF25 contained FTF coding and interspersed DNA sequences corresponding to the exon 3–6 domain in Fig.1A. Intronic oligonucleotides were used for further screening of a mouse P1 library, which yielded an insert extending 35 kb upstream from ZF25. The P1 insert revealed additional exons and a cluster of transcription factor recognition sites 5′-flanking exon 1 and suggestive of a promoter domain (Fig.1B). Intron/exon boundaries mapped from λ or P1 clones all conformed to the GT/AG splicing rule. Mouse FTF exons 3–6 correspond to SF1 exons 1–4 (18Ikeda Y. Lala D.S. Luo X. Kim E. Moisan M.-P. Parker K.L. Mol. Endocrinol. 1993; 7: 852-860Crossref PubMed Google Scholar), and two additional 5′ FTF exons encode the longer FTF N-terminal (A/B) domain (11Galarneau L. Paré J.-F. Allard D. Hamel D. Lévesque L. Tugwood J.D. Green S. Bélanger L. Mol. Cell. Biol. 1996; 16: 3853-3865Crossref PubMed Google Scholar); the conserved exon/intron junctions of SF1 and FTF predict that the FTF 3′-gene domain contains three more exons (7–9 in the mouse) encoding C terminus amino acids 390–560 (11Galarneau L. Paré J.-F. Allard D. Hamel D. Lévesque L. Tugwood J.D. Green S. Bélanger L. Mol. Cell. Biol. 1996; 16: 3853-3865Crossref PubMed Google Scholar). mFTF exons 1 and 4–6 are similar in size to zebrafishff1 exons 1 and 3–5 (17Lin W.-W. Wang H.-W. Sum C. Liu D. Hew C.L. Chung B.-C. Biochem. J. 2000; 348: 439-446Crossref PubMed Scopus (36) Google Scholar). The mFTF gene transcription initiation site was identified by primer extension of adult mouse liver RNA using antisense primer +61/+37 (underlined in Fig. 1B); a predominant transcription start site was mapped (Fig. 1B) at a G residue 79 nt upstream from the previously reported 5′-end of mouse FTF cDNA and 238 nt upstream from the first translation initiation codon (11Galarneau L. Paré J.-F. Allard D. Hamel D. Lévesque L. Tugwood J.D. Green S. Bélanger L. Mol. Cell. Biol. 1996; 16: 3853-3865Crossref PubMed Google Scholar) (other extension primers downstream of +79 bp yielded smears of reverse transcription products terminating around +79; some mRNA structure perhaps blocked polymerase extension but alternative start sites around +79 cannot be excluded). Chromosomal assignment of the mFTF gene was carried out by fluorescence in situ hybridization with a P1-FTF genomic probe minimizing cross-hybridization with the mouse FTF pseudogene. Out of 80 metaphases analyzed, 72 showed single specific labeling of the middle portion of chromosome 1 (identified by 4,6-diamidino-2-phenylindole staining and cohybridization with a specific telomeric probe); measurements of chromosomal distances on 10 fluorescence in situ hybridization metaphases located the mouse FTF gene at chromosome 1 band E4 (Fig. 1C). Functional analyses were initiated with mFTF gene construct 4F-CAT carrying FTF gene segment −4 kb/+79 bp. Transfection assays showed strong reporter gene activity in HepG2 and Hep3B hepatoma cells (which express endogenous FTF) (21Gilbert S. Galarneau L. Lamontagne A. Roy S. Bélanger L. J. Virol. 2000; 74: 5032-5039Crossref PubMed Scopus (44) Google Scholar) and no detectable activity in (FTF-negative) HeLa or F9 cells (inset, Fig.2B). Reporter CAT activity was also higher in Hep3B than HepG2 cells, correlating with higher expression of endogenous FTF in Hep3B cells (21Gilbert S. Galarneau L. Lamontagne A. Roy S. Bélanger L. J. Virol. 2000; 74: 5032-5039Crossref PubMed Scopus (44) Google Scholar). These results thus indicated that mFTF gene segment −4 kb/+79 bp correctly reproduced hepatic specificity and relative activities of endogenous FTF gene promoter functions. Electromobility shift assays were then conducted with presumptive HNF4 and FTF recognition sequences located at +36 and −257 (Fig. 1B), slightly divergent from consensus binding sites. Radiolabeled DNA probe +36/+50 (oligonucleotide FH4, Table II) yielded a single retarded liver complex that was competed by cold oligo FH4 and more efficiently by an optimized HNF4-binding site (oligo DR1) (Fig. 2A, lanes 1–7) but not at all by 100-fold excess of a mutated FH4 sequence (oligo mFH4 in Table II) (Fig. 2A, lane 10). Furthermore, the retarded complex was completely supershifted by anti-HNF4α antibodies (Fig. 2A, lane 11). These results showed that FTF promoter segment +36/+50 is a highly specific HNF4α recognition site, of lower affinity than a canonical HNF4 DR1 element. Similar assays using as a probe the strong FTF-binding element of the AFP gene promoter (10Bernier D. Thomassin H. Allard D. Guertin M. Hamel D. Blaquière M. Beauchemin M. LaRue H. Estable-Puig M. Bélanger L. Mol. Cell. Biol. 1993; 13: 1619-1633Crossref PubMed Google Scholar, 11Galarneau L. Paré J.-F. Allard D. Hamel D. Lévesque L. Tugwood J.D. Green S. Bélanger L. Mol. Cell. Biol. 1996; 16: 3853-3865Crossref PubMed Google Scholar) (oligonucleotide FTFα in Table II) indicated that mFTF gene segment −254/−268 (oligo FF, Table II) can also efficiently bind FTF, with lower affinity than the FTFα site (Fig. 2A, lanes 12–17). mFTF gene activation by HNF4α or FTF was tested by transient transfection in Hep3B and HepG2 cells. Cotransfection of construct 4F-CAT with an HNF4α expression vector resulted in 5–6-fold enhancement of reporter gene activity in both cell lines (Fig.2B, lane 3); cotransfection with an FTF expression vector also resulted in significant increase of CAT activity in Hep3B cells (Fig. 2B, lane 4) but not in HepG2 cells (perhaps because HepG2 cells express abundant SF1, which binds and activates the same DNA motif as FTF) (11Galarneau L. Paré J.-F. Allard D. Hamel D. Lévesque L. Tugwood J.D. Green S. Bélanger L. Mol. Cell. Biol. 1996; 16: 3853-3865Crossref PubMed Google Scholar, 21Gilbert S. Galarneau L. Lamontagne A. Roy S. Bélanger L. J. Virol. 2000; 74: 5032-5039Crossref PubMed Scopus (44) Google Scholar). Three consensus GATA-binding sites (WGATAR) were conspicuous in the proximal mFTF gene domain −80/−138 (Fig.1B), and we tested activatory effects of GATA expression vectors including the GATA6 and GATA4 variants expressed in the foregut endoderm and liver primordium (22Bossard P. Zaret K.S. Development. 1998; 125: 4909-4917Crossref PubMed Google Scholar, 23Koutsourakis M. Langeveld A. Patient R. Beddington R. Grosveld F. Development. 1999; 126: 723-732Crossref Google Scholar). Cotransfection of GATA6 or GATA4 (and GATA1, GATA2, or GATA5 as well) raised 4F-CAT activity 2–3-fold in HepG2 or Hep3B cells (Fig. 2B, lane 2); coexpression of GATA6 or GATA4 with HNF4α showed additive or cooperative effects enhancing FTF promoter activity as much as 12-fold in Hep3B cells (Fig. 2B, lane 6). We also found significant activation of the 4F-CAT construct with the Nkx2.5 expression vector (Fig. 2B, lane 5), which is consistent with the recognition of a cognate high affinity (24Chen C.Y. Schwartz R.J. J. Biol. Chem. 1995; 270: 15628-15633Crossref PubMed Scopus (268) Google Scholar) Nkx2.5-binding element at −99/−105 bp (Fig. 1B); coexpression of GATA4 and Nkx2.5 enhanced 4F-CAT activity only slightly more than each factor alone (not shown). No stimulation of mFTF promoter activity was detected with expression vectors encoding SP1, HNF1α, HNF3α, HNF3β, HNF6, or C/EBPα (Fig. 2B, lanes 7–12) (no binding sequences for these factors were apparent in the mFTF promoter sequence), nor with c-Myc, USF1, or USF2 expression vectors (not shown). Generally similar results were obtained in cotransfections using the shorter FTF gene construct pF-CAT carrying only 280 bp of 5′-flanking DNA (Fig. 2B, lanes 13–17); this suggested a direct action of the activating factors on their cognate DNA motifs clustered around the cap site. Basal activity of pF-CAT was similar to that of 4F-CAT and provided no indication for regulatory components operating between −280 bp and −4 kb. To prove their role in mFTF promoter activity, point mutations were introduced in the HNF4, GATA, and Nkx elements in the natural 4-kb context of construct 4F-CAT; mutations were also introduced in tandem E box motifs at −66 and −58 bp. Mutation of the HNF4 sequence (FmH4 in Table I) reduced basal mFTF promoter activity in HepG2 or Hep3B cells (Fig. 3B, lane 2), and in cotransfection assays it abolished mFTF promoter activation by HNF4α (Fig. 3C, lanes 1 versus 4). Mutations targeting the three GATA elements (mutant mG123) or only the proximal GATA site (mG3) also reduced basal FTF promoter activity (Fig.3B, lanes 3 and 5), and mG3 attenuated the response to cotransfected GATA4 (Fig. 3C, lanes 2 versus 7), whereas mutation of the distal GATA sites (mG12) abolished all induction by GATA4 (Fig. 3C,lane 8). Mutation of the Nkx element also eliminated promoter activation by Nkx2.5 (Fig. 3C, lanes 3 versus 11). These combined data showed that mFTF promoter activation in cotransfection assays using HNF4α, GATA, and Nkx2.5 vectors resulted from a direct action of these factors on their cognate DNA-binding elements surrounding the FTF cap site. Notably, in contrast to the proximal GATA and HNF4 mutants (mG3, mH4), the distal GATA and Nkx mutants (mG12, mNk) showed significant increase of basal promoter activity in both HepG2 and Hep3B cells (Fig. 3B, lanes 2 and 3 versus 4 and6).
Referência(s)