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Identification of a Murine TEF-1-related Gene Expressed after Mitogenic Stimulation of Quiescent Fibroblasts and during Myogenic Differentiation*

  • Debbie K.W. Hsu
    Footnotes
    Affiliations
    Department of Molecular Biology, Holland Laboratory, American Red Cross, Rockville, Maryland 20855
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  • Yan Guo
    Affiliations
    Department of Molecular Biology, Holland Laboratory, American Red Cross, Rockville, Maryland 20855
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  • Gregory F. Alberts
    Affiliations
    Department of Molecular Biology, Holland Laboratory, American Red Cross, Rockville, Maryland 20855
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  • Neal G. Copeland
    Affiliations
    Mammalian Genetics Laboratory, ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, and the
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  • Debra J. Gilbert
    Affiliations
    Mammalian Genetics Laboratory, ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, and the
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  • Nancy A. Jenkins
    Affiliations
    Mammalian Genetics Laboratory, ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, and the
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  • Kimberly A. Peifley
    Affiliations
    Department of Molecular Biology, Holland Laboratory, American Red Cross, Rockville, Maryland 20855
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  • Jeffrey A. Winkles
    Correspondence
    To whom reprint requests and correspondence should be addressed: Dept. of Molecular Biology, Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-738-0655; Fax: 301-738-0465;
    Affiliations
    Department of Molecular Biology, Holland Laboratory, American Red Cross, Rockville, Maryland 20855

    Department of Biochemistry and Molecular Biology, George Washington University Medical Center, Washington, D. C. 20037
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Research Grant HL-39727 (to J. A. W.) and by the National Cancer Institute under contract with ABL (to N. G. C. and N. A. J.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U51743, U51744, U51745.
    § Supported in part by National Institutes of Health Training Grant HL-07698.
Open AccessPublished:June 07, 1996DOI:https://doi.org/10.1074/jbc.271.23.13786
      Fibroblast growth factor (FGF)-1 binding to cell surface receptors stimulates an intracellular signaling pathway that ultimately promotes the transcriptional activation of specific genes. We have used a mRNA differential display method to identify FGF-1-inducible genes in mouse NIH 3T3 fibroblasts. Here, we report that one of these genes, FGF-regulated (FR)-19, is predicted to encode a member of the transcriptional enhancer factor (TEF)-1 family of structurally related DNA-binding proteins. Specifically, the deduced FR-19 amino acid sequence has ~89, 77, and 68% overall identity to chicken TEF-1A, mouse TEF-1, and mouse embryonic TEA domain-containing factor, respectively. Gel mobility shift experiments indicate that FR-19, like TEF-1, can bind the GT-IIC motif found in the SV40 enhancer. The FR-19 gene maps in the distal region of mouse chromosome 6, and analysis of several FR-19 cDNA clones indicates that at least two FR-19 isoforms may be expressed from this locus. FGF-1 induction of FR-19 mRNA expression in mouse fibroblasts is first detectable at 4 h after FGF-1 addition and is dependent on de novo RNA and protein synthesis. FGF-2, calf serum, platelet-derived growth factor-BB, and phorbol 12-myristate 13-acetate can also induce FR-19 mRNA levels. We have also found that FR-19 mRNA expression increases during mouse C2C12 myoblast differentiation in vitro. The FR-19 gene is expressed in vivo in a tissue-specific manner, with a relatively high level detected in lung. These results indicate that increased expression of a TEF-1-related protein may be important for both mitogen-stimulated fibroblast proliferation and skeletal muscle cell differentiation.

      INTRODUCTION

      Fibroblast growth factor (FGF)
      The abbreviations used are: FGF
      fibroblast growth factor
      FGFR
      FGF receptor
      bp
      base pair(s)
      EGF
      epidermal growth factor
      ETF
      embryonic TEA domain-containing factor
      IGF
      insulin-like growth factor
      kb
      kilobase(s)
      PDGF
      platelet-derived growth factor
      PMA
      phorbol 12-myristate 13-acetate
      RACE
      rapid amplification of cDNA ends
      PCR
      polymerase chain reaction
      RT-PCR
      reverse transcription-polymerase chain reaction
      SH2
      Src homology 2
      TEF
      transcriptional enhancer factor
      TEFR1
      TEF-1-related protein 1
      TGF
      transforming growth factor
      M-CAT
      muscle-CAT heptamer CATTCCT.
      -1 (acidic FGF) and FGF-2 (basic FGF) belong to a family of heparin-binding proteins that promote cellular proliferation, migration and differentiation (
      • Burgess W.H.
      • Winkles J.A.
      ,
      • Miyamoto M.
      • Naruo K.
      • Seko C.
      • Matsumoto S.
      • Kondo T.
      • Kurokawa T.
      ). They can stimulate vascular endothelial cell and smooth muscle cell mitogenesis in vivo (
      • Edelman E.R.
      • Nugent M.A.
      • Smith L.T.
      • Karnovsky M.J.
      ,
      • Nabel E.G.
      • Yang Z.-Y.
      • Plautz G.
      • Forough R.
      • Zhan X.
      • Haudenschild C.C.
      • Maciag T.
      • Nabel G.J.
      ,
      • Lindner V.
      • Majack R.A.
      • Reidy M.A.
      ,
      • Lindner V.
      • Lappi D.A.
      • Baird A.
      • Majack R.A.
      • Reidy M.A.
      ,
      • Bjornsson T.D.
      • Dryjski M.
      • Tluczek J.
      • Mennie R.
      • Ronan J.
      • Mellin T.N.
      • Thomas K.A.
      ) and are potent angiogenic factors (
      • Edelman E.R.
      • Nugent M.A.
      • Smith L.T.
      • Karnovsky M.J.
      ,
      • Nabel E.G.
      • Yang Z.-Y.
      • Plautz G.
      • Forough R.
      • Zhan X.
      • Haudenschild C.C.
      • Maciag T.
      • Nabel G.J.
      ,
      • Pu L.-Q.
      • Sniderman A.D.
      • Brassard R.
      • Lachapelle K.J.
      • Graham A.M.
      • Lisbona R.
      • Symes J.F.
      ,
      • Cuevas P.
      • Gonzalez A.M.
      • Carceller F.
      • Baird A.
      ,
      • Asahara T.
      • Bauters C.
      • Zheng L.P.
      • Takeshita S.
      • Bunting S.
      • Ferrara N.
      • Symes J.F.
      • Isner J.M.
      ); thus, they may play an important role in the pathogenesis of various diseases, including atherosclerosis, rheumatoid arthritis, and cancer. FGF-1 and FGF-2 stimulate cellular responses by binding to cell surface tyrosine kinase receptors. Four structurally related receptors, designated FGFR-1, −2, −3, and −4, have been identified (
      • Burgess W.H.
      • Winkles J.A.
      ,
      • Johnson D.E.
      • Williams L.T.
      ). Multiple isoforms of the FGFR-1, −2, and −3 proteins can arise via alternative splicing of primary transcripts; in some cases, variants of a single receptor type can have distinct ligand-binding specificities. FGF-1 can bind with high affinity to all of the FGFR family members and splice variants identified to date; in contrast, FGF-2 binds preferentially to the FGFR-1 and FGFR-2 splice variants containing Ig domain IIIc and to FGFR-4 (
      • Dionne C.A.
      • Crumley G.
      • Bellot F.
      • Kaplow J.M.
      • Searfoss G.
      • Ruta M.
      • Burgess W.H.
      • Jaye M.
      • Schlessinger J.
      ,
      • Ornitz D.M.
      • Leder P.
      ,
      • Miki T.
      • Bottaro D.P.
      • Fleming T.P.
      • Smith C.L.
      • Burgess W.H.
      • Chan A.M.L.
      • Aaronson S.A.
      ,
      • Ron D.
      • Reich R.
      • Chedid M.
      • Lengel C.
      • Cohen O.E.
      • Chan A.M.-L.
      • Neufeld G.
      • Miki T.
      • Tronick S.R.
      ,
      • Chellaiah A.T.
      • McEwen D.G.
      • Werner S.
      • Xu J.
      • Ornitz D.M.
      ).
      The mechanisms by which FGF-1 or FGF-2 binding to cell surface receptors promotes a mitogenic response are unclear. It has been established that FGF binding induces FGFR dimerization, activation of protein-tyrosine kinase activity, and autophosphorylation (
      • Bellot F.
      • Crumley G.
      • Kaplow J.M.
      • Schlessinger J.
      • Jaye M.
      • Dionne C.A.
      ,
      • Hou J.
      • McKeehan K.
      • Kan M.
      • Carr S.A.
      • Huddleston M.J.
      • Crabb J.W.
      • McKeehan W.L.
      ,
      • Mohammadi M.
      • Honegger A.M.
      • Rotin D.
      • Fischer R.
      • Bellot F.
      • Li W.
      • Dionne C.A.
      • Jaye M.
      • Rubinstein M.
      • Schlessinger J.
      ). In addition, the FGF-dependent phosphorylation of numerous cellular proteins including phospholipase C-γ (
      • Mohammadi M.
      • Honegger A.M.
      • Rotin D.
      • Fischer R.
      • Bellot F.
      • Li W.
      • Dionne C.A.
      • Jaye M.
      • Rubinstein M.
      • Schlessinger J.
      ,
      • Burgess W.H.
      • Dionne C.A.
      • Kaplow J.
      • Mudd R.
      • Friesel R.
      • Zilberstein A.
      • Schlessinger J.
      • Jaye M.
      ,
      • Vainikka S.
      • Joukov V.
      • Wennstrom S.
      • Bergman M.
      • Pelicci P.G.
      • Alitalo K.
      ,
      • Wang J.-K.
      • Gao G.
      • Goldfarb M.
      ,
      • Shaoul E.
      • Reich-Slotky R.
      • Berman B.
      • Ron D.
      ), Shc (
      • Vainikka S.
      • Joukov V.
      • Wennstrom S.
      • Bergman M.
      • Pelicci P.G.
      • Alitalo K.
      ,
      • Wang J.-K.
      • Gao G.
      • Goldfarb M.
      ,
      • Klint P.
      • Kanda S.
      • Claesson-Welsh L.
      ), Raf-1 (
      • Vainikka S.
      • Joukov V.
      • Wennstrom S.
      • Bergman M.
      • Pelicci P.G.
      • Alitalo K.
      ,
      • Huang J.
      • Mohammadi M.
      • Rodrigues G.A.
      • Schlessinger J.
      ), mitogen-activated protein kinase (
      • Vainikka S.
      • Joukov V.
      • Wennstrom S.
      • Bergman M.
      • Pelicci P.G.
      • Alitalo K.
      ,
      • Wang J.-K.
      • Gao G.
      • Goldfarb M.
      ,
      • Shaoul E.
      • Reich-Slotky R.
      • Berman B.
      • Ron D.
      ,
      • Huang J.
      • Mohammadi M.
      • Rodrigues G.A.
      • Schlessinger J.
      ), and 80K-H (
      • Goh K.C.
      • Lim Y.P.
      • Ong S.H.
      • Siak C.B.
      • Cao X.
      • Tan Y.H.
      • Guy G.R.
      ) has been demonstrated. Phosphorylation and activation of phospholipase C-γ is not essential for FGF-stimulated mitogenesis (
      • Mohammadi M.
      • Dionne C.A.
      • Li W.
      • Li N.
      • Spivak T.
      • Honegger A.M.
      • Jaye M.
      • Schlessinger J.
      ,
      • Peters K.G.
      • Marie J.
      • Wilson E.
      • Ives H.E.
      • Escobedo J.
      • Delrosario M.
      • Mirda D.
      • Williams L.T.
      ). We have shown that FGF-1 treatment of serum-starved NIH 3T3 fibroblasts induces the expression of several genes, including the proto-oncogenes c-fos, c-jun, and c-myc (
      • Burgess W.H.
      • Shaheen A.M.
      • Ravera M.
      • Jaye M.
      • Donohue P.J.
      • Winkles J.A.
      ,
      • Winkles J.A.
      • Donohue P.J.
      • Hsu D.K.W.
      • Guo Y.
      • Alberts G.F.
      • Peifley K.A.
      ). Recently, we used an RT-PCR-based method to identify cDNA clones representing additional FGF-1-inducible genes in NIH 3T3 cells (
      • Winkles J.A.
      • Donohue P.J.
      • Hsu D.K.W.
      • Guo Y.
      • Alberts G.F.
      • Peifley K.A.
      ). Three immediate-early (
      • Hsu D.K.W.
      • Donohue P.J.
      • Alberts G.F.
      • Winkles J.A.
      ,
      • Donohue P.J.
      • Alberts G.F.
      • Guo Y.
      • Winkles J.A.
      ) and three delayed-early (
      • Hsu D.K.W.
      • Donohue P.J.
      • Alberts G.F.
      • Winkles J.A.
      ,
      • Donohue P.J.
      • Alberts G.F.
      • Hampton B.S.
      • Winkles J.A.
      ,
      • Hsu D.K.W.
      • Guo Y.
      • Alberts G.F.
      • Peifley K.A.
      • Winkles J.A.
      ) response genes have been described to date.
      In this paper, we report that the murine FR-19 gene is a mitogen-inducible delayed-early response gene predicted to encode a TEF-1-related transcription factor. TEF-1 family members contain a highly conserved DNA-binding motif, the TEA domain (
      • Burglin T.R.
      ). The first TEA domain-containing protein identified, human TEF-1, was initially described as a HeLa cell transcriptional activator which could bind the GT-IIC and Sph enhansons of the SV40 enhancer (
      • Xiao J.H.
      • Davidson I.
      • Matthes H.
      • Garnier J.-M.
      • Chambon P.
      ,
      • Hwang J.-J.
      • Chambon P.
      • Davidson I.
      ). Subsequently, it was shown to be involved in transactivation of the SV40 late promoter by large T antigen (
      • Gruda M.C.
      • Zabolotny J.M.
      • Xiao J.H.
      • Davidson I.
      • Alwine J.C.
      ) and to bind to the human papilloma virus type 16 E6/E7 upstream regulatory region (
      • Ishiji T.
      • Lace M.J.
      • Parkkinen S.
      • Anderson R.D.
      • Haugen T.H.
      • Cripe T.P.
      • Xiao J.-H.
      • Davidson I.
      • Chambon P.
      • Turek L.P.
      ). Recently, it has been demonstrated that chicken (
      • Stewart A.F.R.
      • Larkin S.B.
      • Farrance I.K.G.
      • Mar J.H.
      • Hall D.E.
      • Ordahl C.P.
      ) and mouse (
      • Shimizu N.
      • Smith G.
      • Izumo S.
      ) TEF-1 can bind the M-CAT element, suggesting that these proteins may be involved in cardiac- and skeletal muscle-specific gene regulation. The function of FR-19 is presently unknown; however, our results indicate it may play a role in both mammalian cell growth and differentiation. After the original submission of this manuscript, Yockey et al. (
      • Yockey C.E.
      • Smith G.
      • Izumo S.
      • Shimizu N.
      ) reported the cDNA cloning and characterization of this same gene product, which they have named TEFR1.

      DISCUSSION

      The binding of polypeptide growth factors to cell surface receptors triggers a cascade of intracellular biochemical responses, including the transcriptional activation of specific nuclear genes (
      • Williams G.T.
      • Abler A.S.
      • Lau L.F.
      ,
      • Muller R.
      • Mumberg D.
      • Lucibello F.C.
      ). It has been demonstrated that at least some of the proteins encoded by mitogen-inducible genes perform functions necessary for cell cycle progression (
      • Furukawa Y.
      • Piwnica-Worms H.
      • Ernst T.J.
      • Kanakura Y.
      • Griffin J.D.
      ,
      • Kovary K.
      • Bravo R.
      ,
      • Baldin V.
      • Lukas J.
      • Marcote M.J.
      • Pagano M.
      • Draetta G.
      ). Thus, we have begun to identify and characterize FGF-1-inducible genes in NIH 3T3 fibroblasts in order to gain further insight into mammalian cell growth control. Here, we report that the delayed-early response gene FR-19 encodes a protein with ~89, 77, and 68% overall sequence identity to chicken TEF-1A (
      • Stewart A.F.R.
      • Larkin S.B.
      • Farrance I.K.G.
      • Mar J.H.
      • Hall D.E.
      • Ordahl C.P.
      ), human (
      • Xiao J.H.
      • Davidson I.
      • Matthes H.
      • Garnier J.-M.
      • Chambon P.
      ) or mouse (
      • Shimizu N.
      • Smith G.
      • Izumo S.
      ,
      • Blatt C.
      • DePamphilis M.L.
      ,
      • Chen Z.
      • Friedrich G.A.
      • Soriano P.
      ) TEF-1, and mouse ETF (
      • Yasunami M.
      • Suzuki K.
      • Houtani T.
      • Sugimoto T.
      • Ohkubo H.
      ), respectively. These proteins, in combination with Drosophila Scalloped (
      • Campbell S.
      • Inamdar M.
      • Rodrigues V.
      • Raghavan V.
      • Palazzolo M.
      • Chovnick A.
      ), Saccharomyces TEC1 (
      • Laloux I.
      • Dubois E.
      • Dewerchin M.
      • Jacobs E.
      ) and Aspergillus AbaA (
      • Andrianopoulos A.
      • Timberlake W.E.
      ), define a family of transcription factors that share a highly conserved DNA-binding motif, the TEA domain (
      • Burglin T.R.
      ). After the original submission of this manuscript, Yockey et al. (
      • Yockey C.E.
      • Smith G.
      • Izumo S.
      • Shimizu N.
      ) reported the isolation and characterization of murine cDNA clones predicted to encode two TEF-1-related protein isoforms, which they called TEFR1a and TEFR1b. These cDNAs were isolated by low stringency screening of an adult mouse cardiac cDNA library with a human TEF-1 cDNA probe. The FR-19 and TEFR1 cDNAs have >99% nucleotide sequence identity and encode identical proteins, except for a single amino acid difference in the 43-amino acid insertion found in FR-19A (TEFR1a).
      The initial FR-19 cDNA fragment was identified using an RT-PCR approach that is conceptually similar to the mRNA differential display and fingerprinting methods used by others to identify differentially expressed genes (
      • McClelland M.
      • Mathieu-Daude F.
      • Welsh J.
      ). In our method, (i) cDNA is synthesized using random primers, (ii) PCR assays are performed using pairs of oligonucleotide primers designed to amplify cDNA templates encoding proteins with particular structural motifs, and (iii) amplification products are displayed using agarose gel electrophoresis and ethidium bromide staining. We have found that ~50% of the genes identified using this method are in fact FGF-1-regulated; however, cDNA sequence analysis has indicated that most of the genes do not encode proteins with the targeted structural motif(s). In most instances the primers, many of which were degenerate, annealed to regions of low sequence identity. In the case of FR-19, PCR was performed with a sense SH2 domain primer and a degenerate antisense leucine zipper domain primer; however, only the former primer participated in FR-19 cDNA amplification. In addition, under the PCR conditions used, the SH2 domain primer annealed to two regions of minimal sequence identity, one of which was actually in the 3′-untranslated region of the FR-19 cDNA template. It is possible that we can increase the likelihood of successfully identifying certain classes of growth-regulated genes by re-designing some of our primers and/or by changing the PCR conditions.
      The predicted FR-19 amino acid sequence has the greatest identity (~89%) to chicken TEF-1 (
      • Stewart A.F.R.
      • Larkin S.B.
      • Farrance I.K.G.
      • Mar J.H.
      • Hall D.E.
      • Ordahl C.P.
      ); thus, the FR-19 gene may be the murine homologue of this chicken locus. However, the two genes do not have similar tissue expression patterns; the chicken TEF-1 gene, but not the mouse FR-19 gene, is expressed at a relatively high level in liver and heart (
      • Stewart A.F.R.
      • Larkin S.B.
      • Farrance I.K.G.
      • Mar J.H.
      • Hall D.E.
      • Ordahl C.P.
      ). The deduced FR-19 and chicken TEF-1 proteins have a similar degree of sequence identity, ~77%, to human (
      • Xiao J.H.
      • Davidson I.
      • Matthes H.
      • Garnier J.-M.
      • Chambon P.
      ) or mouse (
      • Shimizu N.
      • Smith G.
      • Izumo S.
      ,
      • Blatt C.
      • DePamphilis M.L.
      ,
      • Chen Z.
      • Friedrich G.A.
      • Soriano P.
      ) TEF-1. It appears that at least three chicken TEF-1 isoforms may be expressed from a single gene (
      • Stewart A.F.R.
      • Larkin S.B.
      • Farrance I.K.G.
      • Mar J.H.
      • Hall D.E.
      • Ordahl C.P.
      ). In comparison to chicken TEF-1A, the TEF-1B and TEF-1C isoforms contain insertions of 13 and 3 amino acid residues, respectively, in two distinct regions that are both carboxyl-terminal to the TEA domain. Both chicken TEF-1A and TEF-1B can bind the GT-IIC motif recognized by human TEF-1 (
      • Xiao J.H.
      • Davidson I.
      • Matthes H.
      • Garnier J.-M.
      • Chambon P.
      ), as well as the M-CAT motif, a cis-acting element of similar sequence which is found in several muscle-specific gene promoters (
      • Stewart A.F.R.
      • Larkin S.B.
      • Farrance I.K.G.
      • Mar J.H.
      • Hall D.E.
      • Ordahl C.P.
      ). We have found that at least two FR-19 mRNA splice variants are expressed in murine fibroblasts and skeletal muscle cells, in agreement with the recent report by Yockey et al. (
      • Yockey C.E.
      • Smith G.
      • Izumo S.
      • Shimizu N.
      ). The 43-amino acid insertion predicted to be present in the FR-19A isoform is located at the same position as the 13-amino acid insertion in chicken TEF-1B as well as a 4-amino acid insertion found in the mouse TEF-1 sequence reported by Shimizu et al. (
      • Shimizu N.
      • Smith G.
      • Izumo S.
      ). Taken together, these results indicate that distinct chicken TEF-1, mouse TEF-1, and mouse FR-19 isoforms may be expressed via alternative splicing of their respective primary transcripts. These isoforms may have unique functional properties; indeed, chicken TEF-1B, but not TEF-1A, can transactivate a heterologous reporter gene (
      • Stewart A.F.R.
      • Larkin S.B.
      • Farrance I.K.G.
      • Mar J.H.
      • Hall D.E.
      • Ordahl C.P.
      ). Furthermore, Yockey et al. (
      • Yockey C.E.
      • Smith G.
      • Izumo S.
      • Shimizu N.
      ) found that mouse TEFR1b (FR-19B) is a stronger transactivator than TEFR1a (FR-19A).
      Our experimental results, in combination with previous reports, indicate that there are at least three TEF-1-related genes in the mouse genome: FR-19, TEF-1 (
      • Shimizu N.
      • Smith G.
      • Izumo S.
      ,
      • Blatt C.
      • DePamphilis M.L.
      ,
      • Chen Z.
      • Friedrich G.A.
      • Soriano P.
      ), and ETF (
      • Yasunami M.
      • Suzuki K.
      • Houtani T.
      • Sugimoto T.
      • Ohkubo H.
      ). The FR-19 locus is located on chromosome 6 but the positions of TEF-1 and ETF have not yet been reported. We have compared our interspecific map of chromosome 6 with a composite mouse linkage map that reports the map location of many uncloned mouse mutations (provided from Mouse Genome Database, a computerized data base maintained at The Jackson Laboratory, Bar Harbor, ME). Fr19 mapped in a region of the composite map that lacks mouse mutations with a phenotype that might be expected for an alteration in this locus (data not shown). The distal region of mouse chromosome 6 shares a region of homology with human chromosome 12p; in particular, Fgf6 has been placed on human 12p13. The tight linkage between Fgf6 and Fr19 in mouse suggests that Fr19 will reside on 12p13 as well.
      The amino acid sequence identity between FR-19 and either TEF-1 or ETF is ~77 and 68%, respectively. TEF-1 and ETF have ~68% amino acid sequence identity. The predicted mouse and human TEF-1 proteins have ~99% amino acid sequence identity and thus are encoded by homologous genes. Mouse TEF-1 can bind to the A element (which includes an M-CAT motif) found in the rabbit myosin heavy chain-β promoter (
      • Shimizu N.
      • Smith G.
      • Izumo S.
      ,
      • Yockey C.E.
      • Smith G.
      • Izumo S.
      • Shimizu N.
      ). This finding, in combination with a report demonstrating that disruption of the mouse TEF-1 gene results in heart defects and embryonic lethality (
      • Chen Z.
      • Friedrich G.A.
      • Soriano P.
      ), suggests that mouse TEF-1 may primarily be involved in cardiac-specific regulation of M-CAT-dependent promoters.
      ETF, the most divergent member of the vertebrate TEF-1 family, may be involved in transcriptional control during neural development (
      • Yasunami M.
      • Suzuki K.
      • Houtani T.
      • Sugimoto T.
      • Ohkubo H.
      ). The ETF gene is regulated during mouse embryogenesis and is primarily expressed in the hindbrain region of 10-day-old embryos (
      • Yasunami M.
      • Suzuki K.
      • Houtani T.
      • Sugimoto T.
      • Ohkubo H.
      ). Mouse ETF (
      • Yasunami M.
      • Suzuki K.
      • Houtani T.
      • Sugimoto T.
      • Ohkubo H.
      ), like human TEF-1 (
      • Xiao J.H.
      • Davidson I.
      • Matthes H.
      • Garnier J.-M.
      • Chambon P.
      ) as well as chicken TEF-1A and TEF-1B (
      • Stewart A.F.R.
      • Larkin S.B.
      • Farrance I.K.G.
      • Mar J.H.
      • Hall D.E.
      • Ordahl C.P.
      ), has been shown to bind GT-IIC sequence motifs in vitro. All four of these proteins, as well as mouse TEF-1 (
      • Shimizu N.
      • Smith G.
      • Izumo S.
      ,
      • Blatt C.
      • DePamphilis M.L.
      ,
      • Chen Z.
      • Friedrich G.A.
      • Soriano P.
      ), have an identical TEA domain sequence. Previous studies have indicated that the TEA domain found in human TEF-1 is necessary and sufficient for specific DNA binding (
      • Xiao J.H.
      • Davidson I.
      • Matthes H.
      • Garnier J.-M.
      • Chambon P.
      ,
      • Hwang J.-J.
      • Chambon P.
      • Davidson I.
      ). In comparison to the TEA domain of these other TEF-1-related proteins, the FR-19 TEA domain contains five amino acid substitutions. However, three of these substitutions are conservative changes and one other substitution, where a tyrosine residue at position 77 is replaced by a histidine, is located in a region that is not critical for DNA-binding (
      • Hwang J.-J.
      • Chambon P.
      • Davidson I.
      ). Accordingly, it is likely that FR-19A and FR-19B are also DNA-binding proteins that recognize similar sequence motifs. In support of this possibility, we have found that in vitro translated FR-19B can bind to the GT-IIC motif in gel mobility shift assays. Additionally, Yockey et al. (
      • Yockey C.E.
      • Smith G.
      • Izumo S.
      • Shimizu N.
      ) have demonstrated that both TEFR1a (FR-19A) and TEFR1b (FR-19B) can form specific complexes with the M-CAT motif in vitro.
      The mouse FR-19, TEF-1, and ETF genes are differentially expressed in adult tissues; thus, they are likely to have unique physiological functions in vivo. We observed that FR-19 mRNA was expressed at the highest levels in lung, in agreement with the results of Yockey et al. (
      • Yockey C.E.
      • Smith G.
      • Izumo S.
      • Shimizu N.
      ). TEF-1 mRNA is more widely expressed, with highest levels present in skeletal and cardiac muscle, kidney and lung (
      • Shimizu N.
      • Smith G.
      • Izumo S.
      ). In contrast, the ETF gene is not expressed to any significant degree in those adult mouse tissues that have been examined to date (brain, testis, liver, kidney, thymus, and heart) (
      • Yasunami M.
      • Suzuki K.
      • Houtani T.
      • Sugimoto T.
      • Ohkubo H.
      ).
      In regard to mouse FR-19, TEF-1, or ETF gene regulation in tissue culture cells, we found that several mitogenic agents and PMA, a tumor-promoting phorbol ester, could induce FR-19 mRNA expression in NIH 3T3 fibroblasts. In FGF-1-stimulated cells, FR-19 mRNA levels are significantly increased at 4 h while in calf serum-stimulated cells, elevated FR-19 mRNA levels are detected earlier, at 2 h post-stimulation. This difference could reflect a more rapid cell cycle transit time following serum treatment; alternatively, serum may contain factors that alter FR-19 gene expression by a mechanism distinct from that used by FGF-1. FGF-1-induced FR-19 mRNA accumulation does not occur in the presence of actinomycin D or cycloheximide. This indicates that FGF-1 treatment promotes the synthesis of a transcription factor required for FR-19 gene activation; however, we have not ruled out the possibility that the actinomycin D is preventing the synthesis of a labile protein that stabilizes pre-existing FR-19 mRNA. Although it has been demonstrated that the mouse TEF-1 gene is expressed in Balb/c 3T3 fibroblasts cultured in vitro (
      • Shimizu N.
      • Smith G.
      • Izumo S.
      ,
      • Yockey C.E.
      • Smith G.
      • Izumo S.
      • Shimizu N.
      ), there have been no reports describing the regulation of TEF-1 expression in fibroblast cell lines.
      We have also demonstrated by Northern blot hybridization and RT-PCR analysis that FR-19 mRNA levels increase during C2C12 skeletal muscle cell differentiation in vitro. This finding confirms and extends the data of Yockey et al. (
      • Yockey C.E.
      • Smith G.
      • Izumo S.
      • Shimizu N.
      ), which demonstrated that TEFR1 mRNA expression was higher in Sol8 and C2C12 myotubes than in myoblasts. Shimizu et al. (
      • Shimizu N.
      • Smith G.
      • Izumo S.
      ) have reported that C2C12 myoblasts and myotubes express a similar level of TEF-1 mRNA; thus, the mouse TEF-1 gene does not appear to be regulated during myogenic differentiation. The finding that the FR-19 gene is up-regulated during both growth stimulation (fibroblasts) and growth arrest (myoblasts) is intriguing and suggest that a single TEF-1-related protein may have distinct cell type-specific functions. It is possible that the FR-19 protein expressed during muscle differentiation is involved in the up-regulation of M-CAT-dependent genes. However, in the case of at least one such gene, skeletal α-actin, increased mRNA expression appears to precede FR-19 mRNA accumulation (
      • Thinakaran G.
      • Ojala J.
      • Bag J.
      ). In fibroblasts, FR-19 could function to activate specific genes encoding proteins required for cell cycle progression. The promoter regions of these cellular genes may contain M-CAT motifs or presently undefined sequence motifs that can also be recognized by FR-19. Additional studies that manipulate FR-19 expression levels or inhibit FR-19 function should help to define the role of FR-19 in fibroblast proliferation and myoblast differentiation.

      Acknowledgments

      We thank T. Lanahan for the cDNA library, Dr. W. Burgess for the FGF-1, and Dr. C. Bieberich for the mouse tissue samples. We are also grateful to C. Liu for performing the automated DNA sequence analysis, S. Brown for help with the gel mobility shift assays, D. B. Householder for excellent technical assistance, and K. Wawzinski for excellent secretarial assistance. In addition, we thank Dr. C. Ordahl and Dr. N. Shimizu for sharing experimental results prior to publication.

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