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• W1966500528 abstract "The expression of the cardiac myosin light chain 2 (MLC2) gene is repressed in skeletal muscle as a result of the negative regulation of its transcription. Two regulatory elements, the cardiac specific sequence (CSS) located upstream (−360 base pairs) and a downstream negative modulatory sequence (NMS), which function in concert with each other, are required for repression of the MLC2 promoter activity in skeletal muscle. Individually, CSS and NMS have no effect. Transient transfection analysis with recombinant plasmids indicated that CSS- and NMS-mediated repression of transcription is position- and orientation-dependent and is transferable to heterologous promoters. A minimal conserved motif, GAAG/CTTC, present in both CSS and NMS, is responsible for repression as the mutation in the core CTTC sequence alone was sufficient to abrogate its repressor activity. The DNA binding assay by gel mobility shift analysis revealed that one of the two complexes, CSSBP2, is significantly enriched in embryonic skeletal muscle relative to cardiac muscle. In extracts from adult skeletal muscle, where the cardiac MLC2 expression is suppressed, both complexes, CSSBP1 and CSSBP2, were present, whereas the cardiac muscle extracts contained CSSBP1 alone, suggesting that the protein(s) in the CSSBP2 complex accounts for the negative regulation of cardiac MLC2 in skeletal muscle. A partial cDNA clone (Nished) specific for the candidate repressor factor was isolated by expression screening of the skeletal muscle cDNA library by multimerized CSS-DNA as probe. The recombinant Nished protein binds to the CSS-DNA, but not to ΔCSS-DNA where the core CTTC sequence was mutated. The amino acid sequence of Nished showed a significant structural similarity to the sequence of transcription factor “runt,” a known repressor of gap and pair-rule gene expression in Drosophila. The expression of the cardiac myosin light chain 2 (MLC2) gene is repressed in skeletal muscle as a result of the negative regulation of its transcription. Two regulatory elements, the cardiac specific sequence (CSS) located upstream (−360 base pairs) and a downstream negative modulatory sequence (NMS), which function in concert with each other, are required for repression of the MLC2 promoter activity in skeletal muscle. Individually, CSS and NMS have no effect. Transient transfection analysis with recombinant plasmids indicated that CSS- and NMS-mediated repression of transcription is position- and orientation-dependent and is transferable to heterologous promoters. A minimal conserved motif, GAAG/CTTC, present in both CSS and NMS, is responsible for repression as the mutation in the core CTTC sequence alone was sufficient to abrogate its repressor activity. The DNA binding assay by gel mobility shift analysis revealed that one of the two complexes, CSSBP2, is significantly enriched in embryonic skeletal muscle relative to cardiac muscle. In extracts from adult skeletal muscle, where the cardiac MLC2 expression is suppressed, both complexes, CSSBP1 and CSSBP2, were present, whereas the cardiac muscle extracts contained CSSBP1 alone, suggesting that the protein(s) in the CSSBP2 complex accounts for the negative regulation of cardiac MLC2 in skeletal muscle. A partial cDNA clone (Nished) specific for the candidate repressor factor was isolated by expression screening of the skeletal muscle cDNA library by multimerized CSS-DNA as probe. The recombinant Nished protein binds to the CSS-DNA, but not to ΔCSS-DNA where the core CTTC sequence was mutated. The amino acid sequence of Nished showed a significant structural similarity to the sequence of transcription factor “runt,” a known repressor of gap and pair-rule gene expression in Drosophila. Two distinct factor-binding DNA elements in cardiac myosin light chain 2 gene are essential for repression of its expression in skeletal muscle. Isolation of a cDNA clone for repressor protein Nished.Journal of Biological ChemistryVol. 277Issue 7PreviewPage 18495: In Fig. 7 we presented the nucleotide sequence along with the derived amino acid sequence of Nished cDNA. A repeat of the sequence analysis revealed mistakes that are corrected in the revised sequence with expanded legend shown below. Full-Text PDF Open Access The acquisition of the differentiated phenotype of eukaryotic cells is a consequence of activation of tissue-specific genes and repression of other genes, both of which are precisely controlled during development of multicellular organisms (1Tijan R. Maniatis T. Cell. 1994; 77: 5-8Abstract Full Text PDF PubMed Scopus (955) Google Scholar). Since only a small population of genes is expressed at any given time in the differentiated cell, it is becoming increasingly clear that the mechanisms by which genes are repressed are as important as those that activate them (2Mitchell A.P. Tijan R. Science. 1989; 245: 371-378Crossref PubMed Scopus (2210) Google Scholar, 3Dyan W.S. Cell. 1989; 58: 1-4Abstract Full Text PDF PubMed Scopus (216) Google Scholar, 4Stehle J.H. Foulkes N.S. Molina C.A. Simonneaux V. Pevet P. Sassone-Corsi P. Nature. 1993; 365: 314-320Crossref PubMed Scopus (356) Google Scholar). Repression of transcription is commonly achieved via binding of the negative regulators to cis-elements where the degree of repression is controlled by the location and/or orientation (5Ip Y.T. Kraut R. Levine M. Rushlow C.A. Cell. 1991; 64: 439-446Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 6Mizzen K. Goto M. Masamune Y. Nakanishi Y. 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The coordinate expression of muscle-specific genes during myogenesis in differentiated myocytes suggests the existence of a tightly controlled regulatory program involving a cascade of expression of specific positive and negative transcription factors (19Emerson C. Fischman D. Nadal-Ginard B. Siddiqui M.A.Q. Molecular Biology of Muscle Development, UCLA Symposia on Molecular and Cellular Biology New Series.29. Alan R. Liss, Inc., New York1986Google Scholar). We have shown previously that the tissue-specific expression of the chicken cardiac myosin light chain 2 (MLC2) 1The abbreviations used are: MLC2, myosin light chain 2; CSS, cardiac specific sequence; NMS, negative modulatory sequence; MCK, muscle creatine kinase; bp, base pair(s); CAT, chloramphenicol acetyltransferase; ANG, angiotensinogen; LUC, luciferase; CMV, cytomegalovirus; GMSA, gel mobility shift assay; MOPS, 4-morpholinepropanesulfonic acid; IRE, intron-responsive; IPTG, isopropyl-1-thio-β-d-galactopyranoside; β-Gal, β-galactosidase. gene is regulated by both positively and negatively actingcis-elements and their cognate DNA-binding factors (20Qasba P. Lin E. Zhou M.D. Kumar A. Siddiqui M.A.Q. Mol. Cell. Biol. 1992; 12: 1107-1116Crossref PubMed Scopus (20) Google Scholar, 21Shen R. Goswami S.K. Mascareno E. Kumar A. Siddiqui M.A.Q. Mol. Cell. Biol. 1991; 11: 1676-1685Crossref PubMed Google Scholar, 22Zarraga A.M. Danishefsky K. Deshpande A. Nicholson D. Mendola C. Siddiqui M.A.Q. J. Biol. Chem. 1986; 261: 13852-13860Abstract Full Text PDF PubMed Google Scholar, 23Zhou M.D. Goswami S. Martin M.E. Siddiqui M.A.Q. Mol. Cell. Biol. 1993; 13: 1222-1231Crossref PubMed Scopus (25) Google Scholar, 24Zhou M.D. Wu Y. Siddiqui M.A.Q. Gene Expr. 1992; 2: 127-138PubMed Google Scholar). The regulation is due to the activators CArG box (20Qasba P. Lin E. Zhou M.D. Kumar A. Siddiqui M.A.Q. Mol. Cell. Biol. 1992; 12: 1107-1116Crossref PubMed Scopus (20) Google Scholar, 25Minty A. Kedes L. Mol. Cell. Biol. 1986; 6: 2125-2136Crossref PubMed Scopus (274) Google Scholar) and the myocyte enhancer factor 2 binding sites (26Gossett L.A. Kelvin D.J. Sternberg E.A. Olson E.N. Mol. Cell. Biol. 1989; 9: 5022-5033Crossref PubMed Scopus (449) Google Scholar), and a negative regulatory region, cardiac specific sequence (CSS), responsible for repression of cardiac MLC2 transcription in skeletal muscle (21Shen R. Goswami S.K. Mascareno E. Kumar A. Siddiqui M.A.Q. Mol. Cell. Biol. 1991; 11: 1676-1685Crossref PubMed Google Scholar). Removal of CSS alone restores cardiac MLC2 expression in skeletal muscle without impairing its function in cardiac muscle cells (21Shen R. Goswami S.K. Mascareno E. Kumar A. Siddiqui M.A.Q. Mol. Cell. Biol. 1991; 11: 1676-1685Crossref PubMed Google Scholar). An upstream negative regulatory domain distinct from CSS also exists in the rat MLC2 gene promoter, mutation of which led to ectopic expression of the gene in transgenic animals (27Lee K.J. Hickey R. Zhu H. Chein K.R. Mol. Cell. Biol. 1994; 14: 1220-1229Crossref PubMed Google Scholar). In this report, we have delineated the regulatory domains within CSS essential for repression of the cardiac MLC2 promoter in skeletal muscle. There are three distinct protein binding sites, CSS-A, CSS-B, and CSS-C, each of which contains a common sequence motif, GAAG/CTTC. Mutation in the CTTC motif in CSS-B alone was sufficient to abrogate totally both DNA-protein interaction and the inhibitory function of CSS. CSS-mediated repression, however, requires the presence of another downstream sequence element, the negative modulatory sequence (NMS), which also contains the conserved GAAG motif and serves as the binding site for nuclear proteins. Neither of the two motifs alone can repress transcription. The CSS/NMS-binding protein complex, CSSBP2, is present at a significantly higher level in nuclear extracts from skeletal muscle relative to cardiac muscle, suggesting that CSSBP2 binding to CSS/NMS accounts for repression of the MLC2 promoter function in skeletal muscle. In an attempt to identify the protein(s) involved in the repression mechanism, we have isolated a partial cDNA clone (Nished; Sanskrit for “negative”) by expression screening of the cDNA library derived from chicken skeletal muscle mRNA by multimerized CSS-DNA as a probe. The predicted amino acid sequence of Nished shows a significant similarity to two previously described repressors, runt from Drosophila (28Kania M.A. Bonner A.S. Duffy J.B. Gergen J.P. Genes Dev. 1990; 4: 1701-1713Crossref PubMed Scopus (232) Google Scholar) and SP3 from human (29De Luca P. Majello B. Lania L. J. Biol. Chem. 1996; 271: 8533-8536Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). To construct CSS-containing muscle creatine kinase (MCK) promoter/reporter recombinant, the proximal MLC2 promoter (−160 to +158) in pMLC410CAT was replaced by the 246-bpHindIII fragment containing the MCK basal promoter from plasmid E4 (30Sternberg E.A. Spizz G. Perry W. Vizard M.D. Weil T. Olson E.N. Mol. Cell. Biol. 1988; 8: 2896-2909Crossref PubMed Scopus (175) Google Scholar). MCK enhancer contained within a 300-bpBamHI restriction fragment was then introduced into the unique BamHI site to generate MCKCSS. Likewise, the proximal MLC2 promoter (−160 to +158) of pMLC410CAT was replaced by the 2,500-bp MCK promoter/enhancer fragment (30Sternberg E.A. Spizz G. Perry W. Vizard M.D. Weil T. Olson E.N. Mol. Cell. Biol. 1988; 8: 2896-2909Crossref PubMed Scopus (175) Google Scholar) to construct MCK2.5CSS. The HindIII-PstI restriction fragment from pMLC371CAT that contains the CSS domain was cloned upstream to the pANG700 promoter (31Ohkubo H.R. Kageyama M. Ujihara T. Hirose S. Nakanishi S. Proc. Natl. Acad. Sci. U. S. A. 1983; 80 (2nd Ed.): 2196-2200Crossref PubMed Scopus (210) Google Scholar) and designated as pANG700CSS. To construct pANGCSS, the HindIII-PstI fragment from pMLC371CAT was cloned into the plasmid pBasic CAT (Promega) in theHindIII and PstI sites in the polylinker. The 264-bp NciI-XbaI fragment of ANG proximal promoter was then cloned downstream to CSS in the polylinker atSalI-XbaI to produce pANGCSS. The PstI fragment (−130 bp to +40 bp) from the MLC2 promoter was cloned into the PstI site in pBasic CAT to construct PST. A 160-bpHindIII-PstI fragment containing the CSS domain was spliced in the corresponding sites in the polylinker of pBasic CAT; the resultant plasmid was then linearized with PstI and ligated to the PstI fragment (−160 to +158) containing the MLC2 promoter to create plasmid CSSPST. A synthetic oligonucleotide harboring the NMS sequence (GAAG) flanked by XbaI was inserted downstream of the MLC promoter in plasmid PST to create CSSPSTNMSCAT. MLC2 proximal promoter (−125 to + 158 bp) obtained as anHindIII fragment was cloned into the HindIII site in the polylinker of the basic luciferase reporter plasmid (Promega) to create MLCLUC. An oligonucleotide with HindIII ends, spanning the 50-bp CSS element (5′-agcttccattgtgaaggacgagggggtacttctaccctgaagcaaaagga-3′) was cloned into the HindIII site of polylinker of pBluescript-SK+. Plasmid with the CSS domain in forward and reversed orientations was digested with restriction enzymes SacI and XhoI, and the restriction fragments were cloned in the corresponding sites in the polylinker in MLCLUC. The resultant plasmids were designated MLCCSSLUC and MLC3′CSSLUC, respectively. An oligonucleotide containing the CSS sequence with nucleotides altered to introduce specific mutations and flanked by HindIII and XhoI sites was synthesized. The resultant oligonucleotides were ligated toSmaI and XhoI sites in the polylinker in MLCLUC to ensure directed ligation. The resultant plasmid was designated MLCΔBCSS. Skeletal muscle tissue was collected from the leg (soleus) muscle of 13-day-old chicken embryo, digested with 0.1% pancreatin, and cells were suspended in complete medium (60% Waymouth, 40% Hanks' balanced salt solution medium with 15% horse serum, 2% chicken serum) and filtered through sieves of 90 μm and 45 μm sequentially. Preplating was done three times, for 1 h each, to facilitate differential removal of fibroblasts. Cells were plated at a density of 2 × 106 cells/10-cm dish. Cultures were re-fed with fresh medium before transfection. Transfections were done using calcium phosphate precipitation (32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). CAT enzyme activity in the extracts was assayed as described previously (20Qasba P. Lin E. Zhou M.D. Kumar A. Siddiqui M.A.Q. Mol. Cell. Biol. 1992; 12: 1107-1116Crossref PubMed Scopus (20) Google Scholar, 21Shen R. Goswami S.K. Mascareno E. Kumar A. Siddiqui M.A.Q. Mol. Cell. Biol. 1991; 11: 1676-1685Crossref PubMed Google Scholar, 23Zhou M.D. Goswami S. Martin M.E. Siddiqui M.A.Q. Mol. Cell. Biol. 1993; 13: 1222-1231Crossref PubMed Scopus (25) Google Scholar, 33Goswami S. Qasba P. Ghatpande S. Carleton S. Deshpande A.K. Baig M. Siddiqui M.A.Q. Mol. Cell. Biol. 1994; 14: 5130-5138Crossref PubMed Scopus (29) Google Scholar). Plasmid SV40 luciferase (Promega) was used to normalize against fluctuations in plasmid uptake and expression. Cells were lysed in 1 × lysis buffer (Promega), and the luciferase activity was assessed according to instructions provided by Promega in a monolight luminometer. For normalization in the uptake of luciferase reporter plasmids, β-galactosidase activity under the pCMV promoter was used as an internal control. Tissues from embryonic and adult heart and skeletal muscles were minced finely, and the dissociated cells were lysed in lysis buffer (20 mm Hepes, pH 7.6, 20% glycerol, 10 mm NaCl, 1.5 mmMgCl2, 0.2 mm EDTA, 0.1% Triton X-100), 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and leupeptin (10 μg/ml) with a Wheaton dounce homogenizer. Nuclei were collected, and protein extracts were prepared as described previously (23Zhou M.D. Goswami S. Martin M.E. Siddiqui M.A.Q. Mol. Cell. Biol. 1993; 13: 1222-1231Crossref PubMed Scopus (25) Google Scholar, 33Goswami S. Qasba P. Ghatpande S. Carleton S. Deshpande A.K. Baig M. Siddiqui M.A.Q. Mol. Cell. Biol. 1994; 14: 5130-5138Crossref PubMed Scopus (29) Google Scholar). Double-stranded oligonucleotide was end labeled with [γ-32P]ATP and 0.5 ng of labeled oligonucleotide, incubated with 2 μg of poly(dI·dC), 1–12 μg of protein in 20 mm Hepes, pH 7.5, 3% glycerol, 1.5 mmMgCl2, 1 mm dithiothreitol, 2 mmEDTA, and 50 mm KCl at 4 °C for 30 min. Competitor DNA was added in a 100-fold excess following incubation for 30 min on ice, and the reaction mixtures were analyzed by electrophoresis as described previously (23Zhou M.D. Goswami S. Martin M.E. Siddiqui M.A.Q. Mol. Cell. Biol. 1993; 13: 1222-1231Crossref PubMed Scopus (25) Google Scholar). A footprinting assay was performed using nuclear extract from embryonic and adult skeletal muscle essentially as described earlier (24Zhou M.D. Wu Y. Siddiqui M.A.Q. Gene Expr. 1992; 2: 127-138PubMed Google Scholar). A 160-bpEcoRI-XhoI fragment containing the CSS domain was incubated with nuclear extract (10–60 μg of protein) from embryonic and adult cardiac and skeletal muscle in a 50-μl reaction buffer containing 20 mm Hepes, pH 7.9) 5 mmMgCl2, 0.1 mm EDTA, 50 mm KCl, 0.5 mm dithiothreitol, and 10% glycerol. Following incubation at room temperature freshly diluted DNase-I (1 μg/ml) was added and then allowed to incubate for 60 s. DNA was extracted with phenol/chloroform and analyzed on an 8% sequencing gel. For footprinting of the NMS domain, a PstI-HindIII fragment spanning the NMS region was labeled by end filling. Chicken skeletal muscle cDNA expression library cloned in the λZap expression vector obtained from Stratagene was screened according to Singh et al. (34Singh H. Lebowitz J.H. Baldwin A.S. Sharp P.A. Cell. 1988; 52: 415-423Abstract Full Text PDF PubMed Scopus (419) Google Scholar) using the multimer (4 ×) of CSS-50. For expression of the recombinant protein in Escherichia coli, expression vector pET29B (Novagen) was digested withBamHI and XhoI ligated with cDNA insert obtained as a BamHI-XhoI insert from phagemid Bluescript and transformed into host cells BL(21)DE3. Induction was done according to manufacturer's (Novagen) instructions. Poly(A)+ was made from cardiac and skeletal muscle of an adult chicken following the FastTrack mRNA isolation kit (Invitrogen). Briefly, 1 g of tissue was homogenized in 15 ml of lysis buffer containing RNase protein degrader and incubated at 450 °C for 60 min. The lysate was centrifuged at 4,000 × g for 5 min at room temperature. The supernatant was recovered and 950 μl of 5m NaCl added. The DNA was sheared using a 18–21-gauge needle, and oligo(dT) cellulose pellet was added and incubated with gentle rocking for 60 min at room temperature. Then, the samples were centrifuged at 3,000 × g for 5 min, and the supernatant was carefully removed from the oligo(dT) bed. The beds were washed three times with 20 ml of binding buffer; the supernatant was removed after a spin of 3,000 × g for 5 min, followed by a wash in low salt buffer (three times). The poly(A)+was eluted in 200 μl of elution buffer and then precipitated with cold ethanol and 0.1 volume of 3 m NaOAc. 10 μg of poly(A)+ from cardiac and skeletal muscle was loaded in 1.3% agarose/formaldehyde in 1 × MOPS, as described previously (32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar, 33Goswami S. Qasba P. Ghatpande S. Carleton S. Deshpande A.K. Baig M. Siddiqui M.A.Q. Mol. Cell. Biol. 1994; 14: 5130-5138Crossref PubMed Scopus (29) Google Scholar). Previous studies in our laboratory (21Shen R. Goswami S.K. Mascareno E. Kumar A. Siddiqui M.A.Q. Mol. Cell. Biol. 1991; 11: 1676-1685Crossref PubMed Google Scholar) have identified a negative regulatory region, CSS, located between −371 and −282 bp in the chicken cardiac MLC2 gene promoter, which is required for repression of cardiac MLC2 gene transcription in skeletal muscle cells. To test the potential role of CSS in repressing the transcription of heterologous promoters such as skeletal MCK (30Sternberg E.A. Spizz G. Perry W. Vizard M.D. Weil T. Olson E.N. Mol. Cell. Biol. 1988; 8: 2896-2909Crossref PubMed Scopus (175) Google Scholar) and the non-muscle rat ANG (31Ohkubo H.R. Kageyama M. Ujihara T. Hirose S. Nakanishi S. Proc. Natl. Acad. Sci. U. S. A. 1983; 80 (2nd Ed.): 2196-2200Crossref PubMed Scopus (210) Google Scholar) promoters, we used plasmid pMCKCSS and pANGCSS containing CSS in the respective promoters in a transient transfection assay in skeletal muscle cells in culture. pMCKCSS expression was repressed effectively (80%) compared with that of parent plasmid pMCK without CSS (Fig. 1 A). When CSS was placed 2.5 kilobases upstream to the MCK promoter (see “Materials and Methods”), the activity of the resultant plasmid, MCK2.5CSS, was the same as the parent plasmid pMCK2.5, suggesting that repression of transcription by CSS is position-dependent (Fig.1 A). Similar results were obtained when we tested the rat ANG promoter, which is expressed optimally in liver and at a lower level in skeletal muscle. As shown in Fig. 1 B, pANGCSS activity was repressed significantly (52%) relative to the level of the ANG promoter lacking CSS (pANG). The expression of plasmid containing CSS placed 700 bp upstream in the ANG promoter (pANG700CSS) was not repressed. When CSS was fused to the reporter plasmid carrying the thymidine kinase promoter, no repression was obtained (Fig.1 C), suggesting that the repression mechanism might require other regulatory sequence(s) involved in the CSS-mediated inhibition of transcription (see below). To identify the nucleotide sequence within CSS involved in protein-DNA interaction, a DNase-I footprinting assay was performed using a 160-bp fragment that contains the CSS domain (−410 to −250) and nuclear extracts from adult and embryonic (13-day-old) chicken skeletal (soleus) muscle (Fig.2). Three protected regions (CSS-A: 5′-CCATTGTGAAGGAC-3′; CSS-B: 5′-GATACTTC-3′; and CSS-C: 5′-CTGAAGCAAAGG-3′), which together span between −360 and −310, and at least one hypersensitive site were detected. A common nucleotide sequence motif, GAAG, is present in regions A and C and the complementary sequence, CTTC, in region B. We have denoted this region (−360 to −310) as CSS-50. The sequence GAAG/CTTC was also identified as a regulatory motif in the negative regulatory domains of several other genes (27Lee K.J. Hickey R. Zhu H. Chein K.R. Mol. Cell. Biol. 1994; 14: 1220-1229Crossref PubMed Google Scholar, 35Chow K.L. Schwartz R.J. Mol. Cell. Biol. 1990; 10: 528-538Crossref PubMed Google Scholar, 36Clark A.R. Docherty K. Biochem. J. 1993; 296: 521-541Crossref PubMed Scopus (72) Google Scholar, 37Covitzan P.A. Mitchell A.P. Genes Dev. 1993; 7: 1598-1608Crossref PubMed Scopus (63) Google Scholar, 38Herbst R.S. Boczko E.M. Darnell Jr., J.E. Babiss L.E. Mol. Cell. Biol. 1990; 10: 3896-3905Crossref PubMed Scopus (33) Google Scholar, 39Rincon-Limas D.E. Amaya-Manzanares F. Nino-Rosales M.L. Yu Y. Yang T.P. Patel P.I. Mol. Cell. Biol. 1995; 15: 6561-6571Crossref PubMed Scopus (10) Google Scholar, 40Shio Y. Yamamoto T. Yamaguchi N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5206-5210Crossref PubMed Scopus (126) Google Scholar, 41Tada H. Lashgari S.M. Khalili K. Virology. 1991; 180: 327-338Crossref PubMed Scopus (35) Google Scholar), suggesting that the motif plays a conserved role in negative regulation of transcription. To define further the role of CSS-50, GMSAs were done with nuclear extracts from embryonic heart and skeletal muscles (Fig.3 A). Two well defined complexes, CSSBP1 and CSSBP2, were formed with extracts from both embryonic tissues. The intensity of the fast migrating complex, CSSBP2, was markedly higher in skeletal muscle compared with the corresponding complex in cardiac muscle. Both complexes were competed out by CSS and by an oligonucleotide IRE (intron-responsive) containing an activator element in the first intron of the MLC2 gene, which also contains the GAAG/CTTC motif. When nuclear extracts from the adult (4 weeks old) skeletal and cardiac muscle tissues were compared as above, only CSSBP1 binding activity was present in the cardiac muscle, whereas the skeletal muscle extract contained both CSSBP1 and CSSBP2 complexes (Fig.3 A). Since cardiac MLC2 is down-regulated in skeletal tissue, one may speculate that the relative abundance of CSSBP2 accounts for negative regulation of MLC2 gene in skeletal muscle. A barely visible complex was observed when CTTC in CSS-B was mutated to GGTC (CSSΔB) (Fig. 3 B), suggesting that the binding activity of CSS-50 is primarily due to the CSS-B sequence. To investigate whether the sequence contained in CSS-50 alone is sufficient for repression or whether it acts in concert with other elements, we used the minimal basal MLC2 promoter contained in the −121 to +158 fragment containing the three cis-regulatory elements, CArG box, myocyte enhancer factor 2 site, and TATA box, which are required for its optimal expression in cardiac muscle cells (21Shen R. Goswami S.K. Mascareno E. Kumar A. Siddiqui M.A.Q. Mol. Cell. Biol. 1991; 11: 1676-1685Crossref PubMed Google Scholar,23Zhou M.D. Goswami S. Martin M.E. Siddiqui M.A.Q. Mol. Cell. Biol. 1993; 13: 1222-1231Crossref PubMed Scopus (25) Google Scholar). As shown in Fig. 4, the presence of the CSS-50 alone produced an effective (70%) inhibition of MLC2 transcription, as measured by the luciferase assay. Recombinant plasmid MLCCSS3′LUC, with CSS-50 in reverse orientation, was totally ineffective, suggesting that CSS is not a conventional silencer, as it is both position- and orientation-dependent. Since it was reported that the regulatory sequences of myocyte enhancer factor 2 (44Arnold H. Lohse H. Seidel P.U. Bober E. Eur. J. Biochem. 1988; 178: 53-60Crossref PubMed Scopus (21) Google Scholar), SP1 (45Saidapet C. Khandekar P. Mendola C. Siddiqui M.A.Q. Arch. Biochem. Biophys. 1984; 233: 565-572Crossref PubMed Scopus (13) Google Scholar) and E box (25Minty A. Kedes L. Mol. Cell. Biol. 1986; 6: 2125-2136Crossref PubMed Scopus (274) Google Scholar) have an additive effect on transcription when present in multiple copies, we made recombinants with four copies of CSS, arranged in tandem, placed upstream in plasmid pMLCLUC. However, the presence of multiple copies of CSS in plasmid pMLC4XCSSLUC did not cause additional repression of transcription compared with repression observed with the single CSS copy in pMLCCSSLUC. When a substitution mutation (CTTC → GGTC) was introduced in window B in plasmid pMLCΔBCSSLUC, the repression due to CSS was disrupted, and the expression level of the mutant plasmid reached 80% of the activity of the parent plasmid pMLCLUC. Additional mutations in the reverse complement GAAG sequence in window C exhibit no further loss of repression (data not shown). Since mutation in CSS-B window alone caused a loss of protein binding in CSS-50, it would appear that the conserved sequence CTTC in CSS-B is the primary target for binding the repressor protein(s), which also accounts for its loss of function upon mutation. The plasmid pMLCLUC, used in the experiments above, contains the downstream sequence up to +158. An examination of the downstream sequence revealed that the conserved GAAG/CTTC motif is also present once in the 5′-untranslated region at +60 in the MLC2 gene. To test the potential involvement of this motif in repression, we constructed the plasmids PST and CSSPST, containing the basal promoter alone extending to +42, without and with CSS, respectively (see “Materials and Methods”) (Fig. 5). Plasmid CSSPST, which lacks the downstream sequence containing the GAAG motif but has the upstream CSS element, was surprisingly as active as PST, which lacks both the upstream and downstream elements. This would mean that CSS alone was unable to repress MLC2 transcription in skeletal muscle cells and suggests the requirement of the downstream GAAG motif, which was absent in both constructs. To test this possibility, we inserted the GAAG contained in a short (17-mer) oligonucleotide (5′-tctagacctagaagacttctaga-3′) downstream of the MLC2 promoter in CSSPST (see “Materials and Methods”). The resultant plasmid CSSPSTNMS was act" @default.
• W1966500528 created "2016-06-24" @default.
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• W1966500528 date "1997-07-01" @default.
• W1966500528 modified "2023-09-30" @default.
• W1966500528 title "Two Distinct Factor-binding DNA Elements in Cardiac Myosin Light Chain 2 Gene Are Essential for Repression of Its Expression in Skeletal Muscle" @default.
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