Genomic structure of the chicken slow skeletal muscle troponin T gene

Gene 338 (2004) 243 – 256 www.elsevier.com/locate/gene

Genomic structure of the chicken slow skeletal muscle troponin T gene Chinami Hirao a, Izuru Yonemura b, Jun-Ichi Miyazaki a,* a

Institute of Biological Sciences, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan b Japan Science and Technology Corporation, Komaba, Meguro-ku, Tokyo 153-0041, Japan Received 13 February 2004; received in revised form 6 April 2004; accepted 28 May 2004 Available online 23 July 2004 Received by T. Sekiya

Abstract Troponin T (TnT) is a key protein for Ca2 +-sensitive molecular switching of muscle contraction. In vertebrates, three TnT genes have been identified, which produce isoforms characteristic of cardiac, fast skeletal, and slow skeletal muscles through alternative splicing in a tissue-specific and developmentally regulated manner. The diversification of myofibers into forms with specific metabolic and contractile characteristics is thought to be closely associated with the differential expression of these TnT isoforms. Herein, we determined the nucleotide sequence of the chicken slow skeletal muscle TnT gene and its upstream region. The gene was simpler in structure than the two other chicken genes. The transcription initiation site was positioned 183 bp upstream of the 3Vend of exon 1. Alternative splicing of exon 5 using an internal acceptor site generated two distinct slow skeletal muscle troponin T (sTnT) transcripts. We identified possible regulatory elements, M-CATlike, CACC-box, and E-box (E-box1 to E-box3) motifs in the upstream region and an E-box motif (E-box4) in exon 1. D 2004 Elsevier B.V. All rights reserved. Keywords: Gene structure; Internal acceptor site; cis-Acting element; EMSA; E-box; M-CAT-like

1. Introduction The striated muscle fibers of vertebrates are grouped into cardiac, fast skeletal, and slow skeletal muscle types with respect to metabolic and contractile properties. Contraction of the vertebrate striated muscle is initiated by an interaction between thick and thin filaments. The repeating unit of interdigitating thick and thin filaments forms the sarcomere, which, in turn, is organized into myofibrils. Thick filaments are composed primarily of myosin, and major components of thin filaments include actin, tropomyosin, and troponin. Each of these proteins is produced from multigene families. A large variety of protein isoforms is produced from distinct

Abbreviations: bp, base pair(s); cDNA, DNA complementary to RNA; cTnT, cardiac muscle troponin T; dNTP, deoxyribonucleotide triphosphate; fTnT, fast skeletal muscle troponin T; kb, kilobase(s); nt, nucleotide(s); PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-polymerase chain reaction; SSC, sodium chloride/sodium citrate buffer; sTnT, slow skeletal muscle troponin T; TBE, Tris/borate/EDTA electrophoresis buffer; UTR, untranslated region(s). * Corresponding author. Tel.: +81-298-53-4665; fax: +81-298-53-6614. E-mail address: [email protected] (J.-I. Miyazaki). 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.05.023

genes in these multigene families through alternative splicing of primary transcripts (for review, see Andreadis et al., 1987) and/or the use of alternative promoters (Robert et al., 1984; Concordet et al., 1993). The diversification of myofibers into forms with specific metabolic and contractile characteristics is thought to be closely associated with the differential expression of these isoforms. This differential expression is mediated by the specific interaction of regulatory elements (cis-acting elements) in the proximal promoter region with cognate trans-acting factors, as well as through the specific interaction of the intronic and/or exonic regulatory elements with cognate alternative splicing factors. Therefore, identification of these transcriptional and splicing elements and their cognate trans-acting factors should lead to a better understanding of the mechanism of tissue-specific and developmentally regulated expression of protein isoforms, and may provide insight into the mechanism of muscle diversification. Troponin is a key protein for molecular switching of muscle contraction in response to alteration in the intracellular Ca2 + concentration (Farah and Reinach, 1995; Tobacman, 1996). Troponin is located on the thin filament and consists of three components: the tropomyosin-binding

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subunit, troponin T (TnT), the inhibitory subunit, troponin I, and the Ca2 +-binding subunit, troponin C (for reviews, see Zot and Potter, 1987; Schiaffino and Reggiani, 1996). TnT anchors troponin to thin filaments and transmits its Ca2 +induced conformational change to tropomyosin. In vertebrates, three TnT genes which produce isoforms characteristic of cardiac, fast skeletal, and slow skeletal muscles have been identified (Breitbart et al., 1985; Cooper and Ordahl, 1985; Gahlmann et al., 1987; Smillie et al., 1988; Mesnard et al., 1993; Yonemura et al., 1996, Jin et al., 1998). Several groups have studied the structure of the cardiac muscle troponin T (cTnT) gene and the mechanism of its transcription. The chicken cTnT gene spans about 9 kb and contains 18 exons, one of which (exon 5) is alternatively spliced (Cooper and Ordahl, 1985). This gene is expressed in both cardiac and embryonic skeletal muscles, and its transcription is subject to dual regulation imparted by both common and cardiac muscle-specific regulatory elements (Mar and Ordahl, 1988). The M-CAT motif is an essential cis-acting element involved in the transcription of the cTnT gene in both cardiac and skeletal muscles. The motif interacts with the M-CAT binding factor, TEF-1, and plays an important regulatory role in the transcription of other cardiac muscle genes as well (Mar and Ordahl, 1990; Farrance et al., 1992; Stewart et al., 1994). An additional cis-acting element bound to an unidentified trans-acting factor is indispensable for expression of the cTnT gene in the cardiac muscle. The fast skeletal muscle troponin T (fTnT) gene spans about 34 kb and contains 27 exons (Miyazaki et al., 1999). The gene contains 16 alternative exons that could theoretically produce 215 alternatively spliced transcripts, although far fewer transcripts have actually been detected. Smillie et al. (1988) and Schachat et al. (1995) have described four full-length variants and sixteen 5V-variants of the chicken fTnT DNA complementary to RNA (cDNA), respectively. Additionally, Ogut and Jin (1998) and Jozaki et al. (2002) have found 12 and 19 fTnT transcripts, respectively. Watanabe et al. (1997) has examined the mechanism of transcription of the chicken fTnT gene. Its expression is controlled by the DNA segment between 264 and 44 bp from the most 5V site of transcription initiation, which includes several regulatory elements. The M-CAT-like motif is the most important among these regulatory elements. The motif (CACTCCT) differs by one nucleotide (nt) from the M-CAT motif (CATTCCT) and binds to a factor(s) distinct from TEF-1. Nucleotide sequences homologous to the MCAT-like motif exist in several muscle-specific genes. In contrast, little is known of the structure and transcriptional regulation of the slow skeletal muscle troponin T (sTnT) gene. Variants of the chicken sTnT cDNA that differ by the presence or absence of an additional alanine-encoding codon and by single base substitutions at two positions in the 5V-region and four positions in the 3V-region have been found (Yonemura et al., 2000). Although genomic structures of the mouse (Huang et al., 1999) and human (Barton et al.,

1999) sTnT genes have been reported, the chicken sTnT gene has not yet been studied. Structural and regulatory analyses of the chicken sTnT gene will permit a direct comparison of genomic organization and transcriptional regulation of the three TnT genes from a single species. Such a comparison should lead to an elucidation of the mechanism behind the production of the various protein isoforms and may also provide insight into the mechanism of the evolution of muscle diversification. In this study, we present the complete nucleotide sequence of the chicken sTnT gene. The gene spans only about 3 kb and contains 14 exons, one of which is alternatively spliced. We also present the sequence of the upstream region and map regulatory elements participating potentially in the control of chicken sTnT gene expression. Among these elements, the CACC-box motif in the upstream region and the E-box motif located in exon 1 are possible cis-acting elements for the gene.

2. Materials and methods 2.1. Cloning and sequencing of the chicken sTnT gene Genomic DNA was prepared from liver of adult white leghorn chicken (Gallus domesticus) by proteinase K digestion and phenol/CHCl3 extraction. To clone the full-length chicken sTnT gene, we performed step down polymerase chain reaction (PCR) on genomic DNA using KOD Dash (TOYOBO) and sense primer SP1 (5V-GCAGCCATGTCCGAAGCTGAG-3V, corresponding to nucleotides from 39 to 59 of the sTnT cDNA; Yonemura et al., 1996) and antisense primer AP1 (5V-GGGAATTCGACGAGCAGAGCTTTATTGG-3V, nucleotides 872 – 899 of the sTnT cDNA). Reactions were denatured for 30 s at 95 jC and annealed and extended for 10 min at 85 jC initially to 77 jC with the temperature decreasing by 4 jC in three successive cycles and then at 74 jC for the last 30 cycles. The amplified fragments were separated on a 0.8% Tris/borate/EDTA electrophoresis buffer (TBE) agarose gel, cloned into a pBluescript II KS+ phagemid (Stratagene), and sequenced by primer walking. We performed inverse PCR to clone the upstream region of the sTnT gene. As there are BamHI sites within the sTnT gene and in its upstream region, genomic DNA was digested with BamHI, and the resulting fragments were allowed to self-ligate into circular DNA. The circular DNA was PCRamplified using sense primer SP2 (5V-CATCCACCGGAAGCGCATGGAGAA-3V, nucleotides 251 – 274 of the sTnT cDNA) and antisense primer AP2 (5V-TGTTGGGGCACTCACTCCTCGTATTCC-3V, corresponding to nucleotides from position 38 of exon 2 to position 15 of intron 2) by step down PCR as above. The amplified fragments were separated on a 0.8% TBE agarose gel, cloned, and sequenced. The DDBJ accession number for the chicken sTnT gene is AB07833.

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2.2. Southern blot analysis We treated 50 Ag of chicken genomic DNA separately with ApaI, BamHI, DraI, and KpnI restriction endonucleases. The fragments were separated on a 0.8% agarose gel and transferred to Hybond-N+ (Amersham) in 0.4 M NaOH and 0.6 M NaCl. The membrane was prehybridized at 37 jC in hybridization buffer (35% formamide, 0.75 M NaCl, 75 mM sodium citrate, 65 mM KH2PO4, 5 mM EDTA, 0.02% Denhardt’s solution, 10% [w/v] Dextran sulfate, and 4 ng/ml salmon sperm DNA) and then hybridized to a 32P-labeled and denatured probe (nucleotides 251 –891 of the sTnT cDNA, see Fig. 5) at 42 jC in a shaking water bath. The membrane was washed repeatedly at 60 jC in wash buffer (0.5  sodium chloride/sodium citrate buffer [SSC] and 1% SDS) and subjected to autoradiography on X-ray film (Kodak) with an intensifying screen. 2.3. RNA preparation Total RNA was prepared separately from back muscle, anterior latissimus dorsi (ALD), and liver of adult chicken using Isogen (NIPPON GENE) following the manufacturer’s protocol. Poly(A)+ RNA was isolated from total RNA using Oligotexk-dT30 super (TaKaRa).

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units of SuperScriptk II (GIBCOBRL) at 42 jC for 60 min, and the products were separated on a 6% acrylamide/8 M urea gel along with sequencing reactions of the cloned chicken genomic DNA as a size marker. The gel was fixed in 10% acetic acid and 10% methanol, dried, and subjected to autoradiography. 2.5. Reverse transcriptase-polymerase chain reaction (RTPCR) of sTnT transcripts We performed RT-PCR on sTnT transcripts to confirm the position of the transcription initiation site. Reactions including 2 Ag of poly(A)+ RNA from adult chicken ALD, 0.5 Ag of oligo(dT)12 – 18, and 200 units of SuperScriptk II were incubated at 42 jC for 60 min, and then PCR was performed with KOD Dash (TOYOBO) using four sense primers, up2 (5V-GTCACCCCGACAGCTGCGGGTTTC-3V), up2V-1 (5V-AGCTGCGGGTTTCGGGTGACGGTGAC-3V), up2V-2 (5V-GGGTTTCGGGTGACGGTGACATTTAATGGC-3V), and up3 (5V-GGTGACGGTGACATTTAATGGCATCTGG3V), and an antisense primer (5V-TCATCCTCCCGCATGTGCTCAATG-3V, corresponding to nucleotides 635– 658 of the sTnT cDNA; Yonemura et al., 1996). Amplification of 33 cycles was performed by denaturing at 96 jC for 30 s, annealing at 65 jC for 10 s, and extending at 74 jC for 20 s. The amplified fragments were separated on a 2% TBE agarose gel.

2.4. Primer extension analysis 2.6. Electrophoretic mobility shift assay (EMSA) Antisense primer PE1 (5V-GGTCGGTCCTGTTGGCTCTGC-3V, corresponding to the sequence located in exon 1, see Fig. 6) was labeled at its 5Vend with T4 polynucleotide kinase (NIPPON GENE) and [g-32P]ATP (Amersham Pharmacia Biotech). The labeled primer was added to 2 Ag each of poly(A)+ RNA from adult chicken ALD and liver. The mixtures were incubated at 70 jC for 10 min and quickly chilled on ice. Reverse transcription was carried out with 200

A crude nuclear extract was prepared from whole leg muscle of 1-day-old chicken as described (Mar and Ordahl, 1990), frozen in liquid N2, and stored at 80 jC. The protein concentration of the nuclear extract was determined using the Bio-Rad Protein Assay (Bio-Rad). We synthesized both sense and antisense oligonucleotides containing putative regulatory elements (Table 1). Sense

Table 1 Oligonucleotides used as probes and competitors in EMSA Name

Sequence (sense strand)a

Positionb

Reference

Slow MCL mut slow MCL E-1 mut E-1 CACC mut CACC E-2 mut E-2 E-3 E-4 mut E-4 Fast MCL Mouse E-box Mouse CACC MyLC E-box

5V-CCCCAGCCATCCCAACCCCCCCCCCCCCTT-3V 5V-CCCCAGCGGTCCCAACCCCCCCCCCCCCTT-3V 5V-CCTTTGTCCTCACCCAAATGGTCCCCTG-3V 5V-CCTTTGTCCTCACCCAAAGAGTCCCCTG-3V 5V-TGGAGCCCCCCCCACCCCCCAAGGCTGAG-3V 5V-TGGAGCCCCCCGATATCCCAAGGCTGAG-3V 5V-CCCCTCCCAGCTGACCCCCCCCGTGTCAC-3V 5V-CCCCTCCCAGCGAACCCCCCCCGTGTCAC-3V 5V-TCACCCCGACAGCTGCGGGTTTCGGGTG-3V 5V-TCGGGTGACGGTGACATTTAATGG CATCTGGGGAACAATG-3V 5V-TCGGGTGACGGTGACATTTAATGG CATCGAGGGAACAATG-3V 5V-TCCTTTCCCTCACTCCTCACCCCA-3V 5V-GAAGCTCAGACAGCTGGGAACTCCTGATT-3V 5V-TAACACTGCCCCACCCCCTGCAG-3V 5V-CTCACATCCTCAGCTGCTGCTGCTTT-3V

Chiken sTnT, 267/ 238 Chiken sTnT, 267/ 238 Chiken sTnT, 241/ 214 Chiken sTnT, 241/ 214 Chiken sTnT, 95/ 68 Chiken sTnT, 95/ 68 Chiken sTnT, 46/ 18 Chiken sTnt, 46/ 18 Chiken sTnT, 21/ + 7 Chiken sTnT, + 1/ + 40 Chiken sTnT, + 1/ + 40 Chiken fTnT, 121/ 98 Mouse dystrophin Mouse s/cTnC + 44/ + 56 Chicken MyLc 1980/ 1955

This study This study This study This study This study This study This study This study This study This study This study Watanabe et al., 1997 Marshall et al., 2001 Parmacek et al., 1994 Fujisawa-Sehara et al., 1992

a b

Putative regulatory elements are in bold. Mutated sites are underlined. Positional numbering is relative to the transcriptional initiation site (+ 1).

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without 12 Ag of the nuclear extract. We performed competitive EMSA by adding a 10- or 100-fold molar excess of unlabeled intact and mutant competitor oligonucleotides to the reactions (Table 1). Reaction mixtures were incubated at 30 jC for 30 min and then electrophoresed on a 6% nondenaturing polyacrylamide gel. The gel was fixed in 10% acetic acid and 10% methanol, dried, and subjected to autoradiography.

3. Results 3.1. Cloning of the chicken sTnT gene and its upstream region To isolate the entire sTnT gene and its upstream region, we performed genomic PCR (Fig. 1A) and inverse PCR (Fig. 1B) using genomic DNA from chicken liver and SP1/AP1 and SP2/AP2 primer sets, respectively. Genomic PCR amplified a specific fragment of about 3 kb (arrowhead in the lower panel of Fig. 1A), which we cloned into the pBlue-

Fig. 1. Genomic PCR and inverse PCR for cloning of the full-length chicken sTnT gene and its upstream region. (A) Genomic PCR was performed on chicken liver DNA using SP1 and AP1 primers, which are indicated by arrows in the schema of the sTnT cDNA (upper). The product was separated on a 0.8% TBE agarose gel (lower). No fragment was amplified using SP1 or AP1 only, but a fragment of about 3 kb (arrowhead) was specifically amplified using both primers (SP1+AP1). (B) Inverse PCR of BamHI-digested and self-ligated genomic DNA was performed using SP2 and AP2 primers, which are indicated by arrows in the schema of the sTnT cDNA (upper). The product was separated on a 0.8% TBE agarose gel (lower). No fragment was amplified using SP2 or AP2 only, but a fragment of about 2 kb (arrowhead) was specifically amplified using both primers (SP2+AP2). M, E /HindIII DNA size marker.

oligonucleotides were labeled at the 5V end with T4 polynucleotide kinase (NIPPON GENE) and [g-32P]ATP (Amersham Pharmacia Biotech) and annealed to the corresponding antisense oligonucleotides as described (Marshall et al., 2001). EMSA was performed in 20 Al reaction buffer containing 3105 cpm of the labeled oligonucleotides, 2 Ag of poly(dI-dC)-(dI-dC), 25 mM HEPES (pH 7.6), 40 mM KCl, 0.1 mM EDTA, 1 mM DTT, and 10% glycerol with or

Fig. 2. Southern hybridization of the chicken sTnT gene with a cDNA probe. Purified chicken liver genomic DNA was separately digested by restriction endonucleases, ApaI, BamHI, DraI, and KpnI, and separated on a 0.8% agarose gel. Fragments were transferred to a nylon membrane and hybridized to a 32P-labeled chicken sTnT cDNA probe (see Fig. 5). After washing repeatedly, the membrane was subjected to autoradiography. Hybridization signals are indicated by arrowheads.

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from a single sTnT gene, we performed Southern blot analysis using a 641-bp probe derived from the 3V-region of the sTnT cDNA clone (outlined in Fig. 5). This probe hybridized to a single DNA fragment in ApaI- or DraIdigested DNA and to two fragments in BamHI- or KpnIdigested DNA (Fig. 2). Given that single BamHI and KpnI sites and no ApaI and DraI sites are present within the 3Vregion of the sTnT gene covered by the probe (see Fig. 5), we conclude that a single copy of the sTnT gene is present per haploid chicken genome. 3.3. Mapping of the transcription initiation site by primer extension and RT-PCR

Fig. 3. Determination of the transcription initiation site of the chicken sTnT gene by primer extension and RT-PCR. (A) Primer extension was performed using a labeled oligonucleotide primer (PE1 in Fig. 6). A primary product of 156 nt was obtained using mRNA from chicken adult ALD as a template (lane 1), whereas no product was synthesized using mRNA from chicken adult liver as a template (lane 2). (B) RT-PCR of chicken adult ALD mRNA was performed with four sense primers (up2, up2V-1, up2V-2, and up3 in Fig. 6) and an antisense primer corresponding to nucleotides 635 – 685 of the sTnT cDNA. The products were electrophoresed on a 2% TBE agarose gel. M, 100 bp DNA size marker.

script II phagemid vector and sequenced (Fig. 4). For inverse PCR, we digested genomic DNA with BamHI and then selfligated the resulting fragments into circular DNA. PCR performed on this circular DNA amplified a fragment of about 2 kb (arrowhead in the lower panel of Fig. 1B), which we also cloned and sequenced as above (Fig. 6). This fragment should contain the nucleotide sequence between the sequences complementary to the SP2 primer and the BamHI site within the sTnT gene and between the sequences complementary to the AP2 primer and an upstream BamHI site. 3.2. A single sTnT gene in the chicken genome Several sTnT cDNA sequences have been reported, which might represent distinct sTnT isoforms in chicken skeletal muscles (Yonemura et al., 2000; Yonemura et al., 2002). To determine whether these transcripts are generated

To identify the transcription initiation site of the sTnT gene, we performed primer extension analysis using antisense 21-mer oligonucleotides derived from the 5V-untranslated region (UTR) of the sTnT cDNA (PE1 in Fig. 6). A primary extension product of 156 nt was observed using poly(A)+ RNA from adult chicken ALD as a template (lane 1 in Fig. 3A). In contrast, no product was produced using poly(A)+ RNA from chicken liver as a template (lane 2 in Fig. 3A), indicating that the 156-nt product was specific to sTnT transcripts. This corresponds to a transcription initiation site 204 bp upstream of the translation initiation site (Fig. 6). To confirm the position of the transcription initiation site, we performed RT-PCR on poly(A)+ RNA from adult chicken ALD using four sense primers (up2, up2V-1, up2V-2, and up3 in Fig. 6) and an antisense primer corresponding to nucleotides 635– 658 of the sTnT cDNA. We observed a reaction product of about 800 bp, which is consistent with the size expected from the cDNA sequence (2V-1, 2V-2, and 3 in Fig. 3B). However, up2 produced no RT-PCR product and up2V-1 gave rise to a lesser amount of the reaction product (2 and 2V-1 in Fig. 3B). These results support the position of the transcription initiation site determined by primer extension, because up2 is not predicted to hybridize to sTnT transcripts, and only about half of the sequence of up2V-1 hybridizes to sTnT transcripts, leading to reduced affinity for the primer. 3.4. The structure of the chicken sTnT gene The complete nucleotide and deduced amino acid sequences of the 3-kb fragment amplified by genomic PCR are shown in Fig. 4. The structural organization of exons and introns was confirmed by direct alignment of the genomic sequence with the cDNA sequences. A diagram depicting the sTnT gene structure and the positions of restriction sites used in the Southern blot analysis is shown in Fig. 5. Also shown is a schema of the sTnT cDNA, which is compartmentalized at exon/intron boundaries. The chicken sTnT gene consists of 14 exons and 13 introns spanning about 3 kb. As with other vertebrate TnT genes, the translation initiation codon, ATG, was located in exon 2 (underlined in Fig. 4) and the 5V-UTR is included in exons 1

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Fig. 5. The chicken sTnT gene structure. The sTnT genomic structure is presented in scale and positions of restriction sites used for inverse PCR and Southern blot analysis (see Figs. 1 and 2, respectively) are also indicated (upper). Filled boxes indicate exons, and lines indicate introns and the upstream region. sTnT cDNA is schematically represented with partition at exon/intron boundaries (lower). A probe of 641 bp used for Southern blot analysis (see Fig. 2) is also shown.

and 2. The termination codon, TGA, was located in exon 14 (underlined in Fig. 4) followed by the poly(A)+ signal, AATAAA (asterisks in Fig. 4). All of the exons were flanked by signature gt and ag sequences corresponding to donor and acceptor splice sites of eukaryotic genes. In addition, the 2-kb fragment specifically amplified by inverse PCR was cloned and sequenced to identify putative cis-acting elements involved in the regulation of sTnT gene transcription. The sequence of the upstream region and exon 1 is shown in Fig. 6. There was a series of consensus sequences for regulatory elements in the upstream region, but no apparent TATA-box upstream of the transcription initiation site. Three upstream E-box motifs were located at 227 to 222 (designated as E-box1), 39 to 34 (Ebox2), and 12 to 7 (E-box3), and one additional downstream E-box motif (E-box4) was located at + 5 to + 10 in exon 1. The E-box motif is a binding site for the myogenic bHLH protein family (Olson, 1990). A CACC-box motif was located at 86 to 78. The CACC-box motif has been shown to be important for the transcription of the slow/ cardiac troponin C (Parmacek et al., 1994) and myoglobin (Bassel-Duby et al., 1992) genes in the striated muscle. The CATCCCA sequence at 260 to 254 differed at two positions from the M-CAT motif (CATTCCT), which is the essential cis-acting element for chicken cTnT gene expression (Mar and Ordahl, 1990; Farrance et al., 1992; Stewart et al., 1994). We designated this sequence the M-CAT-like motif. Other putative regulatory elements matched incompletely with known consensus sequences, and included two CArG-box-like motifs and A/T rich and G/C rich sequences (underlined in Fig. 6).

3.5. Alternative splicing of exon 5 sTnT cDNA sequences have been reported previously that differ in the inclusion or exclusion of an Ala codon (GCA) in their 5V-region (Yonemura et al., 2000). The genomic sequence of the sTnT gene demonstrated that these transcripts were not derived from splicing of two independent alternative exons. Instead, they were generated from alternative acceptor sites at the 5V end of exon 5 (Fig. 7). When the last nucleotide (G) of exon 4 is spliced to the first nucleotide (C) of exon 5, the resulting cDNA includes the codon GCA, encoding alanine. On the other hand, when the first three nucleotides of exon 5 (CAG) are skipped, and the last nucleotide (G) of exon 4 is spliced to the fourth nucleotide (A) of exon 5, the resulting cDNA lacks this codon. The splice acceptor site in the former is ag preceding exon 5 and that in the latter is AG of the second and third nucleotides in exon 5. Alternative splicing using an internal acceptor site has been described in exon 6 of the mouse sTnT gene (Huang et al., 1999), exon 12 of the human sTnT gene (Barton et al., 1999), and exon 13 of the human cardiac cTnT gene (Farza et al., 1998). Utilization of alternative acceptor or donor sites during RNA processing provides proteins with structural variations for functional adaptation without requiring additional exons. 3.6. Comparison of TnT gene structures The structures of the chicken sTnT, fTnT (Miyazaki et al., 1999), and cTnT (Cooper and Ordahl, 1985) genes are schematically presented in Fig. 8A. The chicken sTnT gene

Fig. 4. Complete nucleotide and deduced amino acid sequences of the chicken sTnT gene. The product of genomic PCR was cloned and sequenced by primer walking. The nucleotide sequence of the chicken sTnT gene is shown together with the deduced amino acid sequence (below the corresponding nucleotide sequence). The DNA sequence is numbered on the right margin ( + 1, the transcription initiation site). Exonic and intronic sequences are depicted with bold capital letters and lower letters, respectively. All of the exon/intron boundaries were characterized by gt and ag signature sequences for donor and acceptor splice sites. The translation initiation codon, ATG, in exon 2 and the stop codon, TGA, in exon 14 are underlined. Asterisks indicate the poly(A)+ signal. The accession number for the chicken sTnT genomic sequence (3690 bp) is AB078333.

250 C. Hirao et al. / Gene 338 (2004) 243–256 Fig. 6. The nucleotide sequence of the upstream region of the sTnT gene and putative regulatory elements. The product of inverse PCR was cloned and sequenced. The sequence of the upstream region and exon 1 (bold letter) is numbered on the right margin ( + 1, the transcription initiation site). Putative regulatory elements are boxed (E-box1-4, M-CAT-like, and CACC-box) or underlined (CArG-box-like, A/T rich, and G/ C rich). Arrows denote sense primers utilized in RT-PCR (up2, up2V-1, up2V-2, and up3) and the antisense primer (PE1) utilized in primer extension (see Fig. 3).

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Fig. 7. Alternative splicing of exon 5. The chicken sTnT genomic sequence revealed that the inclusion/exclusion of 3 bp found in the 5V-region of chicken sTnT cDNA (Yonemura et al., 2000) was not caused by alternative splicing of separate exons, but was generated by using different acceptor sites around the boundary of intron 4 and exon 5. The partial sequence of exon 4 to exon 5 (capital and lower letters for exons and introns, respectively) is shown with the corresponding amino acid sequence (upper). When the acceptor site preceding exon 5 (underlined) is utilized, the resulting cDNA has the codon (GCA) encoding alanine (asterisk). On the other hand, when the internal acceptor site in exon 5 (double-underlined) is utilized, the resulting cDNA lacks the codon for alanine. Alternative splicing using the internal acceptor site in exon 6 of the mouse sTnT gene is shown for comparison (lower, Huang et al., 1999).

is relatively compact, including 14 exons in the length of about 3 kb. Exon 5 alone is an alternative exon with the internal acceptor site for splicing. The huge chicken fTnT gene of 34 kb has 27 exons, 16 of which can be alternatively

spliced. In the 5V-region, 14 exons (4, w, 5, xa to xg, 6, 7, y, and 8) are combinatorially spliced and 2 exons (16 and 17) in the 3V-region are spliced in a mutually exclusive manner. The chicken cTnT gene of 9 kb has 18 exons and exon 5 is

Fig. 8. Comparison of TnT gene structures. (A) Diagrams represent structures of the chicken sTnT, fTnT (Miyazaki et al., 1999), and cTnT (Cooper and Ordahl, 1985) genes in scale. Exons are indicated by bars. Different types of alternative splicing are distinguished as follows; +, internal acceptor site type; *, cassette (combinatorial) type; #, mutually exclusive type. The positions of the translation initiation codon (ATG) and the stop codon (STOP) are indicated. (B) Diagrams represent structures of the chicken and mouse (Huang et al., 1999) sTnT genes. The numbers above exons (boxes) represent those of amino acids encoded by the corresponding exons. The positions of the translation initiation codon (ATG) and the stop codon (STOP) are indicated by arrows. The genomic organization is highly conserved between the chicken and mouse sTnT genes.

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combinatorially spliced. The fTnT gene potentially produces 215 transcripts through alternative splicing, while the sTnT and cTnT genes produce just two transcripts each. Therefore, the heterogeneity in sTnT cDNA sequences reported by Yonemura et al. (2000) is attributable not only to alternative splicing, but also to intraspecific variations in exonic sequences. In addition, we compared the structures of the chicken and mouse (Huang et al., 1999) sTnT genes (Fig. 8B). The mouse sTnT gene (11 kb) is 3.6-fold larger than its chicken counterpart (3 kb) and contains 14 exons, two of which (exons 5 and 6) are alternatively spliced. The numbers of amino acids encoded by the genes are substantially identical (264 for the chicken gene and 261 for the mouse gene; Jin et al., 1998). Thus, the avian and mammalian genes have similar structures despite the large difference in size, supporting the idea that genes of the same muscular type in different species are much more conserved than genes of different muscular types in a single species. 3.7. Identification of putative cis-acting elements To identify elements in the upstream region of the sTnT gene that bind possible regulatory factors, we synthesized oligonucleotide probes (Table 1) corresponding to the MCAT-like motif (slow MCL), E-box1-4 motifs (E-1 to -4), and the CACC-box motif (CACC) and incubated them with nuclear extracts from whole leg muscle of 1-day-old chicken. Reactions were then subjected to electrophoretic mobility shift assays (Fig. 9). sTnT expression in the leg muscle

was confirmed by RT-PCR using the sTnT-specific primer set (data not shown). In addition, oligonucleotide probes corresponding to the fTnT M-CAT-like motif (fast MCL; Watanabe et al., 1997), the mouse dystrophin E-box motif (mouse E-box; Marshall et al., 2001), and the mouse slow/ cardiac troponin C CACC-box motif (mouse CACC; Parmacek et al., 1994) were also synthesized (Table 1). In the presence of nuclear extract (+), slow MCL and E-2 apparently generated two protein-DNA complexes (lanes 2 and 8). E-1, E-4, and CACC generated two complexes of similar mobility (lanes 4, 6, and 12) and fast MCL generated three protein-DNA complexes (lane 14). In contrast, E-3, mouse E-box, and mouse CACC did not form stable complexes with proteins in the extract (lanes 10, 16, and 18), suggesting that the E-box3 motif does not participate in the regulation of sTnT gene transcription and that formation of protein-DNA complexes derived from heterogeneous sources (chicken vs. mouse) was not preferable. To determine whether nuclear proteins bound specifically to these putative regulatory elements, we performed EMSA in the presence of intact and mutant competitor oligonucleotides. The sequences of the mutant probes are shown in Table 1. An excess of unlabeled slow MCL (lanes 3 and 4 in Fig. 10A) effectively inhibited formation of the two proteinDNA complexes formed on the labeled slow MCL probe (lane 2). However, mut slow MCL, containing a mutant sTnT M-CAT-like motif, also competed with the labeled probe for complex formation (lanes 5 and 6), while fast MCL failed to compete for binding (lanes 7 and 8). These

Fig. 9. EMSA with oligonucleotide probes containing putative regulatory elements. Radiolabeled, double-stranded synthetic oligonucleotides, slow MCL, E-1 to -4, CACC, fast MCL, mouse E-box, and mouse CACC, were incubated with (+) or without ( ) nuclear extracts from whole leg muscle of 1-day-old chicken. The resulting complexes were resolved by electrophoresis on a nondenaturing 6% polyacrylamide gel. All the chicken oligonucleotide probes, except for E-3, formed protein – DNA complexes (lanes 2, 4, 6, 8, 12, and 14), while E-3 (lane 10) and the mouse oligonucleotides (lanes 16 and 18) were inefficient at forming complexes. The nucleotide sequences of the probes are described in Table 1.

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Fig. 10. Competitive EMSA with intact and mutant competitors. Radiolabeled synthetic oligonucleotides, slow MCL and CACC (lanes 2 and 10 in A) and E-2 and E-4 (lanes 2 and 10 in B), were incubated with nuclear extracts from whole leg muscle of 1-day-old chicken. Arrows or arrowheads indicate bands of protein – DNA complexes. Asterisks indicate nonspecific bands. Unlabeled intact and mutant competitors were added at 10- or 100-fold molar excess. The competitors used are slow MCL, mut slow MCL, and fast MCL (lanes 3 – 8 in A), CACC, mut CACC, and mouse CACC (lanes 11 – 16 in A), E-2, mut E-2, and MyLC E-box (lanes 3 – 8 in B), and E-4, mut E-4, and MyLC E-box (lanes 11 – 16 in B). Formation of all of the complexes was inhibited by intact unlabeled competitor oligonucleotides. Formation of the slow MCL, faster-migrating CACC, and E-2 complexes was also inhibited by mutant competitor oligonucleotides. The nucleotide sequences of the mutant competitors are described in Table 1.

results suggest that nuclear factors bind to the slow MCL probe, but not to the sTnT M-CAT-like motif, CATCCCA, and that these factors do not bind to the fTnT M-CAT-like motif. The formation of the two complexes on the CACC probe (lane 10 in Fig. 10A) was inhibited by an excess of unlabeled CACC (lanes 11 and 12). Formation of the faster-migrating complex (C2), but not the more slowly migrating complex (C1), was inhibited by inclusion of unlabeled mut CACC containing a mutant CACC-box motif (lanes 13 and 14). These results suggest that a nuclear protein(s) involved in the formation of the more slowly migrating complex might be a putative trans-acting fac-

tor(s). Mouse CACC failed to compete for binding to chicken CACC (lanes 15 and 16). As for the E-box2 motif, the formation of two complexes (lane 2 in Fig. 10B) was inhibited by inclusion of an excess of unlabeled E-2 (lanes 3 and 4), as well as mut E2, containing a mutant E-box2 motif (lanes 5 and 6), but not by MyLC E-box, containing a chicken myosin light chain E-box motif (lanes 7 and 8; Fujisawa-Sehara et al., 1992). The complexes formed by E-1 were also competed out by an excess of unlabeled E-1 and mut E-1, but not by MyLC E-box (data not shown). These results indicated that the nuclear factors that bound to E-1 and E-2 did not recognize the E-box motif, CANNTG (Olson, 1990), and

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that sequences flanking the motifs and/or conformations assumed by the sequences were possibly involved in complex formation. E-4 formed complexes of relatively low mobility (lane 10 in Fig. 10B), because this probe (40 bp) was longer than the other probes (see Table 1). Formation of the complexes was inhibited by an excess of unlabeled E-4 (lanes 11 and 12), but not by an excess of mut E-4 (lanes 13 and 14) or MyLC E-box (lanes 15 and 16). These results suggest that the Ebox4 motif does not form complexes with bHLH family proteins, but that other trans-acting factors must bind to this motif.

4. Discussion 4.1. The structure of the slow skeletal muscle TnT gene and evolution of the TnT gene family In this study, we determined the complete nucleotide sequence and structure of the chicken sTnT gene (Figs. 4 and 5). The chicken sTnT gene (3 kb, 14 exons) is considerably more compact than the chicken fTnT (34 kb, 27 exons; Miyazaki et al., 1999) and cTnT (9 kb, 18 exons, Cooper and Ordahl, 1985) genes. Surprisingly, the size of the sTnT gene was approximately equivalent to that of intron 1 of the fTnT gene (Fig. 8). It would be of interest to investigate the evolution of the TnT gene family, because the gene size and the exon number are quite different among the avian TnT genes. We hypothesize two possible evolutionary processes in the TnT gene family on the basis of our knowledge of the similarity of small tandem exons in the 5’region of the chicken fTnT gene, which proposes relatively recent duplication events. (1) The potential prototype of the TnT gene family was of a size similar to that of the chicken sTnT gene (3 kb), and the ancestral gene diverged into the three types, accompanied by exon duplication and intron insertion for the fTnT and cTnT genes. (2) Alternatively, the potential prototype was of a greater size, similar to that of the chicken cTnT gene (9 kb) and similar to the general sizes of the mammalian TnT genes (10 kb). The gene diverged into the three types, accompanied by exon/intron amplification (gene expansion) to produce the fTnT gene and exon/intron deletion (gene reduction) to produce the sTnT gene. Structural studies on more evolutionarily distant TnT genes in fishes, amphibians, and reptiles are necessary to resolve the evolution of the TnT gene family. The chronology of TnT gene divergence and the relevance of this divergence for muscle diversification also remain to be investigated. 4.2. The promoter region of the chicken sTnT gene Examination of the nucleotide sequence of the upstream region (Fig. 6) revealed that it was highly G/C-rich (66.7% from 360 to + 183) and that it lacked a canonical TATA-

box, as do some housekeeping and constitutively expressed genes. The muscular TEF-1 (Boam et al., 1995) and carnitine palmitoyltransferase I h (Moore et al., 2001) genes also lack TATA-boxes. Instead, their promoters use G/C-rich sequences (GC-box) that bind to Sp1 proteins to support basal transcription. Several genes whose expression is specific to neurons lack TATA-boxes, and instead rely on GC-boxes and their binding factors of the Sp1 family for their transcription (Ross et al., 2002; Ohkawara et al., 2000; Galvagni et al., 2001). Therefore, we suggest that transcription of the TATA-less chicken sTnT gene might be controlled by interaction of the Sp1 transcription factor(s) with the G/C-rich Sp1-binding site. The human trans-acting factor, TFIID, binds to a sequence about 30 bp upstream of the transcription initiation site of TATA-less genes and plays a role in determining both the precise site(s) and the efficiency of transcription initiation (Wiley et al., 1992). The E-2 probe used for EMSA comprised bases 46 to 18 of the upstream region (Table 1). Although nuclear proteins from whole leg muscle of 1-day-old chicken bound to this probe, they did not bind at the E-box motif ( 39 to 34, Fig. 10). Nevertheless, the proteins binding to bases around position 30 of the sTnT gene might contribute to the initiation of transcription. Since the transcription initiation complex only forms within the proper context of other elements upstream and downstream of the initiation site, further studies are required to elucidate the mechanism of sTnT gene transcription. 4.3. Identification of cis-acting elements in the upstream region of the sTnT gene Transcription factors and signaling events responsible for myogenic determination and differentiation have been defined in detail (Chin et al., 1998; Olson and Williams, 2000). In contrast, the molecular mechanisms by which myofibers assume one of several highly specialized phenotypes are much less well understood. The nucleotide sequence of the proximal upstream region is now available for all the chicken TnT genes. Identification of the regulatory elements of the sTnT gene should provide a better understanding of the regulation of the transcription of TnT gene family members. Previous studies have shown that the most important cis-acting elements of the cTnT and fTnT genes are the M-CAT motif (CATTCCT; Mar and Ordahl, 1990; Farrance et al., 1992; Stewart et al., 1994) and the M-CAT-like motif (CACTCCT; Watanabe et al., 1997), respectively. Although the motifs differ only by a single nucleotide at the third position, they play absolutely different roles. If the M-CAT motif of the cTnT gene is mutated at the third position from T to C, the mutated motif (CACTCCT), which is completely identical with the M-CAT-like motif of the fTnT gene, can no longer activate cTnT gene expression (Farrance et al., 1992). Moreover, the fTnT M-CAT-like motif binds to a

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protein that is distinct from the cTnT M-CAT binding factor, TEF-1 (Watanabe et al., 1997). We identified an M-CAT-like motif (CATCCCA) in the upstream region of the sTnT gene. Because of the similarity, we suggested that the motif and its binding protein(s) regulate sTnT gene expression. EMSA in the present study suggested that the slow M-CAT-like motif was not a cis-acting element responsible for the regulation of the sTnT gene transcription. Instead, flanking regions of the motif probably bound to the nuclear proteins. EMSA also demonstrated that the E-box4 motif (CATCTG) located in exon 1 specifically bound to nuclear proteins. It is interesting that only the E-box4 motif formed specific complexes, and that the other E-box motifs upstream of the transcription initiation site did not. A previous study has shown that the E-box motif of the mouse myoglobin gene is located in exon 1 and functions as a negative regulatory element, contributing to the regulation of myoglobin expression in myofibers (Yan et al., 2001). Thus, the E-box4 motif of the sTnT gene may play a different regulatory role from that performed by the typical E-box motif. The existence of the intragenic E-box4 motif raises the possibility that other regulatory elements are also present within the sTnT gene. It is known that enhancers can activate gene expression in the rage of about 3 kb from the transcription initiation site. Since the sTnT gene is only about 3 kb in size (Fig. 4), it is reasonable to consider whether other transcription regulatory elements exist within the sTnT gene. In a previous study, the intronic regulatory factor (fast intronic regulatory element [FIRE]), which is an important cis-acting element for fiber type-specific regulation, has been found in the quail fast skeletal TnI gene (Yutzey et al., 1989). In the sTnT gene, five E-box motifs and one CACC-box motif were additionally found (data not shown). In order to specify which regulatory elements in the upstream and intragenic regions contribute directly to the transcriptional regulation of the sTnT gene, further investigations using CAT and luciferase reporter genes are required. Our preliminary study using the upstream region sequentially deleted and combined with the CAT reporter gene showed that only a portion including E-box4 could enhance expression of the reporter gene in a myogenic cell line. It is conceivable that expression of trans-acting factors and alternative splicing factors are closely associated with muscle diversification into myofiber subtypes that have specific metabolic and contractile characteristics, because they direct muscle tissue-specific and developmentally regulated mRNA expression of muscular genes. It is not known what controls expression of these factors. It may be a regulatory protein(s) encoded by gene(s) subordinate to the MyoD family genes. Alternatively, it may be nerve stimulation or active muscle contraction. Future studies to isolate trans-acting and alternative splicing factors and identify a regulator(s) for their expression are needed in order to elucidate the mechanism of muscle diversification.

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