Characterization of a Cardiac-specific Enhancer, Which Directs α-Cardiac Actin Gene Transcription in the Mouse Adult Heart
2004; Elsevier BV; Volume: 279; Issue: 53 Linguagem: Inglês
10.1074/jbc.m411082200
ISSN1083-351X
AutoresMarguerite Lemonnier, Margaret Buckingham,
Tópico(s)Congenital heart defects research
ResumoExpression of the mouse α-cardiac actin gene in skeletal and cardiac muscle is regulated by enhancers lying 5′ to the proximal promoter. Here we report the characterization of a cardiac-specific enhancer located within -2.354/-1.36 kbp of the gene, which is active in cardiocytes but not in C2 skeletal muscle cells. In vivo it directs reporter gene expression to the adult heart, where the proximal promoter alone is inactive. An 85-bp region within the enhancer is highly conserved between human and mouse and contains a central AT-rich site, which is essential for enhancer activity. This site binds myocyte enhancer factor (MEF)2 factors, principally MEF2D and MEF2A in cardiocyte nuclear extracts. These results are discussed in the context of MEF2 activity and of the regulation of the α-cardiac actin locus. Expression of the mouse α-cardiac actin gene in skeletal and cardiac muscle is regulated by enhancers lying 5′ to the proximal promoter. Here we report the characterization of a cardiac-specific enhancer located within -2.354/-1.36 kbp of the gene, which is active in cardiocytes but not in C2 skeletal muscle cells. In vivo it directs reporter gene expression to the adult heart, where the proximal promoter alone is inactive. An 85-bp region within the enhancer is highly conserved between human and mouse and contains a central AT-rich site, which is essential for enhancer activity. This site binds myocyte enhancer factor (MEF)2 factors, principally MEF2D and MEF2A in cardiocyte nuclear extracts. These results are discussed in the context of MEF2 activity and of the regulation of the α-cardiac actin locus. α-Cardiac actin is a member of a highly conserved multigene family, found in all eukaryotic cells, of which the isoforms are differentially expressed in a tissue-specific and developmentally regulated manner (1Vandekerckhove J. Weber K. Differentiation. 1979; 14: 123-133Crossref PubMed Scopus (366) Google Scholar). In mouse, α-cardiac actin is expressed in the early myocardium and in somites from embryonic day 8.5. as the skeletal muscle of the myotome begins to form (2Sassoon D. Garner I. Buckingham M. Development (Camb.). 1988; 104: 155-164Crossref PubMed Google Scholar). After birth, the α-cardiac actin gene remains expressed at a high level in the heart and is down-regulated in skeletal muscles at the same time as α-skeletal actin remains expressed in skeletal muscle and is down-regulated in the heart (3Gunning P. Ponte P. Blau H.M. Kedes L. Mol. Cell. Biol. 1983; 3: 1985-1995Crossref PubMed Google Scholar, 4Buckingham M.E. Essays Biochem. 1985; 20: 77-109PubMed Google Scholar, 5Garner I. Sassoon D. Vandekerkhove J. Alonso S. Buckingham M.E. Dev. Biol. 1989; 134: 236-245Crossref PubMed Scopus (33) Google Scholar). Several lines of genetic evidence suggest that α-cardiac actin is essential for normal structure and function of adult cardiac myocytes. In BALB/c mice, a 5′-partial duplication of the α-cardiac actin gene is associated with the down-regulation of the α-cardiac actin mRNA and protein (6Garner I. Minty A.J. Alonso S. Barton P.J. Buckingham M.E. EMBO J. 1986; 5: 259-267Crossref PubMed Scopus (48) Google Scholar), leading to enhanced expression of α-skeletal actin mRNA and protein after birth through a compensatory mechanism (5Garner I. Sassoon D. Vandekerkhove J. Alonso S. Buckingham M.E. Dev. Biol. 1989; 134: 236-245Crossref PubMed Scopus (33) Google Scholar). However, the hearts of adult BALB/c mice present functional alterations with increased contractility (7Hewett T.E. Grupp I.L. Grupp G. Robbins J. Circ. Res. 1994; 74: 740-746Crossref PubMed Scopus (120) Google Scholar). When a null mutation is introduced into the α-cardiac actin gene, the mice either do not survive to term or die within 2 weeks of birth because of extensive loss of thin filaments and sarcomere disorganization leading to heart failure (8Kumar A. Crawford K. Close L. Madison M. Lorenz J. Doetchman T. Pawlowski S. Duffy J. Neumann J. Robbins J. Boivin G.P. O'Toole B.A. Lessard J.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4406-4411Crossref PubMed Scopus (174) Google Scholar). Attempts to rescue the deficient cardiocytes by transgenic expression of a non-cardiac actin in such mutant mice causes heart enlargement and dysfunction (8Kumar A. Crawford K. Close L. Madison M. Lorenz J. Doetchman T. Pawlowski S. Duffy J. Neumann J. Robbins J. Boivin G.P. O'Toole B.A. Lessard J.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4406-4411Crossref PubMed Scopus (174) Google Scholar) resembling human idiopathic dilated cardiomyopathy. In humans, missense mutations of the α-cardiac actin gene predispose affected cardiocytes to mechanical injury leading to idiopathic dilated cardiomyopathy (9Olson T.M. Michels V.V. Thibodeau S.N. Tai Y.S. Keating M.T. Science. 1998; 280: 750-752Crossref PubMed Scopus (593) Google Scholar) and eventual cell death. Taken together, these results indicate that both the type of actin expressed in the adult heart and the levels at which it is expressed are important for correct contractile function. Several regulatory sequences involved in the expression of the α-cardiac actin gene during muscle cell differentiation in vitro and the development of mammalian striated muscles in vivo have been identified. Transcriptional activation in differentiating skeletal muscle cells depends on critical sites in the proximal promoter of the human and mouse genes, notably a CArG-box binding serum response factor (10Sartorelli V. Webster K.A. Kedes L. Genes Dev. 1990; 4: 1811-1822Crossref PubMed Scopus (231) Google Scholar, 11Tuil D. Clergue N. Montarras D. Pinset C. Kahn A. Phan-Dinh-Tuy F. J. Mol. Biol. 1990; 213: 677-686Crossref PubMed Scopus (38) Google Scholar), an E-box binding myogenic regulatory factors (10Sartorelli V. Webster K.A. Kedes L. Genes Dev. 1990; 4: 1811-1822Crossref PubMed Scopus (231) Google Scholar, 11Tuil D. Clergue N. Montarras D. Pinset C. Kahn A. Phan-Dinh-Tuy F. J. Mol. Biol. 1990; 213: 677-686Crossref PubMed Scopus (38) Google Scholar, 12Skerjanc I.S. McBurney M.W. Dev. Biol. 1994; 163: 125-132Crossref PubMed Scopus (35) Google Scholar, 13Biesiada E. Hamamori Y. Kedes L. Sartorelli V. Mol. Cell. Biol. 1999; 19: 2577-2584Crossref PubMed Scopus (73) Google Scholar), and an SpI site (10Sartorelli V. Webster K.A. Kedes L. Genes Dev. 1990; 4: 1811-1822Crossref PubMed Scopus (231) Google Scholar, 13Biesiada E. Hamamori Y. Kedes L. Sartorelli V. Mol. Cell. Biol. 1999; 19: 2577-2584Crossref PubMed Scopus (73) Google Scholar). In cardiocyte cultures, the same sites seem to be involved (14Pari G. Jardine K. McBurney M.W. Mol. Cell. Biol. 1991; 11: 4796-4803Crossref PubMed Scopus (73) Google Scholar, 15Sartorelli V. Hong N.A. Bishopric N.H. Kedes L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4047-4051Crossref PubMed Scopus (71) Google Scholar), although the role of the E-box is controversial (12Skerjanc I.S. McBurney M.W. Dev. Biol. 1994; 163: 125-132Crossref PubMed Scopus (35) Google Scholar). Interestingly, it has been shown that the multiple CArG-boxes found in the α-cardiac actin promoters of several species (16Minty A. Kedes L. Mol. Cell. Biol. 1986; 6: 2125-2136Crossref PubMed Scopus (274) Google Scholar, 17Mohun T.J. Garrett N. Gurdon J.B. EMBO J. 1986; 5: 3185-3193Crossref PubMed Scopus (102) Google Scholar) are sites of serum response factor and Nkx-2.5 interaction (18Chen C.Y. Schwartz R.J. J. Biol. Chem. 1995; 270: 15628-15633Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar) acting to promote high levels of activity (19Chen C.Y. Schwartz R.J. Mol. Cell. Biol. 1996; 16: 6372-6384Crossref PubMed Google Scholar). However, previous observations on the BALB/c mouse line suggested that there might be other levels of regulation operating on this gene (6Garner I. Minty A.J. Alonso S. Barton P.J. Buckingham M.E. EMBO J. 1986; 5: 259-267Crossref PubMed Scopus (48) Google Scholar). Two DNase I hypersensitive sites (HSp 1The abbreviations used are: HSp, proximal DNase1 hypersensitive site; HSd, distal DNase1 hypersensitive site; MEF, myocyte enhancer factor; POX, proximal promoter of the α-cardiac actin gene; TkCAT, thymidine kinase promoter driving the chloramphenicol acetyltransferase gene; TkLacZ, thymidine kinase promoter driving the β-galactosidase gene; RSVLuc, Rous sarcoma virus long repeat driving the luciferase gene; MCK muscle creatine kinase; contig, group of overlapping clones; EnC, new enhancer region; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; n, nuclear localization sequence.1The abbreviations used are: HSp, proximal DNase1 hypersensitive site; HSd, distal DNase1 hypersensitive site; MEF, myocyte enhancer factor; POX, proximal promoter of the α-cardiac actin gene; TkCAT, thymidine kinase promoter driving the chloramphenicol acetyltransferase gene; TkLacZ, thymidine kinase promoter driving the β-galactosidase gene; RSVLuc, Rous sarcoma virus long repeat driving the luciferase gene; MCK muscle creatine kinase; contig, group of overlapping clones; EnC, new enhancer region; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; n, nuclear localization sequence. and HSd) were identified upstream of the mouse α-cardiac actin gene in the C2 skeletal muscle cell line. When these sequences where tested for activity in different cell lines with homologous and heterologous promoters they were shown to act as striated muscle-specific enhancers (20Biben C. Kirschbaum B. Garner I. Buckingham M. Mol. Cell. Biol. 1994; 14: 3504-3513Crossref PubMed Scopus (25) Google Scholar). Analysis of the more distal sequence, at ∼-7 kbp from the gene, showed that its activity depends on an E-box, a target of myogenic regulatory factors, and also on an AT-rich 3′ sequence, which binds the homeodomain protein Emb in interaction with MEF2D and p300 (21Molinari S. Relaix F. Lemonnier M. Kirschbaum B. Schafer B. Buckingham M. Mol. Cell. Biol. 2004; 24: 2944-2957Crossref PubMed Scopus (21) Google Scholar). This complex potentially plays a role in opening chromatin at the α-cardiac actin locus, rendering the E-box and downstream regulatory regions accessible; in transgenic mice when this sequence is present robust expression is seen (22Biben C. Hadchouel J. Tajbakhsh S. Buckingham M. Dev. Biol. 1996; 173: 200-212Crossref PubMed Scopus (50) Google Scholar). The proximal promoter itself is a weak regulatory element directing expression to embryonic skeletal muscle and to the embryonic heart in a few transgenic lines. The distal sequence HSd acts as a skeletal muscle enhancer in vivo. Expression in the adult heart is seen with transgenes containing ∼-5 kbp of α-cardiac actin DNA, which includes the proximal DNase I hypersensitive site, consistent with a role for this proximal enhancer region in the transcription of the gene in cardiac muscle. In this study, we have examined in more detail this aspect of α-cardiac actin expression. We report on a third enhancer located within -2.354 kbp of the gene that directs transgene expression in the adult heart but not in skeletal muscle. In the embryo, it directs expression in the skeletal muscle of the somites, as well as the heart. However, unlike the previously described distal and proximal enhancers, it is not active in the C2 skeletal muscle cell line derived from postnatal muscle but only in cardiocytes. Its activity depends on a MEF2 site. Reporter Constructs and Site-directed Mutagenesis—A 2.473-kbp BamHI fragment derived from the clone λIG 10 of the murine α-cardiac actin gene (6Garner I. Minty A.J. Alonso S. Barton P.J. Buckingham M.E. EMBO J. 1986; 5: 259-267Crossref PubMed Scopus (48) Google Scholar) containing the -2.354, +0.119-kbp region of the gene was inserted into the BamHI site of the promoterless plasmid, pBLCAT6 (23Boshart M. Klüppel M. Schmidt A. Schütz G. Luckow B. Gene (Amst.). 1992; 110: 129-130Crossref PubMed Scopus (230) Google Scholar) generating the construct EnC proximal promoter region (POX). Deletions in this sequence were generated either by using convenient restriction sites in the 5′ region or by using partial digestion. Fragments were also cloned into a CAT construct (20Biben C. Kirschbaum B. Garner I. Buckingham M. Mol. Cell. Biol. 1994; 14: 3504-3513Crossref PubMed Scopus (25) Google Scholar) in which the POX (-0.667, + 0.119 kbp) of the α-cardiac actin gene had been cloned into the promoterless plasmid pBLCAT6. Mutations of the MEF2 site were introduced separately, by site directed mutagenesis (transformer site-directed mutagenesis kit or QuikChange site-directed mutagenesis, Stratagene), in the -2.3- to -1.4-kbp region cloned in BlueScript. The two mutations substituted for the native MEF2 site were, respectively, TTTTCTACCCGGGACTGGT (m1) and TTTTCGATTCTTAACTGGT (m2). L. Bold characters indicate mutated nucleotides. Mutated DNA fragments were subsequently sequenced and transferred into POX (-0.669 kbp) or TkCAT. Multimerized sequences of the native or mutated α-cardiac actin MEF2 box were cloned into TkLacZ at a SfiI site introduced previously (provided by F. Relaix), and confirmed by sequencing. Cell Culture, DNA Transfection, and Assays—The C2/7 line, a subclone of the C2 cell line derived from the skeletal muscle of adult C3H mice, was grown as reported previously (24Catala F. Wanner R. Barton P. Cohen A. Wright W. Buckingham M.E. Mol. Cell. Biol. 1995; 15: 4585-4596Crossref PubMed Scopus (83) Google Scholar). For DNA transfection, the cells were cultured in 6-cm diameter dishes with 20% fetal calf serum and allowed to differentiate in 2% fetal calf serum. The embryonic mouse fibroblast cell line C3H10T1/2 was grown in 10% fetal calf serum (24Catala F. Wanner R. Barton P. Cohen A. Wright W. Buckingham M.E. Mol. Cell. Biol. 1995; 15: 4585-4596Crossref PubMed Scopus (83) Google Scholar). Ventricular cardiocytes were prepared from hearts of 18 embryonic day Wistar rat fetuses according to the protocol of (25Kelly R.G. Lemonnier M. Zaffran S. Munk A. Buckingham M.E. J. Cell Sci. 2003; 116: 5005-5013Crossref PubMed Scopus (7) Google Scholar). Hearts were trimmed of the atria and outflow tract, and the ventricular cells were dissociated under gentle shaking in 1× trypsin (T4674 Sigma) in ADS medium complemented with glucose. Trypsin was inhibited by adding decomplemented newborn calf serum (Invitrogen). Cells were recovered by centrifugation and kept at 37 °C in newborn calf serum. Fresh enzyme solution was added to the tissue until complete dissociation was reached. Cells were plated in 6-cm dishes in plating medium and grown in 5% CO2. Cardiocytes and myogenic cells were transfected by the calcium phosphate transfection technique (26Graham F.L. Van der Eb A.J. Virology. 1973; 52: 456-467Crossref PubMed Scopus (6490) Google Scholar). The embryonic fibroblast cell line, C3H10T1/2, was transfected with superfect (Qiagen) under the conditions described by the manufacturer. 9 μg of CAT reporter construct and 0.5 μg of a reporter gene containing the Rous sarcoma long terminal repeat linked to the luciferase gene (RSVluc) were added to each dish. Myotubes were rinsed in phosphate-buffered saline and collected after being maintained 48 h in Dulbecco's modified Eagle's medium supplemented with 2% fetal calf serum as reported elsewhere (24Catala F. Wanner R. Barton P. Cohen A. Wright W. Buckingham M.E. Mol. Cell. Biol. 1995; 15: 4585-4596Crossref PubMed Scopus (83) Google Scholar). Cardiocytes were rinsed with phosphate-buffered saline for 60 h following transfection, collected in 40 mm Tris-HCl (pH 7.5), 150 mm NaCl, and 1 mm EDTA, and lysed by three freeze-thaw cycles. Co-transfection was carried out with 15.5 μg of total DNA consisting of 0.5 μg of RSVLuc, 10 μg of a TkLacZ construct regulated by a concatamerized MEF2 site (x4) from the mouse muscle creatine kinase (MCK) or α-cardiac actin genes and 5 μg of MEF2A, MEF2C, or MEF2D expression vectors (27Martin J.F. Miano J.M. Hustad C.M. Copeland N.G. Jenkns N.A. Olson E.N. Mol. Cell. Biol. 1994; 14: 1647-1656Crossref PubMed Scopus (186) Google Scholar). As a control an empty expression vector was used. CAT activity was measured by the simple phase extraction procedure (28Seed B. Sheen J.Y. Gene (Amst.). 1988; 67: 271-277Crossref PubMed Scopus (830) Google Scholar), and luciferase activity was measured as described previously (20Biben C. Kirschbaum B. Garner I. Buckingham M. Mol. Cell. Biol. 1994; 14: 3504-3513Crossref PubMed Scopus (25) Google Scholar). β-Galactosidase activity was measured with a galactolight kit (Tropix). The resulting CAT activity of the different constructs was normalized to the corresponding luciferase activity. All transfections were carried out in duplicate, and three different DNA preparations of each construct were tested. Preparation of Nuclear Extracts and Gel Mobility Shift Assays—Nuclear extracts from ventricles of adult rat hearts were prepared according to the procedure of Mar and Ordahl (29Mar J.H. Ordahl C.P. Mol. Cel. Biol. 1990; 10: 4271-4283Crossref PubMed Scopus (148) Google Scholar) with slight modifications. Hearts were trimmed of the atria and outflow tract, washed in sterile phosphate-buffered saline, and homogenized in a Tissue Mizer for 12 s at half-power in homogenization buffer (10 mm Hepes, pH 7.6, 25 mm KCl, 1 mm EDTA, 0.15 mm spermine, 0.5 mm spermidine, 0.4 mm phenylmethylsulfonyl fluoride, 1.8 m sucrose, 5% glycerol), with added protease inhibitor mixture (Roche Applied Science, number 1697498). Liberation of nuclei was checked with trypan blue dye, and the homogenate was centrifuged on a pad of homogenization buffer at 25K for 60 min at 4 °C. Nuclei were transferred to a Dounce homogenizer in lysis buffer (10 mm Hepes, pH 7.6, 100 mm KCl, 0.1 mm EDTA, 3 mm MgCl2, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, 10% glycerol), with added protease inhibitor mixture. Following 6 strokes with an A pestle, NaCl was added to a 0.5 m final volume. The homogenate was left for 30 min on a slow rotating table and then spun for 60 min at 35,000 rpm. The supernatant was dialyzed against 25 mm Hepes, pH 7.6, 100 mm KCl, 0.1 mm EDTA, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, and 10% glycerol, with added protease inhibitor mixture, and stored in liquid nitrogen. Protein concentration was determined using the Bio-Rad protein assay. Gel mobility shift assays were performed with a 10-μl reaction volume containing 1 μg of poly(dI-dC) (Amersham Biosciences), 1 μl of buffer (50 mm Tris-HCl, pH 8, 5 mm EDTA, 5 mm dithiothreitol, 250 mm NaCl, and 10% Ficoll), 0.5 ng of labeled DNA probe, and 3-6 μg of protein in crude nuclear extracts (24Catala F. Wanner R. Barton P. Cohen A. Wright W. Buckingham M.E. Mol. Cell. Biol. 1995; 15: 4585-4596Crossref PubMed Scopus (83) Google Scholar). The assay mixtures were incubated for 20 min at room temperature and run on 5% polyacrylamide gels (acrylamide/bisacrylamide ratio 49/1). The nucleotide sequences of the sense strand of probe and competitor DNAs used in the binding assays were as follows, α-cardiac actin, 5′-CAGTTTTCTATTTTTAACTGGTGTG; α-cardiac actin m2, 5′-CAGTTTTCGATTCTTAACTGGTGTG; MEF2 MCK, 5′-GCTCGCTCTAAAAATAACCCTGTC. Where indicated, antibodies or competing oligonucleotides (20-100-fold molar excess) were added to the incubation 10 min before adding the probe. Polyclonal preimmune and anti-MEF2 antibody were obtained from Santa Cruz Biotechnology. Polyclonal antibodies to the three MEF2 isoforms (MEF2A, MEF2C, and MEF2D) (30Han T.H. Prywes R. Mol. Cell. Biol. 1995; 15: 2907-2915Crossref PubMed Scopus (167) Google Scholar) were used at a 1:20 final dilution. The mixture was directly loaded on a 5% polyacrylamide gel in 0.5× Tris-buffered EDTA, prerun for 1 h. Then the gels were fixed, dried in a vacuum desiccator, and exposed to x-ray film overnight. Generation of Transgenic Mice, Screening of the Transgenic Lines, and Histochemical Staining for β-Galactosidase—The 2.4-kbp BamHI fragment was inserted at the BamHI site of pAC1SDKnLacZ (n represents a nuclear localization sequence) (22Biben C. Hadchouel J. Tajbakhsh S. Buckingham M. Dev. Biol. 1996; 173: 200-212Crossref PubMed Scopus (50) Google Scholar). The insert was extracted by cutting with SacII and XhoI, eluted, purified, resuspended, and injected as described previously (22Biben C. Hadchouel J. Tajbakhsh S. Buckingham M. Dev. Biol. 1996; 173: 200-212Crossref PubMed Scopus (50) Google Scholar). A 0.5-cm tail biopsy was digested, and DNA was prepared as reported before (22Biben C. Hadchouel J. Tajbakhsh S. Buckingham M. Dev. Biol. 1996; 173: 200-212Crossref PubMed Scopus (50) Google Scholar). In the case of founder animals, ∼10 μg of DNA were digested with EcoRI, separated in an 0.8% agarose gel and transferred onto a Hybond N+ membrane, which was treated according to standard procedures (31Maniatis T. Sambrook J. Fritsch E.F. Molecular Cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 52-55Google Scholar). Probes (recognizing the β-galactosidase sequence or 5′ regions of the α-cardiac actin gene) were labeled using Amersham Biosciences Megaprime RPN 1606 and purified on a Chromaspin 100 column. Filters were hybridized in 50 mm Na2HPO4, pH 7.6, 5× SSC, 5× Denhardt's solution, 0.1% SDS, 100 μg/ml salmon sperm DNA, and washed in 0.1× SSC, 0.1% SDS at 65 °C. Transgenic litters were detected by amplification of a 0.9-kbp fragment encompassing the 3′-terminal of the α-cardiac actin promoter and the 5′-end of the nLacZ gene using the primers α-cardiac actin, 5′-GCTGCTCCAACTGACCCCGTCCATCAGAGAG and nLacZ, 5′-CGCATCGTAACCGTGCATCTGCCAGTTTGAG. The reaction was carried out for 25 cycles (94° 30 s, 57° 1 min, 72° 45 s) followed by 1 cycle of 72° for 10 min in 30 μl o f 1× PCR buffer, 2.5 mm MgCl2, 0.2 mm dNTP mix, 0.5 μm primer mix, 1 unit of Taq DNA polymerase (Invitrogen). Embryos or organs were dissected, fixed (22Biben C. Hadchouel J. Tajbakhsh S. Buckingham M. Dev. Biol. 1996; 173: 200-212Crossref PubMed Scopus (50) Google Scholar), and stained (32Sanes J.R. Rubenstein J.L.R. Nicolas J-F. EMBO J. 1986; 5: 3133-3142Crossref PubMed Scopus (944) Google Scholar) for various lengths of time. Thee copy number was estimated by Southern blotting (22Biben C. Hadchouel J. Tajbakhsh S. Buckingham M. Dev. Biol. 1996; 173: 200-212Crossref PubMed Scopus (50) Google Scholar). The transgenic line shown here had a copy number of 2-3. Comparison of Mouse with Human Genomic Sequences—A 150-kbp contig of human genomic sequence was extracted from the human genome data base using the promoter sequence of the human α-cardiac actin gene (16Minty A. Kedes L. Mol. Cell. Biol. 1986; 6: 2125-2136Crossref PubMed Scopus (274) Google Scholar). Different alignments of parts of this sequences with the 2-kbp cognate sequence of the mouse gene were performed. Two methods were used with the DNA Strider TM 1.3f8 program. DNA matrix analysis was performed with variable windows and stringencies. DNA alignment was performed by the blocks method with variable mismatch and gap penalties. Identification of a Cardiac Muscle Enhancer—The 5′-flanking region of the mouse α-cardiac actin gene was re-examined for cardiac as well as skeletal muscle regulatory elements by transfection experiments in primary cultures of cardiac muscle cells (cardiocytes), as well as the C2 skeletal muscle cell line, previously used to identify muscle enhancers (20Biben C. Kirschbaum B. Garner I. Buckingham M. Mol. Cell. Biol. 1994; 14: 3504-3513Crossref PubMed Scopus (25) Google Scholar). The POX of -770 bp is active in both cell types but is a weak regulatory element in vivo where it does not direct transgene expression to the adult heart. The activity of the CAT reporter gene driven by POX was taken as 1 to compare the effect of upstream sequences, cloned in front of the POX transgene (Fig. 1). The distal enhancer region (HSd), which is active in differentiated skeletal muscle myotubes, shows very low activity in cardiocytes. The proximal enhancer, HSp, is active in cardiocytes as well as in C2/7 myotubes. In this deletion analysis a new enhancer region (EnC), which is active only in cardiocytes, is now identified in the -2.354- to -0.669-kbp region immediately 5′ of the proximal promoter. We had previously shown that ∼-5.7 kbp of α-cardiac actin upstream sequence (construction T3, (22Biben C. Hadchouel J. Tajbakhsh S. Buckingham M. Dev. Biol. 1996; 173: 200-212Crossref PubMed Scopus (50) Google Scholar)) contained the necessary regulatory elements to direct nlacZ transgene expression to the adult heart. We how tested whether -2.354 kbp was sufficient for this transcriptional activity in vivo. Of four founder mice, one had a rearranged transgene, as shown by Southern blotting, and one was infertile, the two others gave rise to transgenic lines, which expressed the nlacZ transgene in the adult heart. In one case β-galactosidase activity was patchy in both atria and the ventricles, with prior expression in the embryonic heart also (results not shown); this expression was lost in subsequent generations. In the other line, all aspects of transgene expression were stable over the four generations examined. Between embryos/mice within a single litter, the pattern was also maintained, with minor variations in the extent of labeling in the right ventricle. Strong expression was seen in the embryonic (Fig. 2, a and b) and adult (Fig. 2, c and d) heart. In the adult both atria show intense X-gal staining. Most of the left ventricular myocardium is labeled. Part of the right ventricle is positive, with negative areas of myocardium ventrally. This was also the case at birth and during postnatal development (3 weeks, 3 months, results not shown). This is similar to the expression profile of the T3 transgenic lines, where -5.746 kbp, which included the HSp and EnC enhancers, was driving the transgene. The results shown here demonstrate that the EnC with the POX promoter is sufficient for expression in the adult heart. Expression in somites, in the skeletal muscle of the myotome, is also seen with this transgene in the strongly expressing line (Fig. 2, a and b). This is probably because of the proximal promoter, which can direct expression in embryonic muscle but not in the adult heart (22Biben C. Hadchouel J. Tajbakhsh S. Buckingham M. Dev. Biol. 1996; 173: 200-212Crossref PubMed Scopus (50) Google Scholar). No expression was detected in postnatal (3 weeks) or adult skeletal muscles (results not shown). Sequence Conservation in the 5′-Flanking Region of the α-Cardiac Actin Gene—Comparison of DNA genomic sequences located 8.6 kbp 5′ to the CAP site of the mouse and human α-cardiac actin genes was carried out using the DNA matrix program (Fig. 3a). A genomic contig of 150 kbp was identified in which the human gene was located. The sequence comparison shows regions of homology at ∼-8 to -7 kbp and -5 to -4 kbp, where the HSd and HSp sites are located, respectively. It also identifies a conserved region lying within -2 kbp from the CAP site potentially corresponding to the new enhancer region (EnC), in addition to sequences in the POX, which have been shown previously to play a role in its activity (16Minty A. Kedes L. Mol. Cell. Biol. 1986; 6: 2125-2136Crossref PubMed Scopus (274) Google Scholar). The mouse -2 kbp fragment does not display significant homology with unrelated fragments of the human contig, using the matrix program (results not shown). Similar results were obtained using a larger (15 kbp) fragment of human genomic DNA (results not shown). We then focused on the conserved region at about -1.4 kbp from the CAP site. This relative location is conserved between human and mouse, and the homology is maintained at high stringency. Using the blocks method of Martinez (51Martinez H.M. Nucleic Acids Res. 1983; 11: 4629-4634Crossref PubMed Scopus (93) Google Scholar), DNA alignment shows that the conserved sequence is located between -1.432 and -1.350 kbp from the CAP site, with 85% identity and two gaps, and with most mismatches located at the extremities of the region (Fig. 3b). The functional characteristics of the conserved 82-bp sequence were tested by transfection experiments in primary cardiocyte cultures, in myotubes of the skeletal muscle C2/7 cell line and in the C3H 10T1/2 embryonic fibroblast line (Fig. 4). Upstream sequences were introduced in front of the POX directing a CAT reporter sequence. The activity of POX in the different cell cultures was taken as 1. As reported in Fig. 1 the complete -2.354-kbp sequence upstream of the α-cardiac actin CAP site was active in cardiocytes only. A series of deletions demonstrate that activity in cardiocytes depends on sequences lying 5′ to the SpeI site at -1.35 kbp from the gene. The BamHI/SpeI fragment (-2.354 to -1.364 kbp) was cloned in front of the Tk promoter driving the CAT reporter and shown to be active in cardiocytes, with some activity also in C2/7 myotubes (Table I). Activity in cardiac muscle cells was retained in the reverse orientation, and we therefore concluded that the sequence acts as an enhancer. Full activity is retained with a subfragment, which extends from the SpeI site to the HincII site a further 500 bp upstream (Fig. 4). The highly conserved 85-bp region alone does not exert more than about 2-fold activity, with incremental rises in activity as sequences extending to the HincII site are included. Between the HincII and the XbaI site, there is an Ebox, and its mutation results in some loss of activity, but we have not identified any single site that is essential for activity in contrast to the MEF2 site, which is essential for activity in the context of the larger fragment (BamHI/SpeI fragment). There are potential sites for cardiac regulators, some of which had a minor effect on activity.Table IThe MEF2 site is required for enhancer activityCardiocytesC2/7 MyotubesFibroblastsTkCAT11ND((S)/B-S) sense TkCAT17.15.5ND(S
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