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• W2058693181 abstract "The B creatine kinase gene is regulated by an array of positive and negative cis-elements in the 5′-flanking DNA that function in both muscle and nonmuscle cells. In C2C12 myogenic cells M and B creatine kinase mRNAs are coordinately up-regulated in the early stages of myogenesis and then undergo distinct regulatory programs. The B creatine kinase gene is down-regulated in the late stages of myogenesis as M creatine kinase becomes the predominant species in mature myotubes. Sequences between −92 and +80 of the B creatine kinase gene confer a regulated pattern of expression to chimeric plasmids that closely resembles the time course of expression of the endogenous B creatine kinase gene in C2C12 cells undergoing differentiation. We show that sequences within the first exon of the B creatine kinase gene are important for the developmental regulation of the gene in C2C12 cells and that these sequences bind a nuclear protein that shows a similar tissue-specific distribution and developmentally regulated expression to that of the endogenous B creatine kinase gene. The B creatine kinase gene is regulated by an array of positive and negative cis-elements in the 5′-flanking DNA that function in both muscle and nonmuscle cells. In C2C12 myogenic cells M and B creatine kinase mRNAs are coordinately up-regulated in the early stages of myogenesis and then undergo distinct regulatory programs. The B creatine kinase gene is down-regulated in the late stages of myogenesis as M creatine kinase becomes the predominant species in mature myotubes. Sequences between −92 and +80 of the B creatine kinase gene confer a regulated pattern of expression to chimeric plasmids that closely resembles the time course of expression of the endogenous B creatine kinase gene in C2C12 cells undergoing differentiation. We show that sequences within the first exon of the B creatine kinase gene are important for the developmental regulation of the gene in C2C12 cells and that these sequences bind a nuclear protein that shows a similar tissue-specific distribution and developmentally regulated expression to that of the endogenous B creatine kinase gene. Creatine kinase (CK)1( 1The abbreviations used are: CKcreatine kinaseCATchloramphenicol acetyltransferase. 1The abbreviations used are: CKcreatine kinaseCATchloramphenicol acetyltransferase.)catalyzes the reversible phosphorylation of ADP and creatine and is important in the regulation and maintenance of cellular energy metabolism. The M and B creatine kinase genes encode highly homologous M and B subunit proteins that associate in the cytoplasm to form three dimeric cytoplasmic isoenzymes (MM, MB, and BB). B CK is expressed in many tissues, is regulated by steroid hormones(1Reiss N.A. Kaye A.M. J. Biol. Chem. 1981; 256: 5741-5749Abstract Full Text PDF PubMed Google Scholar, 2Somjen D. Weisman Y. Binderman I. Kaye A.M. Biochem. J. 1984; 219: 1037-1041Crossref PubMed Scopus (36) Google Scholar), and undergoes developmental regulation in the lens of the eye(3Friedman D.L. Hejtmancik J.F. Hope J.N. Perryman M.B. Exp. Eye Res. 1989; 49: 445-457Crossref PubMed Scopus (12) Google Scholar), in cartilage(4Shapiro I.M. Debolt K. Funanage V.L. Smith S.M. Tuan R.S. J. Bone Miner. Res. 1992; 7: 493-500Crossref PubMed Scopus (21) Google Scholar), and in osteoblastic cells in culture(5Ch'ng J.L.C. Ibrahim B. J. Biol. Chem. 1994; 269: 2336-2341Abstract Full Text PDF PubMed Google Scholar). B CK is a marker for certain histologic types of lung cancer (6Kaye F.J. McBride O.W. Battey J.F. Gazdar A.F. Sausville E.A. J. Clin. Invest. 1987; 79: 1412-1420Crossref PubMed Scopus (23) Google Scholar) and for brain damage(7Chandler W.L. Clayson K.J. Longstreth Jr., W.T. Fine J.S. Clin. Chem. 1984; 30: 1804-1806Crossref PubMed Scopus (31) Google Scholar, 8Pfeiffer F.E. Homburger H.A. Yanagihara T. Arch. Neurol. 1984; 41: 1175-1178Crossref PubMed Scopus (19) Google Scholar). B-containing isoenzymes (MB and BB) increase in skeletal muscle during chronic exercise training (9Apple F.S. Rogers M.A. Casal D.C. Sherman W.M. Ivy J.L. J. Appl. Physiol. 1985; 59: 149-153Crossref PubMed Scopus (67) Google Scholar) and in the myocardium in response to hypertrophy, acute myocardial ischemia, and heart failure (10Ingwall J.S. Circulation. 1993; 87: VII-58-VII-62Google Scholar, 11Sharkey S.W. Elsperger K.J. Murakami M. Apple F.S. Am. J. Physiol. 1989; 256: H508-H514PubMed Google Scholar, 12Sharkey S.W. Murakami M.M. Smith S.A. Apple F.S. Circulation. 1991; 84: 333-340Crossref PubMed Scopus (31) Google Scholar) in adaptation to conditions of decreased energy reserve(10Ingwall J.S. Circulation. 1993; 87: VII-58-VII-62Google Scholar). creatine kinase chloramphenicol acetyltransferase. creatine kinase chloramphenicol acetyltransferase. Like other multigene families expressed in muscle the CK genes undergo an isoenzyme switch during development(13Eppenberger H.M. Eppenberger M. Richterich R. Aebi H. Dev. Biol. 1964; 10: 1-16Crossref PubMed Scopus (160) Google Scholar, 14Ingwall J.S. Kramer M.F. Friedman W.F. Jacobus W.E. Ingwall J.S. Developmental Changes in Heart Creatine Kinase. Williams & Wilkins, Baltimore1979: 9-17Google Scholar, 15Lough J. Bischoff R. Dev. Biol. 1977; 57: 330-344Crossref PubMed Scopus (55) Google Scholar, 16Rosenberg U.B. Eppenberger H.M. Perriard J.C. Eur. J. Biochem. 1981; 116: 87-92Crossref PubMed Scopus (34) Google Scholar). B CK is expressed in immature proliferating myogenic cells (myoblasts). Muscle differentiation is characterized by down-regulation of the B gene, which is switched off during myogenesis, and induction of M CK, which becomes the major isoform present in both cardiac and skeletal muscle (17, 18). With the use of M- and B- specific cDNA probes we showed that coordinate up-regulation of M and B mRNA occurs in the early stages of myogenesis in C2C12 cells (19Ritchie M.E. Trask R.V. Fontanet H.L. Billadello J.J. Nucleic Acids Res. 1991; 19: 6231-6240Crossref PubMed Scopus (21) Google Scholar) and in the developing heart (20Trask R.V. Billadello J.J. Biochim. Biophys. Acta. 1990; 1049: 182-188Crossref PubMed Scopus (89) Google Scholar) prior to down-regulation of B mRNA during the final stages of differentiation as M mRNA becomes the predominant species. This fetal pattern of expression of the CK genes is recapitulated in heart in response to acute pressure overload(21Fontanet H.L. Trask R.V. Haas R.C. Strauss A.W. Abendschein D.R. Billadello J.J. Circ. Res. 1991; 68: 1007-1012Crossref PubMed Scopus (38) Google Scholar). Interestingly, the developmental expression of B CK mRNA is very similar to that of cardiac actin mRNA, which is also up-regulated in the early stages of differentiation of C2C12 cells and down-regulated as it is replaced by skeletal actin mRNA in the late stages of myogenesis(22Bains W. Ponte P. Blau H. Kedes L. Mol. Cell. Biol. 1984; 4: 1449-1453Crossref PubMed Scopus (127) Google Scholar). Thus, the pattern of expression of certain fetal isoforms such as B CK and cardiac actin may represent an evolutionarily conserved developmental program in muscle. This program is different from that of other fetal isoforms such as β and γ actin, which are expressed in myoblasts and are down-regulated during all subsequent stages of myogenesis(23Bergsma D. Hayward L. Grichnik J. Schwartz R. Molecular Biology of Muscle Development. Alan R. Liss, Inc., 1986: 531-546Google Scholar). We showed that the human B CK gene is regulated by an array of positive and negative cis-elements in the 5′-flanking DNA that function in both muscle and nonmuscle cells and that sequences between −92 and +80 confer expression to chimeric plasmids that resembles that of the endogenous B CK gene in C2C12 cells undergoing differentiation(19Ritchie M.E. Trask R.V. Fontanet H.L. Billadello J.J. Nucleic Acids Res. 1991; 19: 6231-6240Crossref PubMed Scopus (21) Google Scholar). We now show that sequences within the first exon of the B CK gene are important for the developmental regulation of the gene in C2C12 cells and that these sequences bind a nuclear protein that may play a role in the developmentally regulated expression of the B CK gene. The plasmid BCKCAT92 (Fig. 1A) that contains 92 base pairs of 5′-flanking DNA, the first exon (untranslated), and the first 12 base pairs of the first intron of the human B CK gene inserted in the HindIII site of pSVOCAT was described previously(19Ritchie M.E. Trask R.V. Fontanet H.L. Billadello J.J. Nucleic Acids Res. 1991; 19: 6231-6240Crossref PubMed Scopus (21) Google Scholar). To determine whether sequences within the first exon and first intron are important for expression of BCKCAT92 we prepared BCKCAT92del (+3 to +80), a construct that contains the first 92 base pairs of upstream DNA and the cap site (Fig. 1B). This construct was prepared by synthesizing oligonucleotides representing the sense and antisense strands of the sequence from −92 to +2 designed to reconstitute HindIII-compatible ends after annealing to facilitate subcloning into the HindIII site of pSVOCAT. We also prepared a 3′-deletion series of constructs with identical 5′-ends containing sequences from −92 to +2 and different 3′-ends designed to delete select sequences from the first exon and intron of the B CK gene (Fig. 1, C-F). The constructs were prepared by ligating annealed double-stranded oligonucleotides representing the desired sequences into the HindIII site of pSVOCAT(24Hawker K.J. Billadello J.J. BioTechniques. 1993; 14: 764-765PubMed Google Scholar). All plasmids were sequenced in both strands to ensure a single copy of the desired insert was present in the correct orientation(25Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4767-4771Crossref PubMed Scopus (1677) Google Scholar). Because the neomycin resistance (neo) gene encodes a very stable transcript (26Schuler G.D. Cole M.D. Cell. 1988; 55: 1115-1122Abstract Full Text PDF PubMed Scopus (154) Google Scholar) we prepared BCKneo reporter plasmids to facilitate detection of transcripts in transiently transfected cells. BCKCAT92 and BCKCAT92del(+3 to +80) were digested completely with BamHI and then partially with HindIII to release the chloramphenicol acetyltransferase (CAT) gene from the plasmid. The neo gene obtained by digesting pSV2neo with BamHI and HindIII was ligated to BamHI/HindIII-digested BCKCAT92 and BCKCAT92del (+3 to +80) resulting in the constructs BCKneo92 and BCKneo92del (+3 to +80). The murine skeletal muscle cell line C2C12 (ATCC #CRL1772) was maintained in an atmosphere of 8% CO2, 92% air in growth medium (Dulbecco's modified Eagle's medium) supplemented with 20% fetal calf serum, penicillin (50 μg/ml), and streptomycin (50 μg/ml)). Differentiation medium was Dulbecco's modified Eagle's medium supplemented with 10% horse serum and antibiotics. C2C12 cells were plated 24 h before transfection at a density of 3.5 × 105 cells/60-mm dish in 3 ml of growth medium. Transfections were performed by the calcium phosphate coprecipitation method(19Ritchie M.E. Trask R.V. Fontanet H.L. Billadello J.J. Nucleic Acids Res. 1991; 19: 6231-6240Crossref PubMed Scopus (21) Google Scholar). Precipitates contained a total of 20 μg of DNA comprised of 15 μg of test plasmid and 5 μg of pMSVβgal as an internal standard to correct for transfection efficiency. After a 4-h incubation with the precipitate, cells were subjected to a 3-min glycerol shock and harvested 24-48 h later. Other dishes of cells were fed with 3 ml of differentiation medium after the transfected cells had become fully confluent and harvested 60 h later as fully differentiated myotubes. Cell extracts were prepared, and assays for β-galactosidase and chloramphenicol acetyltransferase were performed as described previously(19Ritchie M.E. Trask R.V. Fontanet H.L. Billadello J.J. Nucleic Acids Res. 1991; 19: 6231-6240Crossref PubMed Scopus (21) Google Scholar). The amount of extract used for chloramphenicol acetyltransferase assays was based on the results of the β-galactosidase assay and contained 25-100 μg of protein. The assays were terminated after 60 min. C2C12 cells plated as described above were transfected with 15 μg of either BCKneo92 or BCKneo92del(+3 to +80) and 5 μg of pCMVCAT as an internal standard to control for transfection efficiency. The cells were harvested 28 h after transfection, and Poly(A)+ mRNA was purified directly from transfected cells with the use of a Micro-FastTrack mRNA isolation kit (Invitrogen). Northern blots were prepared with 7 μg of Poly(A)+ mRNA and Nytran membranes (Schleicher and Schuell) as recommended by the supplier. The probes were a 1321-base pair HindIII/SmaI fragment of the neo gene and a 550-base pair HindIII/NcoI fragment of the CAT gene radiolabeled with [32P]dCTP (Amersham Corp.) as described(19Ritchie M.E. Trask R.V. Fontanet H.L. Billadello J.J. Nucleic Acids Res. 1991; 19: 6231-6240Crossref PubMed Scopus (21) Google Scholar). Autoradiograms prepared with Kodak XAR film and Cronex intensifying screens were analyzed with an LKB Ultroscan XL laser densitometer. Extracts were prepared from C2C12 cells and tissues by a modification of the method described by Heberlein et al.(27Heberlein U. Tjian R. Nature. 1988; 331: 410-415Crossref PubMed Scopus (74) Google Scholar). The protein concentration was determined by the method of Bradford(28Bradford M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211941) Google Scholar). A DNA fragment comprising the first exon and first 13 base pairs of the first intron of the B CK gene was used as the template in a polymerase chain reaction with primers designed to include bases +1 to 19, the reverse complement of +64 to +80, and with [32P]dCTP at a final concentration of 0.32 μM(29Sandler M.A. Zhang J.-N. Westerhausen Jr., D.R. Billadello J.J. J. Biol. Chem. 1994; 269: 21500-21504Abstract Full Text PDF PubMed Google Scholar). Gel mobility shift assays were performed as described previously (30Trask R.V. Koster J.C. Ritchie M.E. Billadello J.J. Nucleic Acids Res. 1992; 20: 2313-2320Crossref PubMed Scopus (13) Google Scholar) with 1.0 × 104 dpm of double-stranded 32P-labeled probe and 10 μg of nuclear protein extract. Nuclear extract was incubated with 19.2 milliunits of potato acid phosphatase (Sigma) or with buffer (50 mM Tris HCl, pH = 9.3, 1.0 mM MgCl2, 0.1 mM ZnCl2, and 1.0 mM spermidine) and 2.0 units of calf intestinal alkaline phosphatase (Promega) for 30 min at 37°C. After phosphatase treatment 32P-body-labeled probe was added to the extract, and gel mobility shift assays were performed. We showed that sequences within −92 to +80 of the B CK gene confer a regulated pattern of expression to CAT reporter plasmids in C2C12 cells that resembles the time course of expression of B CK mRNA(19Ritchie M.E. Trask R.V. Fontanet H.L. Billadello J.J. Nucleic Acids Res. 1991; 19: 6231-6240Crossref PubMed Scopus (21) Google Scholar). The plasmid BCKCAT92 (Fig. 1A) showed peak expression 24-48 h after transfection and was not expressed above background in fully differentiated myotubes. In nonmyogenic cells that express B CK (HeLa cells, Hep G2 cells, and NIH3T3 cells) BCKCAT92 expression was above background 48-108 h after transfection(19Ritchie M.E. Trask R.V. Fontanet H.L. Billadello J.J. Nucleic Acids Res. 1991; 19: 6231-6240Crossref PubMed Scopus (21) Google Scholar). These results show that the decrease in expression of BCKCAT92 in C2C12 cells is due to differentiation and does not simply reflect the time course of expression of a plasmid in a transient transfection experiment. To determine the importance of exonic and intronic sequences for expression of BCKCAT92 in C2C12 cells we prepared a construct in which the exonic and intronic sequences were deleted (BCKCAT92del (+3 to +80), Fig. 1B). When compared to the expression of BCKCAT92, which was approximately 5-fold above background in myoblasts, deletion of exon I and intron I sequences from BCKCAT92 resulted in a plasmid that was not expressed above background in either myoblasts or myotubes (Fig. 1B). These results were unexpected and showed that sequences from +2 to +80 were critical for expression of BCKCAT92. Accordingly, to determine the sequences from +2 to +80 that regulate expression of BCKCAT92 we prepared a 3′-deletion series (Fig. 1, C-F). Deletion of sequences from +26 to +80 had no effect on expression of the resultant plasmid (Fig. 1, C-D). Although expression of the plasmid BCKCAT92del (+26 to +80) was higher than that of BCKCAT92 (Fig. 1, A and D) the difference was not statistically significant (Student's t test). However, deletion of sequences from +18 to +25 resulted in a construct that was not expressed above background in myoblasts or myotubes (Fig. 1, E and F). In contrast, the plasmid MCKCAT2620 that contains the human M CK gene enhancer (30Trask R.V. Koster J.C. Ritchie M.E. Billadello J.J. Nucleic Acids Res. 1992; 20: 2313-2320Crossref PubMed Scopus (13) Google Scholar) was inactive in myoblasts and was expressed 19-fold above background in fully differentiated myotubes (Fig. 1G). These results show that sequences from +18 to +25 are important for expression of BCKCAT chimeric plasmids in C2C12 myoblasts. BCKCAT mRNA transcripts derived from the constructs shown in Fig. 1, A-F are different. This suggests expression may vary with translational efficiency of BCKCAT mRNAs. Accordingly, we sought to determine whether the observed difference in CAT activity in cells transfected with these constructs correlated with CAT mRNA levels. We performed Northern blot analysis of mRNA extracted from C2C12 cells transfected with BCKCAT92 and BCKCAT92del(+3 to +80). Because CAT mRNA was not detectable with this technique we prepared constructs in which the CAT gene was replaced with the neo gene, which encodes a more stable transcript. C2C12 cells were transfected with BCKneo92 and BCKneo92del(+3 to +80), and neo mRNA transcripts were analyzed by Northern blot hybridization. The steady-state level of neo mRNA directed by BCKneo92 was 9-fold greater than that of BCKneo92del(+3 to +80) when normalized to CAT mRNA encoded by the internal standard pCMVCAT (Fig. 2). These results show that sequences within B CK exon I modulate expression of chimeric plasmids at the level of mRNA accumulation. Effects at the level of transcription as well as mRNA processing and stability are all formal possibilities. To characterize the trans-acting factors that interact with sequences within the first exon of the B CK gene we prepared nuclear extracts from C2C12 myoblasts and myotubes at select developmental stages and performed gel mobility shift assays. We were particularly interested in factors that are expressed in myoblasts and are down-regulated with differentiation that could be important mediators of B CK gene expression. A representative result from two independent preparations of extracts is shown in Fig. 3. Three nuclear protein-DNA complexes are depicted with arrows. Complex 1 exhibited marked down-regulation with differentiation. Complex 2 was not consistently detected in different preparations of extract tested, and complex 3 was not regulated with differentiation. These results identify a nuclear protein complex that is expressed in myoblasts and is down-regulated with differentiation resembling the expression of B CK in myogenic cells in culture. To determine whether the DNA-protein complexes shown in Fig. 3 represented the specific interaction of nuclear proteins with sequences within B CK exon I shown to be important for expression of chimeric plasmids in transfected cells, additional gel mobility shift experiments were performed with competitor DNA (Fig. 4). Unlabeled DNA including sequences from +1 to +25 was added to the gel mobility shift reaction in molar excess to 32P-labeled exon I probe. The cold DNA competed with the probe for binding to nuclear protein present in complex 1 but not complex 3 (Fig. 4, lanes 1-4). In comparison, unlabeled DNA that included sequences from +1 to +17 did not compete with the probe for binding to complex 1 (Fig. 4, lanes 5-7). These results show that sequences from +18 to +25 that are critical for expression of chimeric plasmids in transfected cells also represent the binding site for complex 1. We used gel mobility shift assays to evaluate the expression of protein 1 in select tissues. We prepared nuclear extracts from brain, a tissue in which B CK is expressed, and from heart and skeletal muscle, tissues in which the CK genes undergo developmental regulation(20Trask R.V. Billadello J.J. Biochim. Biophys. Acta. 1990; 1049: 182-188Crossref PubMed Scopus (89) Google Scholar, 31Seraydarian M.W. Yamada T. Adv. Exp. Med. Biol. 1986; 194: 41-53Crossref PubMed Scopus (4) Google Scholar). The results showed that expression of protein 1 was abundant in brain (Fig. 5, lane BR) and expressed at a lower level in heart, a tissue in which the MB isoenzyme of CK is found and in which B CK mRNA is present (Fig. 5, lane H). Mature skeletal muscle, which expresses only trace amounts of B mRNA, did not show detectable amounts of protein 1 (Fig. 5, lane M). These results show that the expression of protein 1 in the tissues evaluated correlated well with that of B CK mRNA. In contrast, band 3 was detected in all tissues. In skeletal muscle and heart, B CK mRNA is down-regulated with development. To determine whether the developmental regulation of protein 1 in heart and skeletal muscle resembled that of B CK mRNA, we prepared nuclear protein extracts from tissues obtained from 1-day-old neonatal mice, a stage at which B CK mRNA expression is abundant relative to that of mature tissue(20Trask R.V. Billadello J.J. Biochim. Biophys. Acta. 1990; 1049: 182-188Crossref PubMed Scopus (89) Google Scholar). The results of gel mobility shift assays showed that protein 1 was abundant in both neonatal tissues and was down-regulated with development, correlating well with the developmental regulation of B CK mRNA in these tissues (Fig. 6). In contrast, band 3 was not developmentally regulated.Figure 6:Developmental expression of band 1 protein in tissues. 32P-labeled B CK probe was incubated with nuclear extract prepared from heart and skeletal muscle from day 1 neonatal (N) and adult (A) mice and evaluated in a standard gel mobility shift assay.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Developmental down-regulation of band 1 protein could be due to either a decrease in synthesis or a post-translational modification of the protein resulting in lower affinity for the target DNA. Because phosphorylation has been shown to modulate the DNA binding activity of many transcription factors(32Hunter T. Karin M. Cell. 1992; 70: 375-387Abstract Full Text PDF PubMed Scopus (1115) Google Scholar), we determined the effect of treatment of extracts with phosphatases on binding of the probe by band 1 protein. Treatment of extract with either enzyme resulted in marked inhibition of formation of complex 1 (Fig. 7). These results suggest that phosphorylation of band 1 protein may be an important post-transcriptional mechanism that mediates affinity for the target DNA. To determine the molecular weight of protein 1 we performed a gel mobility shift assay with the B CK probe and nuclear protein extract from C2C12 myoblasts. Band 1 was excised from a wet gel after a brief exposure to Kodak XAR film to facilitate localization of the band in the gel. The protein was separated from the gel with the use of electroelution, analyzed by electrophoresis in a 7.5% SDS-polyacrylamide gel, and detected with the silver reagent (Bio-Rad). A single protein of approximate molecular weight 150 kDa (n = 6 gels) was seen consistently (Fig. 8). This band was not seen when free DNA probe was separated from a band shift gel by electroelution, subjected to SDS-polyacrylamide gel electrophoresis, and stained with silver reagent. Autoradiography of the gel showed that the band represented protein, and not protein complexed to [32P]DNA probe. The creatine kinase gene family represents an interesting model of coordinate regulation of isoproteins that can be studied in convenient cell culture systems. A great deal of attention has been given to elucidating the molecular mechanisms that regulate the M CK gene. The regulatory elements that are essential for expression of this gene have been characterized by transfection experiments in cell culture (33-35), direct gene injection into heart and skeletal muscle(36Amacher S.L. Buskin J.N. Hauschka S.D. Mol. Cell. Biol. 1993; 13: 2753-2764Crossref PubMed Scopus (134) Google Scholar, 37Vincent C.K. Gualberto A. Patel C.V. Walsh K. Mol. Cell. Biol. 1993; 13: 1264-1272Crossref PubMed Scopus (86) Google Scholar) and in transgenic animals(38Johnson J.E. Wold B.J. Hauschka S.D. Mol. Cell. Biol. 1989; 9: 3393-3399Crossref PubMed Scopus (158) Google Scholar). The M CK gene is regulated by a tissue-specific enhancer that contains two MEF-1 motifs or E-boxes (CANNTG) that bind myogenic factors (MyoD, myogenin, Myf-5, and MRF-4) and an MEF-2 motif that binds a MADS box transcription factor(39Yu Y.-T. Breitbart R.E. Smoot L.B. Lee Y. Mahdavi V. Nadal-Ginard B. Genes & Dev. 1992; 6: 1783-1798Crossref PubMed Scopus (380) Google Scholar). In contrast, regulatory mechanisms that control genes that are turned off during myogenesis have received less attention. The down-regulation of β-actin mRNA during myogenesis is controlled at the level of transcription by conserved sequences in the 3′-nontranslated region of the gene(40DePonti-Zilli L. Seiler-Tuyns A. Paterson B.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1389-1393Crossref PubMed Scopus (67) Google Scholar). The cardiac α-actin gene is regulated by the interaction of MyoD1, the serum response factor, and Sp1 with promoter elements(41Sartorelli V. Webster K.A. Kedes L. Genes & Dev. 1990; 4: 1811-1822Crossref PubMed Scopus (231) Google Scholar). The helix-loop-helix protein Id is expressed in C2C12 myoblasts and is down-regulated with differentiation(42Benezra R. Davis R.L. Lockshon D. Turner D.L. Weintraub H. Cell. 1990; 61: 49-59Abstract Full Text PDF PubMed Scopus (1785) Google Scholar). Id associates specifically with MyoD and attenuates its ability to trans-activate muscle-specific genes such as the M CK gene. There is no evidence that Id or other helix-loop-helix proteins play a direct or indirect role in the regulation of the B CK gene. The regulation of the B CK gene has been studied in a number of nonmuscle systems. A 61-base pair sequence between −98 and −37 that contains both the CCAAT and TATA sequences is important for efficient in vitro transcription from a minimal B CK promoter (43-46). The results of transfection experiments have identified 5′-upstream sequence elements and sequences within the first (untranslated) exon that are important for expression in HeLa cells (47Hobson G.M. Molloy G.R. Benfield P.A. Mol. Cell. Biol. 1990; 10: 6533-6543Crossref PubMed Scopus (37) Google Scholar) and neuroblastoma cells(48Mariman E. Wieringa B. Gene (Amst.). 1991; 102: 205-212Crossref PubMed Scopus (17) Google Scholar). The B CK gene promoter has strong sequence similarity to the adenovirus E2E gene promoter and like the E2E gene is regulated by the viral activator Ela. This finding may be of significance for the metabolic energy-requiring events that take place after oncogenic activation(49Kaddurah-Daouk R. Lillie J.W. Daouk G.H. Green M.R. Kingston R. Schimmel P. Mol. Cell. Biol. 1990; 10: 1476-1483Crossref PubMed Scopus (40) Google Scholar). In osteoblastic cells transcriptional up-regulation of the gene during differentiation is associated with the formation of a nuclear protein-DNA complex that binds a cis-element between +1 and +228 of the gene(5Ch'ng J.L.C. Ibrahim B. J. Biol. Chem. 1994; 269: 2336-2341Abstract Full Text PDF PubMed Google Scholar). In U937 cells the highly conserved 3′-NTR of B CK mRNA is important for translational regulation of B CK protein(50Ch'ng J.L.C. Shoemaker D.L. Schimmel P. Holmes E.W. Science. 1990; 248: 1003-1006Crossref PubMed Scopus (56) Google Scholar). We present evidence that sequences within the first exon are important for regulation of expression of the B CK gene in transfected myogenic cells in culture. This sequence does not contain recognition sites for any previously described transcription factor (Genetics Computer Group (GCG) transcription factor recognition sites release 6.5). The presence of regulatory sequences within the first exon of a gene is an unusual finding but is not unique to the B CK gene. Important regulatory sequences within the first exon have been described in the human tissue-type plasminogen activator gene(51Medcalf R.L. Ruegg M. Schleuning W.-D. J. Biol. Chem. 1990; 265: 14618-14626Abstract Full Text PDF PubMed Google Scholar), the skeletal troponin I gene(52Nikovits W. Mar J.H. Ordahl C.P. Mol. Cell. Biol. 1990; 10: 3468-3482Crossref PubMed Scopus (26) Google Scholar), and a human nonmuscle myosin heavy chain gene(53Kawamoto S. J. Biol. Chem. 1994; 269: 15101-15110Abstract Full Text PDF PubMed Google Scholar). Interestingly, none of the exonic regulatory sequences important for expression of other genes in muscle cells share significant identity with the sequence we describe. Protein phosphorylation is an important control mechanism for regulation of the myogenic developmental program. Both MyoD and myogenin are phosphoproteins(54Li L. Zhou J. James G. Heller-Harrison R. Czech M.P. Olson E.N. Cell. 1992; 71: 1181-1194Abstract Full Text PDF PubMed Scopus (280) Google Scholar, 55Tapscott S.J. Davis R.L. Thayer M.J. Cheng P.-F. Weintraub H. Lassar A.B. Science. 1988; 242: 405-411Crossref PubMed Scopus (576) Google Scholar). Phosphatase treatment of MyoD attenuates specific binding of MyoD-E12 heterodimers to the M CK gene enhancer(56Nakamura S. J. Biol. Chem. 1993; 268: 11670-11677Abstract Full Text PDF PubMed Google Scholar). Fibroblast growth factor inhibits myogenesis by inactivating myogenic helix-loop-helix proteins by phosphorylating a site in the DNA binding domain of myogenin(54Li L. Zhou J. James G. Heller-Harrison R. Czech M.P. Olson E.N. Cell. 1992; 71: 1181-1194Abstract Full Text PDF PubMed Scopus (280) Google Scholar). Treatment of C2C12 myoblasts with the protein phosphatase inhibitor okadaic acid inhibits skeletal muscle cell differentiation and MyoD expression and induces expression of the id gene (57Kim S.-J. Kim K.Y. Tapscott S.J. Winokur T.S. Park K. Fujiki H. Weintraub H. Roberts A.B. J. Biol. Chem. 1992; 267: 15140-15145Abstract Full Text PDF PubMed Google Scholar). We describe a nuclear protein that binds specifically to sequence elements that are important for expression of B CK chimeric constructs in C2C12 myoblasts. Evidence that the protein we describe is important for regulation of the B CK gene includes specific binding to sequences within the first exon that confer regulated expression to the CAT gene in transfected C2C12 cells, expression in tissues that correlates with that of B CK mRNA and protein, and developmental expression in myogenic cells and in heart and skeletal muscle that resembles that of B CK mRNA. Our results suggest that expression of B CK in cells and tissues may be regulated through a mechanism that alters phosphorylation of this DNA binding protein. Further investigation may provide insight into these mechanisms that mediate the developmental down-regulation of genes during myogenic development. We thank Kimberly Goodwin, Kimberly Hawker, and Nancy Brada for technical assistance, Kelly Hall for secretarial assistance, and Michael Ritchie for contributions to the early stage of this project." @default.
• W2058693181 created "2016-06-24" @default.
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• W2058693181 creator A5058429480 @default.
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• W2058693181 date "1995-07-01" @default.
• W2058693181 modified "2023-09-28" @default.
• W2058693181 title "Characterization of a Nuclear Protein That Interacts with Regulatory Elements in the Human B Creatine Kinase Gene" @default.
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