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• W2006468675 abstract "General transcription factor 3 (GTF3) binds specifically to the bicoid-like motif of the troponin Islow upstream enhancer. This motif is part of a sequence that restricts enhancer activity to slow muscle fibers. GTF3 contains multiple helix-loop-helix domains and an amino-terminal leucine zipper motif. Here we show that helix-loop-helix domain 4 is necessary and sufficient for binding the bicoid-like motif. Moreover, the affinity of this interaction is enhanced upon removal of amino-terminal sequences including domains 1 and 2, suggesting that an unmasking of the DNA binding surface may be a precondition for GTF3 to bind DNAin vivo. We have also investigated the interactions of six GTF3 splice variants of the mouse, three of which were identified in this study, with the troponin enhancer. The γ-isoform lacking exon 23, and exons 26–28 that encode domain 6, interacted most avidly with the bicoid-like motif; the α- and β- isoforms that include these exons fail to bind in gel retardation assays. We also show that GTF3 polypeptides associate with each other via the leucine zipper. We speculate that cells can generate a large number of GTF3 proteins with distinct DNA binding properties by alternative splicing and combinatorial association of GTF3 polypeptides. General transcription factor 3 (GTF3) binds specifically to the bicoid-like motif of the troponin Islow upstream enhancer. This motif is part of a sequence that restricts enhancer activity to slow muscle fibers. GTF3 contains multiple helix-loop-helix domains and an amino-terminal leucine zipper motif. Here we show that helix-loop-helix domain 4 is necessary and sufficient for binding the bicoid-like motif. Moreover, the affinity of this interaction is enhanced upon removal of amino-terminal sequences including domains 1 and 2, suggesting that an unmasking of the DNA binding surface may be a precondition for GTF3 to bind DNAin vivo. We have also investigated the interactions of six GTF3 splice variants of the mouse, three of which were identified in this study, with the troponin enhancer. The γ-isoform lacking exon 23, and exons 26–28 that encode domain 6, interacted most avidly with the bicoid-like motif; the α- and β- isoforms that include these exons fail to bind in gel retardation assays. We also show that GTF3 polypeptides associate with each other via the leucine zipper. We speculate that cells can generate a large number of GTF3 proteins with distinct DNA binding properties by alternative splicing and combinatorial association of GTF3 polypeptides. general transcription factor 3 slow upstream regulatory element fetal bovine serum extensor digitorum longus helix-loop-helix basic helix-loop-helix bicoid-like motif general transcription factor II-I Williams syndrome leucine zipper electrophoretic mobility shift assay days in vitro reverse transcription The establishment and maintenance of mature fast- and slow-twitch muscle fibers require the expression of distinct sets of genes for contractile proteins, metabolic enzymes, and ion channels. These expression patterns are mainly controlled at the level of transcription. Fiber type specificity of muscle genes can be recapitulated in transgenic reporter mouse models or in vivotransfection assays by using respective transcription control regions (see, for example, Refs. 1Donoghue M.J. Alvarez J.D. Merlie J.P. Sanes J.R. J. Cell Biol. 1991; 115: 423-434Google Scholar, 2Shield M.A. Haugen H.S. Clegg C.H. Hauschka S.D. Mol. Cell. Biol. 1996; 16: 5058-5068Google Scholar, 3Banerjee-Basu S. Buonanno A. Mol. Cell. Biol. 1993; 13: 7019-7028Google Scholar, 4Hallauer P.L. Hastings K.E. Peterson A.C. Mol. Cell. Biol. 1988; 8: 5072-5079Google Scholar, 5Salminen M. Maire P. Concordet J.P. Moch C. Porteu A. Kahn A. Daegelen D. Mol. Cell. Biol. 1994; 14: 6797-6808Google Scholar, 6McCarthy J.J. Fox A.M. Tsika G.L. Gao L. Tsika R.W. Am. J. Physiol. 1997; 272: R1552-R1561Google Scholar, 7Jerkovic R. Vitadello M. Kelly R. Buckingham M. Schiaffino S.J. Muscle Res. Cell Motil. 1997; 18: 369-373Google Scholar, 8Lupa-Kimball V.A. Esser K.A. Am. J. Physiol. 1998; 274: C229-C235Google Scholar). Over the past years, signaling proteins such as calcineurin and Ras, and transcription factors GTF31/MusTRD1, MEF-3, MEF-2, NFAT, and PGC-1α were implicated in the regulation of fiber type-specific expression in adult muscle (9Serrano A.L. Murgia M. Pallafacchina G. Calabria E. Coniglio P. Lomo T. Schiaffino S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13108-13113Google Scholar, 10Torgan C.E. Daniels M.P. Mol. Biol. Cell. 2001; 12: 1499-1508Google Scholar, 11Calvo S. Vullhorst D. Venepally P. Cheng J. Karavanova I. Buonanno A. Mol. Cell. Biol. 2001; 21: 8490-8503Google Scholar, 12O'Mahoney J.V. Guven K.L. Lin J. Joya J.E. Robinson C.S. Wade R.P. Hardeman E.C. Mol. Cell. Biol. 1998; 18: 6641-6652Google Scholar, 13Murgia M. Serrano A.L. Calabria E. Pallafacchina G. Lomo T. Schiaffino S. Nat. Cell Biol. 2000; 2: 142-147Google Scholar, 14Spitz F. Salminen M. Demignon J. Kahn A. Daegelen D. Maire P. Mol. Cell. Biol. 1997; 17: 656-666Google Scholar, 15Wu H. Naya F.J. McKinsey T.A. Mercer B. Shelton J.M. Chin E.R. Simard A.R. Michel R.N. Bassel-Duby R. Olson E.N. Williams R.S. EMBO J. 2000; 19: 1963-1973Google Scholar, 16Chin E.R. Olson E.N. Richardson J.A. Yang Q. Humphries C. Shelton J.M. Wu H. Zhu W. Bassel-Duby R. Williams R.S. Genes Dev. 1998; 12: 2499-2509Google Scholar, 17Lin J. Wu H. Tarr P.T. Zhang C.Y. Wu Z. Boss O. Michael L.F. Puigserver P. Isotani E. Olson E.N. Lowell B.B. Bassel-Duby R. Spiegelman B.M. Nature. 2002; 418: 797-801Google Scholar). In birds and lower vertebrates, the sonic hedgehog signaling pathway was shown to be involved in the specification of primary slow myofibers (18Cann G.M. Lee J.W. Stockdale F.E. Anat. Embryol. 1999; 200: 239-252Google Scholar, 19Blagden C.S. Currie P.D. Ingham P.W. Hughes S.M. Genes Dev. 1997; 11: 2163-2175Google Scholar, 20Du S.J. Devoto S.H. Westerfield M. Moon R.T. J. Cell Biol. 1997; 139: 145-156Google Scholar). However, the transcription factors and signaling pathways that control the establishment of slow and fast fiber phenotypes during mammalian muscle development are not known. The troponin Islow gene (TnIs) is activated during terminal myogenic differentiation in all skeletal muscles regardless of their future fiber type. Its expression is then confined to prospective slow fibers during fetal development (11Calvo S. Vullhorst D. Venepally P. Cheng J. Karavanova I. Buonanno A. Mol. Cell. Biol. 2001; 21: 8490-8503Google Scholar, 21Zhu L. Lyons G.E. Juhasz O. Joya J.E. Hardeman E.C. Wade R. Dev. Biol. 1995; 169: 487-503Google Scholar). The enhancer that confers slow fiber specificity to TnIs expression is located ∼800 bp upstream of the gene and was termed SURE (for slowupstream regulatory element; Refs.3Banerjee-Basu S. Buonanno A. Mol. Cell. Biol. 1993; 13: 7019-7028Google Scholar and 22Nakayama M. Stauffer J. Cheng J. Banerjee-Basu S. Wawrousek E. Buonanno A. Mol. Cell. Biol. 1996; 16: 2408-2417Google Scholar). Using a transgenic approach, we showed that the downstream half of the 128-bp SURE, including binding sites for myogenic regulatory factors (i.e. MyoD and myogenin) and MEF-2, is necessary for general muscle-specific activity, but not sufficient to restrict transcription to specific fiber types (11Calvo S. Vullhorst D. Venepally P. Cheng J. Karavanova I. Buonanno A. Mol. Cell. Biol. 2001; 21: 8490-8503Google Scholar). Rather, a 36-bp upstream region of the SURE is required in addition to downstream sequences to re-establish slow fiber-specific reporter expression. Within this sequence, a bicoid-like motif (BLM; CGGATTAAC) was found in a yeast one-hybrid screen to interact with the general transcription factor GTF3. In a similar approach, the corresponding sequence of the human TnIs upstream enhancer (equivalent to SURE) was used to isolate a cDNA encoding MusTRD1 (12O'Mahoney J.V. Guven K.L. Lin J. Joya J.E. Robinson C.S. Wade R.P. Hardeman E.C. Mol. Cell. Biol. 1998; 18: 6641-6652Google Scholar). GTF3, MusTRD1, GTF2ird1, WBSCR11, CREAM, and the mouse ortholog BEN are synonyms for proteins encoded by the same gene (12O'Mahoney J.V. Guven K.L. Lin J. Joya J.E. Robinson C.S. Wade R.P. Hardeman E.C. Mol. Cell. Biol. 1998; 18: 6641-6652Google Scholar, 23Tassabehji M. Carette M. Wilmot C. Donnai D. Read A.P. Metcalfe K. Eur. J. Hum. Genet. 1999; 7: 737-747Google Scholar, 24Franke Y. Peoples R.J. Francke U. Cytogenet. Cell Genet. 1999; 86: 296-304Google Scholar, 25Osborne L.R. Campbell T. Daradich A. Scherer S.W. Tsui L.C. Genomics. 1999; 57: 279-284Google Scholar, 26Yan X. Zhao X. Qian M. Guo N. Gong X. Zhu X. Biochem. J. 2000; 345: 749-757Google Scholar). GTF3 is ubiquitous in rodent tissues (11Calvo S. Vullhorst D. Venepally P. Cheng J. Karavanova I. Buonanno A. Mol. Cell. Biol. 2001; 21: 8490-8503Google Scholar, 27Bayarsaihan D. Ruddle F.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7342-7347Google Scholar), whereas MusTRD1 has been suggested to be muscle-specific in humans (Ref. 12O'Mahoney J.V. Guven K.L. Lin J. Joya J.E. Robinson C.S. Wade R.P. Hardeman E.C. Mol. Cell. Biol. 1998; 18: 6641-6652Google Scholar; but see Ref. 24Franke Y. Peoples R.J. Francke U. Cytogenet. Cell Genet. 1999; 86: 296-304Google Scholar). As we showed previously, the highest expression of GTF3 in rodent muscle occurs during fetal development, after which it is down regulated to very low levels in mature muscle fibers. In transfected rat muscle, GTF3 significantly reduces the transcriptional activity from the SURE. This is consistent with the idea that a repressive mechanism establishes slow fiber-specific TnIs expression during myogenic development. We therefore proposed that GTF3 is involved in the confinement of TnIs expression to slow-twitch fibers (11Calvo S. Vullhorst D. Venepally P. Cheng J. Karavanova I. Buonanno A. Mol. Cell. Biol. 2001; 21: 8490-8503Google Scholar). A characteristic feature of GTF3, and its paralog TFII-I, is the presence of reiterated helix-loop-helix (HLH) domains, so-called I-repeats (R1–R5/R6; for review, see Ref. 28Roy A.L. Gene (Amst.). 2001; 274: 1-13Google Scholar). Most of these repeats are believed to function as protein-protein interaction surfaces because they lack a basic domain. In TFII-I, a basic motif precedes R2, and its deletion abrogates binding to Vβ Inr and c-Fos promoter sequences (29Roy A.L. Du H. Gregor P.D. Novina C.D. Martinez E. Roeder R.G. EMBO J. 1997; 16: 7091-7104Google Scholar, 30Cheriyath V. Roy A.L. J. Biol. Chem. 2001; 276: 8377-8383Google Scholar). Largely based on protein sequence, MusTRD1 has been suggested to bind to the USE B1 enhancer element of the human TnIs gene via a domain located in its amino-terminal half (12O'Mahoney J.V. Guven K.L. Lin J. Joya J.E. Robinson C.S. Wade R.P. Hardeman E.C. Mol. Cell. Biol. 1998; 18: 6641-6652Google Scholar). However, two lines of evidence indicate that the first two HLH domains of GTF3 are dispensable for binding to the TnIs BLM; (a) most of the GTF3 clones we obtained from the yeast one-hybrid screen lack the sequences encoding R1 and R2, and (b) a partial GTF3 protein containing the carboxyl-terminal half including R3–R5 forms a complex with a SURE-derived oligonucleotide probe in electrophoretic mobility shift assays (EMSAs), whereas a mutant protein encompassing the amino-terminal half including R1 and R2 does not (11Calvo S. Vullhorst D. Venepally P. Cheng J. Karavanova I. Buonanno A. Mol. Cell. Biol. 2001; 21: 8490-8503Google Scholar). In that study, GTF3 cDNAs conforming to either one of two reported human GTF3 transcripts (containing either the long or the short form of exon 19) were isolated, indicating that sequence variability in the region between R3 and R4 encoded by this exon does not appreciably affect DNA binding. However, mice express at least six GTF3 isoforms that differ more extensively in sequence (Ref. 31Bayarsaihan D. Dunai J. Greally J.M. Kawasaki K. Sumiyama K. Enkhmandakh B. Shimizu N. Ruddle F.H. Genomics. 2002; 79: 137-143Google Scholar and this paper). Most notably, both α- and β-GTF3 isoforms contain a sixth HLH domain located between R5 and the carboxyl terminus, plus an additional 27 amino acids between R4 and R5 encoded by mouse exon 23. Their expression pattern and functional properties are not known. GTF3 and TFII-I were mapped along with at least 21 other genes as part of a 1.5-Mb microdeletion in persons with Williams syndrome (WS) (32Francke U. Hum. Mol. Genet. 1999; 8: 1947-1954Google Scholar,33Merla G. Ucla C. Guipponi M. Reymond A. Hum. Genet. 2002; 110: 429-438Google Scholar). Given the potential importance of GTF3 and TFII-I for the pathology of WS, and the suggested role of GTF3 in regulating slow fiber-specific gene expression, a better understanding of the biochemical properties of GTF3 and its functional relation to TFII-I is necessary. Therefore, the goal of this study was to characterize the interactions between GTF3 and the TnI SURE. We mapped the DNA binding domain of GTF3 to HLH domain 4 that lacks a consensus basic region. Interestingly, affinity of GTF3 for the BLM was dramatically augmented upon removal of NH2-terminal sequences. We also show that rodent skeletal muscles express at least five different GTF3 isoforms that exhibit distinct DNA binding properties in EMSAs; three variants (α2, α3, and γ2) are reported here for the first time. We furthermore demonstrate that GTF3 proteins can form dimers via the NH2-terminal leucine zipper (LZ) motif, suggesting that cells can generate a large number of GTF3 transcription factor complexes with potentially different properties and functions. C2C8 myogenic cells were propagated in low glucose Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 20% fetal bovine serum (FBS; Invitrogen) and 2 mm l-glutamine. Cells were maintained at 37 °C in an 8% CO2 environment. Cell density was kept between 20 and 80% confluence to prevent terminal myogenic differentiation. HEK293 cells were propagated in minimal essential medium (Invitrogen) supplemented with 10% FBS and maintained in a 37 °C, 10% CO2environment. Primary myotube cultures were prepared from rat embryonic day 19 hindlimbs and grown for 10 days in 10% FBS/Dulbecco's modified Eagle's medium at 37 °C and 8% CO2 (34Emerson C.P. Sweeney H.L. Methods in Cell Biology: Methods in Muscle Biology. 52. Academic Press, San Diego1997: 85-116Google Scholar). Cytosine β-d-arabinofuranoside was added to the cultures at day 4 to enrich for myotubes. Generation of full-length human (h) GTF3 and hGTF3Δ 12 was described previously (11Calvo S. Vullhorst D. Venepally P. Cheng J. Karavanova I. Buonanno A. Mol. Cell. Biol. 2001; 21: 8490-8503Google Scholar). All PCR products were generated using a mix of proofreading (Tgo) and Taq polymerase (Expand High Fidelity PCR system, Roche). hGTF3.R4 was generated from hGTF3Δ1–3 template DNA and subcloned directly into expression plasmid pCMVSport2 (Invitrogen). All other constructs were amplified from YIH clone 81 template DNA (11Calvo S. Vullhorst D. Venepally P. Cheng J. Karavanova I. Buonanno A. Mol. Cell. Biol. 2001; 21: 8490-8503Google Scholar) and subcloned into shuttle vector pGemT (Promega) for sequence verification before being transferred to pCMVSport2. Gene-specific coding oligonucleotides for hGTF3Δ1–3, hGTF3Δ1–4, hGTF3Δ12C, and hGTF3Δ124 included a SalI linker, Kozak consensus sequences, and a translation initiation codon (underlined). Primer sequences were 5′-GTCGACGCCACCATG GGC TTT CAA GAA AAT TAT GAC GC-3′ and hGTF3Δ1–4, 5′-GTCGACGCCACCATG GAT GAG GAT GAT GCC ACC AGA C-3′, respectively. T7 was used as antisense oligonucleotide. GTF3 inserts in pGemT were released with SalI and NotI and cloned between the corresponding sites of pCMV-Sport2. Coding oligonucleotide was 5′-GTCGACGCCACCATG GAT TCT GGT TAT GGG ATG GAG ATG-3′, and antisense oligonucleotide was hGTF3ΔC (5′-GAA TTC TAT GCA AAG GGT TGG AGC TGG-3′). Coding oligonucleotide was same as for hGTF3Δ12C and hGTF3Δ12 (see above); antisense oligonucleotide was 5′-GAT GAG TCC TTG GAA AGG CGC GTC ATA ATT TTC TTG AAA GCC-3′. Following ligation to pGemT, a SalI-EcoNI fragment was inserted between the corresponding sites of hGTF3Δ12. Coding oligonucleotide sequence was 5′-ACC AGG CCT TTC CAA GGA CTC ATC GCA GAA ATC TGC AAT GAT GCC AAG GTG-3′; antisense primer was T7. Following ligation to pGemT, a StuI fragment was released and used to replace the correspondingStuI fragment in hGTF3Δ12. A SapI fragment including the sequences flanking the deletion was released from hGTF3Δ124 and used to replace the corresponding sequence in full-length hGTF3. Repeat domain 4 was amplified from hGTF3Δ1–3 template DNA using a cytomegalovirus promoter coding oligonucleotide (5′-GGT GAC ACT ATA GAA GGT ACG CCT GC-3′) and noncoding oligonucleotide 5′-CCT ACA AGC TTA CTT TGG GAT GAG TCC TTG GAA AGG-3′. The PCR product was digested with SalI andHindIII and ligated to pCMV-Sport2. Total RNA from mouse tissues and rat primary myotubes (days in vitro (DIV) 10) was extracted using RNA Wiz reagent (Ambion). 2 μg from each preparation were reverse transcribed with Superscript IITM(Invitrogen) using oligo(dT) primer. Oligonucleotides to amplify 3′-sequences from GTF3α and -γ splice isoforms were mGTF3+2346s, 5′-AGC AAC CCA GGC TCG GTA ATC ATT GAA GG-3′ (coding) and mGTF3+3446r, 5′-CCT TTA GTC TTT TGA GTT GAG GTC CTG-3′ (noncoding). Cycling parameters were 95 °C/4-min initial denaturation; 35 cycles of 92 °C/30 s, 64 °C/45 s, 72 °C/1 min; last extension step of 72 °C/10 min. PCR reactions were purified and concentrated on MinElute DNA purification columns (Qiagen) and ligated to pCRScriptTM-Cam (Stratagene). Inserts were sequenced and aligned to GTF3 sequences already deposited with GenBankTMusing BLAST (35Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Google Scholar). Using mGTF3β in pCMV-Sport2 as backbone (IMAGE clone 555547; GenBankTM accession no. AA111609 (Ref. 36Lennon G. Auffray C. Polymeropoulos M. Soares M.B. Genomics. 1996; 33: 151-152Google Scholar)), alternatively spliced GTF3α and GTF3γ sequences were inserted between the internal BstEII site and vector-derivedHindIII or NotI sites, depending on the orientation of the GTF3 insert in the shuttle vector. Full-length and truncated mouse GTF3 expression plasmid were based on I.M.A.G.E. clone 555547 (see above). The generation of carboxyl-terminal deletion mutant mGTF3β.Δ3–6 is described elsewhere (11Calvo S. Vullhorst D. Venepally P. Cheng J. Karavanova I. Buonanno A. Mol. Cell. Biol. 2001; 21: 8490-8503Google Scholar). The amino-terminal deletion mGTF3β.ΔLZ was made by PCR-amplifying the GTF3β cDNA with a coding oligonucleotide located downstream of the leucine zipper motif (5′-TCCAGTCGACGCCACCATG CAG TCA GAC TTC CTC AGG TTC TGC-3′) and that includes a SalI linker, Kozak sequence, and translation initiation codon (underlined). T7 was used as antisense oligonucleotide. The PCR product was digested with SalI andEagI and inserted between the SalI andNotI sites of pCMV-Sport2. A 229-bp SacII fragment including the new translation start site was then excised from this intermediate and used to replace the corresponding sequence in GTF3β. The Pk epitope (Ref. 37Southern J.A. Young D.F. Heaney F. Baumgartner W.K. Randall R.E. J. Gen. Virol. 1991; 72: 1551-1557Google Scholar; also referred to as “V5”) was added on to full-length mGTF3β, mGTF3β.Δ3–6, and mGTF3β.ΔLZ to generate respective affinity-tagged derivatives. Corresponding constructs were made by inserting a double-stranded oligonucleotide coding for this epitope into the NcoI site that overlaps with the translation initiation codon (CCATGG) in GTF3. The sequence of this oligonucleotide (excluding NcoI linker arms) was 5′-GAA GGT AAG CCT ATC CCT AAC CCT CTC CTC GGT CTC GAT TCT ACG AG-3′. Mouse versions of human GTF3Δ1–3 were made by amplifying the coding sequence of the various GTF3 splice variants downstream of HLH repeat 3 using coding oligonucleotide 5′-GTCGACGCCACCATG AAG AGA CAG GGC CTT CAA G-3′;SalI linker, Kozak sequence, and translation initiation codon are underlined) and T7 as the noncoding oligonucleotide. The resulting PCR products were digested withSalI-BstEII and inserted between the corresponding sites of mGTF3 full-length isoform cDNAs in pCMV-Sport2. A full-length TFII-I cDNA (Δ-isoform) in pCMV-Sport6 was obtained as an Expressed Sequence Tag clone (accession no: AW912318; I.M.A.G.E. clone 3157775) from Incyte Genomics. Relative abundance of mouse GTF3α/γ splice variants was determined by a combination of RT-PCR and subsequent hybridization analysis. Mouse total RNA (2 μg) from adult soleus and EDL muscles, as well as from whole brain, was reverse transcribed and PCR-amplified with oligonucleotides mGTF3+2346s and mGTF3+3446r using the conditions described in the previous section. Parallel PCR reactions were subjected to 18, 19, and 20 cycles to ensure that PCR product growth was in the logarithmic phase. PCR products were electrophoresed on a 1.5% agarose gel and blotted onto a charged nylon membrane. For GTF3 isoforms α, α2, and α3, membranes were hybridized at 50 °C in 6× SSC, 1% SDS, 1 mm EDTA (1× SSC = 150 mm NaCl, 15 mm sodium citrate, pH 7.0) with the following [γ-32P]-labeled oligonucleotide probes: α, 5′-CC AAA GCC TG/A AAC CAA ATT-3′; α2, 5′-CC AAA GCC TG/G ACA TGA AGC-3′; α3, 5′-CC AAA GCC TG/A TGA GGA TGA-3′ (“/” indicates the boundary between exon 22 common to all isoforms and the isoform-specific adjacent sequences). Following a stringent wash (2× SSC, 1% SDS at 45 °C for α/α3 and 40 °C for α2), the membrane was exposed to a Storm® phosphorimager screen (Amersham Biosciences). Specificity was monitored, and hybridization signals were normalized, by including known quantities ofBstEII-HindIII cDNA fragments of α–α3 isoforms on the same membrane. For GTF3 isoforms γ and γ2, and to quantify total α- and γ-PCR products, membranes were hybridized with a [α-32P]dCTP-labeledBstEII-HindIII restriction fragment of mouse GTF3γ2 excised from pCMV-Sport2. To normalize for variability related to RNA input and reverse transcription, aliquots of RT samples were amplified in parallel with oligonucleotides specific for a 352-bp fragment of the mouse ribosomal protein L7 transcript (L7s, 5′-AGA TGT ACC GCA CTG AGA TCC-3′; L7a, 5′-ACT TAC CAA GAG ACC GAG CAA-3′; Ref. 38Krowczynska A.M. Coutts M. Makrides S. Brawerman G. Nucleic Acids Res. 1989; 17: 6408Google Scholar). L7 PCR products were electrophoresed and blotted as described above and hybridized against a [α-32P]dCTP-labeled cDNA probe derived from the L7 PCR product. Signals were quantified with the phosphorimager and used to normalize corresponding values for GTF3 splice isoforms. Full-length and partial GTF3 proteins used for EMSAs were generated in vitro from cDNAs subcloned into pCMV-Sport2 (see above). Proteins were synthesized from 0.5 μg of plasmid DNA using 14 μl of TnT® SP6 reticulocyte coupled transcription-translation system (Promega). Relative efficiency of translation was monitored in parallel reactions by the addition of [35S]methionine. Radiolabeled proteins were fractionated on 4–20% gradient SDS-polyacrylamide gels (SDS-PAGE), and gels dried and exposed to autoradiographic film for visualization of proteins. The relative levels of translated protein were determined by quantification using the phosphorimager and normalization for the number of methionines in each GTF3 construct. Double-stranded complementary oligonucleotide used in EMSAs was SURE−842/−815 (5′-TAC CGG ATT AAC ATA GCA GGC ATT GTC T-3′). In some cases, the shorter probe, SURE−844/−827 (5′-GCT ACC GGA TTA ACA TAG-3′) was used (11Calvo S. Vullhorst D. Venepally P. Cheng J. Karavanova I. Buonanno A. Mol. Cell. Biol. 2001; 21: 8490-8503Google Scholar). Probes were generated with [γ-32P]ATP (6,000 Ci/mmol, Amersham Biosciences) and T4 polynucleotide kinase and purified on acrylamide gels. Binding reactions were performed in a 10-μl total volume using 1.5 μl ofin vitro-translated GTF3 proteins, 1 μg of poly(dI-dC), and 32P-labeled probe (15,000 cpm). Binding buffer composition was: 20 mm HEPES, pH 7.9, 50 mmKCl, 4 mm MgCl2, 4% Ficoll, 5% glycerol, 0.2 mm EDTA, 0.5 mm dithiothreitol. 50 ng of single-stranded oligonucleotide SURE 832/808 (5′-ACA TAG CAG GCA TTG TCT TTC TCT G-3′) was included to reduce nonspecific binding of proteins present in the reticulocyte lysate. Reactions were incubated at room temperature for 20 min, and DNA-protein complexes were resolved by electrophoresis at 4 °C on 5% polyacrylamide gels in 0.5× TBE buffer. Gels were dried, visualized by autoradiography, and quantified on the phosphorimager. A cDNA fragment encoding the amino-terminal 130 amino acids of mouse GTF3 was amplified from I.M.A.G.E. clone 555547 using the following primers: GTF3-N.Cod (5′-CAC TAG GAA TTC GGA TCC GCC TTG CTG GGG AAG CAC TGT TGA C-3′) and GTF3-N.NCod (5′-TGG TAC GAA TTC ATC TTC TGC AGC AGG TAC ACA TCC-3′). The PCR product was digested withBamHI and EcoRI and inserted between the corresponding sites of pGEX-2T (Amersham Biosciences). GlutathioneS-transferase fusion protein was expressed inEscherichia coli BL21 cells and extracted from lysates on glutathione-Sepharose (Amersham Biosciences). The immunogen was further purified by preparative SDS-PAGE followed by electroelution of the specific 40-kDa band. This preparation was used to immunize rabbits. Immunoglobulins were purified from whole antiserum on Protein A-Sepharose columns (Pierce). Antibody specificity was confirmed by Western blotting of whole extracts from HEK293 and C2C8 cells transfected with expression constructs for human and mouse GTF3 as well as mouse TFII-I (see above). No cross-reactivity of anti-GTF3 antibodies toward TFII-I was observed. HEK293 cells grown in 35-mm dishes were transfected at 50% confluence using FuGENE 6 transfection reagent (Roche). Crude nuclear extracts were made 48–60 h post-transfection from three or four dishes using the NE-PER reagent kit (Pierce). Affinity-tagged GTF3 proteins were immunoprecipitated with 1.5 μg of anti-Pk antibody (sv5-pk, Serotec) and 25 μl of Protein A-Sepharose (Santa Cruz Biotechnology) from 100 μg of nuclear proteins in 500 μl of binding buffer (150 mm NaCl, 50 mm Tris-Cl, pH 8.0, 1% Triton® X-100). Nuclear extracts and immune complexes were separated by 10% SDS-PAGE, Western-blotted onto nitrocellulose membranes, and probed with anti-GTF3 polyclonal antibody (1:2000) or anti-TFII-I monoclonal antibody (clone 42, 1:1000; BD Transduction Laboratories). A duplicate set of Western blots was probed with anti-Pk antibody (1:5000). C2C8 myoblasts were transfected at 20% confluence with plasmids expressing affinity-tagged GTF3 proteins. The next day, cells were fixed with 4% paraformaldehyde/phosphate-buffered saline, permeabilized with 0.25% Triton® X-100, and blocked in 10% normal goat serum (Sigma). Cells were incubated overnight at 4 °C with the anti-Pk antibody (1:1000). Alexa 488 goat-anti-mouse secondary antibody (Molecular Probes) was used at 1:500. Nuclei were stained with Hoechst 33258. For immunofluorescence cytochemistry of endogenous GTF3 and TFII-I expression, C2C8 cells were prepared as above and incubated overnight at 4 °C with polyclonal anti-GTF3 antibody (1:100) and monoclonal anti-TFII-I antibody (1:25). Secondary antibodies were goat-anti-rabbit Alexa 488 (1:500; Molecular Probes) and goat-anti-mouse Cy3TM (1:100; Jackson Immunoresearch), respectively. We have previously demonstrated that GTF3 interacts specifically with the BLM of the TnIs upstream enhancer (11Calvo S. Vullhorst D. Venepally P. Cheng J. Karavanova I. Buonanno A. Mol. Cell. Biol. 2001; 21: 8490-8503Google Scholar). We concluded that the DNA binding domain of GTF3 must be located downstream of R2 because many GTF3 clones obtained from our yeast one-hybrid screen lacked sequences upstream of R3, and EMSA experiments confirmed that the carboxyl-terminal half of GTF3 (including R3–R5) bound to the BLM, but not the amino-terminal half (including R1 and R2). To map the location of its DNA binding domain, we have generated a series of truncated human GTF3 expression plasmids (Fig. 1). The ability to interact with the BLM was tested in EMSAs using in vitro translated proteins and an oligonucleotide probe that encompasses the sequence between −842 and −815 of the rat TnI SURE (11Calvo S. Vullhorst D. Venepally P. Cheng J. Karavanova I. Buonanno A. Mol. Cell. Biol. 2001; 21: 8490-8503Google Scholar). To ensure that all GTF3 proteins were properly translated, parallel reactions spiked with [35S]methionine were loaded on a 4–20% gradient SDS-PAGE and autoradiographed (data not shown). EMSAs were performed with unlabeled proteins synthesized from the different GTF3 constructs shown in Fig. 1. As shown in Fig. 2 A, full-length GTF3 produced a relatively weak specific shift (lanes 2 and 13) similar to that observed previously (11Calvo S. Vullhorst D. Venepally P. Cheng J. Karavanova I. Buonanno A. Mol. Cell. Biol. 2001; 21: 8490-8503Google Scholar). GTF3 proteins lacking the NH2 terminus plus the first two (hGTF3Δ12, lane 3) or three HLH domains (hGTF3Δ1–3,lane 4) interacted strongly with the probe. In contrast, no shift was observed when R4 was removed in addition to the first three repeats (hGTF3Δ1–4; lanes 5 and 14). Thus, a region between R3 and R4 is necessary for GTF3 to bind to the BLM. Next, we deleted specific regions in the carboxyl-terminal half of GTF3 within the context of hGTF3Δ12. A mutant protein that lacks the carboxyl terminus downstream of R5 efficiently formed a complex with the probe (hGTF3Δ12C, lane 6), indicating that this region, which includes a serine-rich stretch and the nuclear localization signal (26Yan X. Zhao X. Qian M. Guo N. Gong X. Zhu X. Biochem. J. 2000; 345: 749-757Google Scholar), is n" @default.
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• W2006468675 title "Characterization of General Transcription Factor 3, a Transcription Factor Involved in Slow Muscle-specific Gene Expression" @default.
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