Two Distinct Factor-binding DNA Elements in Cardiac Myosin Light Chain 2 Gene Are Essential for Repression of Its Expression in Skeletal Muscle

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 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 (1). 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 (2)(3)(4). 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 (5)(6)(7)(8)(9), structure (10), or copy number (11) of the regulatory elements. Other mechanisms of transcriptional repression involve occupation of the activator binding site (12)(13)(14), squelching of factors via protein-protein interaction (15), and formation of a dimeric complex that binds DNA but lacks the activation domain (16 -18).
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 (19). We have shown previously that the tissue-specific expression of the chicken cardiac myosin light chain 2 (MLC2) 1 gene is regulated by both positively and negatively acting ciselements and their cognate DNA-binding factors (20 -24). The regulation is due to the activators CArG box (20,25) and the myocyte enhancer factor 2 binding sites (26), and a negative regulatory region, cardiac specific sequence (CSS), responsible for repression of cardiac MLC2 transcription in skeletal muscle (21). Removal of CSS alone restores cardiac MLC2 expression in skeletal muscle without impairing its function in cardiac muscle cells (21). 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 (27).
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 (28) and SP3 from human (29).

MATERIALS AND METHODS
Construction of Plasmids-To construct CSS-containing muscle creatine kinase (MCK) promoter/reporter recombinant, the proximal MLC2 promoter (Ϫ160 to ϩ158) in pMLC410CAT was replaced by the 246-bp HindIII fragment containing the MCK basal promoter from plasmid E4 (30). MCK enhancer contained within a 300-bp BamHI restriction fragment was then introduced into the unique BamHI site to generate MCKCSS. Likewise, the proximal MLC2 promoter (Ϫ160 to ϩ158) of pMLC410CAT was replaced by the 2,500-bp MCK promoter/ enhancer fragment (30) to construct MCK2.5CSS. The HindIII-PstI restriction fragment from pMLC371CAT that contains the CSS domain was cloned upstream to the pANG700 promoter (31) and designated as pANG700CSS. To construct pANGCSS, the HindIII-PstI fragment from pMLC371CAT was cloned into the plasmid pBasic CAT (Promega) in the HindIII 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 at SalI-XbaI to produce pANGCSS. The PstI fragment (Ϫ130 bp to ϩ40 bp) from the MLC2 promoter was cloned into the PstI site in pBasic CAT to construct PST. A 160-bp HindIII-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 (Ϫ160 to ϩ158) 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 (Ϫ125 to ϩ 158 bp) obtained as an HindIII 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 (5Ј-agcttccattgtgaaggacgagggggtacttctaccctgaagcaaaagga-3Ј) was cloned into the HindIII site of polylinker of pBluescript-SKϩ. 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 MLC3ЈCSSLUC, 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 to SmaI and XhoI sites in the polylinker in MLCLUC to ensure directed ligation. The resultant plasmid was designated MLC⌬BCSS.
Skeletal Muscle Culture and Transient Transfection-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 m and 45 m sequentially. Preplating was done three times, for 1 h each, to facilitate differential removal of fibroblasts. Cells were plated at a density of 2 ϫ 10 6 cells/10-cm dish. Cultures were re-fed with fresh medium before transfection. Transfections were done using calcium phosphate precipitation (32). CAT enzyme activity in the extracts was assayed as described previously (20,21,23,33). Plasmid SV40 luciferase (Promega) was used to normalize against fluctuations in plasmid uptake and expression. Cells were lysed in 1 ϫ 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, ␤-galactosidase activity under the pCMV promoter was used as an internal control.
Gel Electrophoretic Mobility Shift Assay (GMSA)-Double-stranded oligonucleotide was end labeled with [␥-32 P]ATP and 0.5 ng of labeled oligonucleotide, incubated with 2 g of poly(dI⅐dC), 1-12 g of protein in 20 mM Hepes, pH 7.5, 3% glycerol, 1.5 mM MgCl 2 , 1 mM dithiothreitol, 2 mM EDTA, 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 (23).
DNase-I Footprinting Assay-A footprinting assay was performed using nuclear extract from embryonic and adult skeletal muscle essentially as described earlier (24). A 160-bp EcoRI-XhoI fragment containing the CSS domain was incubated with nuclear extract (10 -60 g of protein) from embryonic and adult cardiac and skeletal muscle in a 50-l reaction buffer containing 20 mM Hepes, pH 7.9) 5 mM MgCl 2 , 0.1 mM EDTA, 50 mM KCl, 0.5 mM dithiothreitol, and 10% glycerol. Following incubation at room temperature freshly diluted DNase-I (1 g/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.
Screening of Expression Library from Skeletal Muscle with CSS-50 -Chicken skeletal muscle cDNA expression library cloned in the Zap expression vector obtained from Stratagene was screened according to Singh et al. (34) using the multimer (4 ϫ) of CSS-50. For expression of the recombinant protein in Escherichia coli, expression vector pET29B (Novagen) was digested with BamHI 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.
Northern Blot Hybridization-Poly(A) ϩ 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 ϫ g for 5 min at room temperature. The supernatant was recovered and 950 l of 5 M 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 ϫ 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 ϫ g for 5 min, followed by a wash in low salt buffer (three times). The poly(A) ϩ was eluted in 200 l of elution buffer and then precipitated with cold ethanol and 0.1 volume of 3 M NaOAc. 10 g of poly(A) ϩ from cardiac and skeletal muscle was loaded in 1.3% agarose/formaldehyde in 1 ϫ MOPS, as described previously (32,33). (21) have identified a negative regulatory region, CSS, located between Ϫ371 and Ϫ282 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 (30) and the non-muscle rat ANG (31) 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. 1A). 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.  1A). 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. 1B, 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. 1C), suggesting that the repression mechanism might require other regulatory sequence(s) involved in the CSS-mediated inhibition of transcription (see below).

CSS Element Represses Transcription of Heterologous Promoters-Previous studies in our laboratory
Identification of Protein Binding Domains in CSS-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 (Ϫ410 to Ϫ250) and nuclear extracts from adult and embryonic (13day-old) chicken skeletal (soleus) muscle (Fig. 2). Three protected regions (CSS-A: 5Ј-CCATTGTGAAGGAC-3Ј; CSS-B: 5Ј-GATACTTC-3Ј; and CSS-C: 5Ј-CTGAAGCAAAGG-3Ј), which together span between Ϫ360 and Ϫ310, 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 (Ϫ360 to Ϫ310) as CSS-50. The sequence GAAG/CTTC was also identified as a regulatory motif in the negative regulatory domains of several other genes (27,(35)(36)(37)(38)(39)(40)(41), 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. 3A). 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 skele-tal muscle extract contained both CSSBP1 and CSSBP2 complexes (Fig. 3A). 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 (CSS⌬B) (Fig. 3B), suggesting that the binding activity of CSS-50 is primarily due to the CSS-B sequence.
The CTTC/GAAG Motif Is Essential for Repression-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 Ϫ121 to ϩ158 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 (21,23). 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 MLCCSS3ЈLUC, 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 (44), SP1 (45) and E box (25) 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 pM-LCLUC. 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 3 GGTC) was introduced in window B in plasmid pMLC⌬BCSSLUC, 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 mu-  tations 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.
CSS-mediated Function Is Dependent upon a Downstream NMS-The plasmid pMLCLUC, used in the experiments above, contains the downstream sequence up to ϩ158. An examination of the downstream sequence revealed that the conserved GAAG/CTTC motif is also present once in the 5Ј-untranslated region at ϩ60 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 ϩ42, 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 (5Ј-tctagacctagaagacttctaga-3Ј) downstream of the MLC2 promoter in CSSPST (see "Materials and Methods"). The resultant plasmid CSSPSTNMS was active in repression of the promoter activity almost to the same extent as the native sequence NMS containing the motif in plasmid pMLC371. The downstream motif alone (PSTNMS) was inactive as repressor. These findings thus confirm the requirement of the downstream GAAG sequence which was supported further when insertion of another cis-regulatory sequence, the IRE region, present in the first intron of the MLC2 gene containing GAAGCTTC motif, also led to repression of the promoter. 2 To test whether NMS is recognized by DNA-binding proteins, we performed the DNase-I footprinting assay with the PstI-HindIII fragment, which includes the NMS region spanning from ϩ40 to ϩ158. As shown in Fig. 6A, there was a protected window corresponding to CTTCATG in the noncoding strand. Although a partial protection also occurred in regions flanking the CTTCTGA sequence, the repression activity in plasmid CSSPSTNMS containing only the protected GAAG sequence (see Fig. 5) was sufficient to restore repression of the MLC2 promoter activity. The sequence flanking the GAAG motif has no resemblance to the native sequence flanking GAAG in the MLC2 gene. Additional analysis of the NMS-DNA-binding protein interaction was done by GMSAs with an oligonucleotide containing the NMS domain (ϩ40 to ϩ70) and nuclear extracts from the embryonic and adult heart and skeletal muscles. As seen in Fig. 6B, the NMS-binding proteins are present in both cardiac and skeletal embryonic tissues. As with the CSS-binding proteins, the NMSBP1 binding activity was abundantly present in the skeletal extracts but to a lesser extent in cardiac extracts. The specificity of the complex formation was established by self-competition, with CSS and another GAAG-containing regulatory domain, IRE, all of which competed out the complex formation. Isolation of a cDNA Clone (Nished) for CSS-binding Protein-As a prelude to identifying the CSS-and NMS-binding proteins and delineating their role in repression of transcription, we isolated a cDNA clone by screening an expression cDNA library made from the adult chicken skeletal muscle mRNA (Stratagene) with a high affinity DNA containing the multimerized CSS-50 element. Of several candidate clones, one with the largest insert was identified as a candidate for CSSspecific protein coding cDNA clone, referred to from here on as Nished, which contains an insert of 1,217 bp in length. The nucleotide and the predicted amino acid sequences are shown in Fig. 7. Plasmid pET29Nished, containing the Nished cDNA in-frame with ATG, was introduced into host strain BL21(DE23) and induced by isopropyl-1-thio-␤-D-galactopyranoside (IPTG). Extracts from IPTG-induced and uninduced bacteria harboring Nished were examined by GMSAs using CSS-50 as a probe. As seen in Fig. 8, a new protein-DNA complex (see arrow) was formed with the IPTG-induced extract which was absent in the uninduced extract. Both extracts showed a nonspecific DNA binding activity (top band). The specificity of binding was demonstrated when CSS⌬B (⌬CSS) used as a probe failed to bind the recombinant protein Nished. A Northern blot using adult skeletal mRNA (Fig. 9) indicated that the Nished mRNA of approximately 1,600 nucleotides was present in skeletal muscle RNA, but a barely detectable signal of approximately the same size was seen in cardiac muscle mRNA. Based on size estimation of the mRNA, the cDNA clone lacks about 300 bp consisting the 5Ј-untranslated and coding sequences. When analyzed against the available data bases (SwissProt) using BLAST program, the Nished sequence showed a sequence homology with two repressors, a zinc finger protein SP3 (29) and the Drosophila melanogaster repressor runt protein (28). There are three regions of similarity between Nished and runt: the amino terminus of runt which is characteristically rich in alanine and glutamine residues, the runt homology domain including the ATP-binding domain, and the carboxyl region of the runt protein which is not highly conserved among the other family members. The positively charged residues are clustered mainly toward the carboxyl end of Nished and overlap with the runt homology domain within Nished, suggesting that this region could serve as the DNA binding motif in the protein. Further characterization of Nished is presently in progress. DISCUSSION The physiological expression of the chicken cardiac MLC2 is restricted to cardiac muscle (44 -46). In this report, we identified the functional sequence elements within the upstream negative regulatory domain (CSS) essential for repression of the cardiac MLC2 gene expression in skeletal muscle (21). In addition, a downstream sequence domain, NMS, was identified which functions in concert with CSS. The conserved CTTC motif in CSS-B alone is essential for CSS-mediated repression of the cardiac MLC2 promoter, as the substitution mutation in this motif caused the loss of DNA-protein interaction and led to a significant reduction in CSS-mediated repression of tran- scription. This motif is also present in the negative regulatory element (HF3) of the rat cardiac MLC2 promoter, mutation of which led to ectopic expression of the reporter luciferase activity in transgenic animals (27). A comparison of the nucleotide sequence of CSS and NMS with other genes indicated that GAAG/CTTC motif common to both elements is present in other known negative regulatory domains of several genes, such as retinoblastoma, human hypoxanthine phosphoribosyltransferase, yeast RME1, mouse albumin, rat MLC2, human neutrotropic papovavirus JC virus late promoter (27,28,37,41). Thus, it appears that the negative regulation of transcription, at least in the genes tested here, is mediated by a conserved sequence element (GAAG/CTTC). The prevalence of this motif in disparate promoters suggests that a common protein, presumably a family of proteins, is responsible for down-regulation of the respective genes. In this context, CSSBP appears to be the member of an active repressor family of proteins that have intrinsic repression activity by inhibition of transcription initiation. Repression is presumably achieved with specific modulation in the level of CSSBPs, most likely of CSSBP2, which exhibits increased binding activity in the adult skeletal muscle where the expression of cardiac MLC2 is optimally repressed.
Different mechanisms are used for the negative regulation of eukaryotic genes (for review, see Ref. 36). One of them involves interference with the binding of activators that recognize sequences that overlap the binding sequences of negative factors. The fact that CSS and NMS do not overlap with any known activator site in the MLC2 gene, nor do they serve as binding sites for other known regulatory factors, suggests that the mechanism of negative regulation based on binding of factors to the same sequence, but with the opposite effect (47-50), or via steric hindrance due to overlapping sites (51), can be excluded. For repressors, as for activators, interaction with the components of the basal transcription machinery is common, although not essential (52). Some factors can repress transcription of certain promoters but not of others, suggesting the involvement of specific intermediary factors or cofactors (16). It is not uncommon for transcription factors to have different effects on transcription based on different locations within the same promoter or in different promoters. Some repressors become active only when they are overexpressed (17, 36, 54 -56). CSS appears to function when positioned proximal to the promoter but requires, at the same time, the downstream GAAG motif. Thus the tissue-specific expression of the MLC2 gene is not the function of CSS alone; it depends on cooperative interaction with NMS.
Cooperation among multiple regulatory domains is, in general, the basis of negative regulation in several genes (36,52,55,(57)(58)(59), although bipartite promoter elements have also been identified in negative regulation (38,60,61). Interaction between distinct domains, presumably by virtue of the folding of DNA, leads to repression of transcription, such as of the insulin-like growth factor I receptor under negative regulation by Wilms' tumor-1 gene (62). The effect of Wilms' tumor-1 depends on the number of Wilms' tumor-1 binding sites located both upstream and downstream of the insulin-like growth factor I receptor transcription initiation site (63). Repressors that pose interference with the function and assembly of initiation complex without binding DNA have also been described (47)(48)(49)(50). However, in our case, at least two protein-binding DNA motifs, located upstream and downstream of the transcription initiation site, are needed for repression, a situation analogous to the requirement for multiple sites for silencing of insulinlike growth factor I receptor or vimentin gene expression (63,64). Interaction of the putative repressor protein(s) with multiple sites (CSS and NMS) located on both sides of the transcription initiation site might be a requirement for the physical and structural rigidity needed for the negative control of transcription initiation.
To understand further the CSS-mediated repression, we obtained a partial 1.2-kilobase cDNA encoding the CSS binding factor, Nished. Although the evidence on its involvement in repression of transcription is not yet available, the primary sequence analysis of the Nished cDNA revealed a similarity to other known repressors, such as runt of Drosophila melanogaster (28,65), and SP3 (29). Runt is required for suppression of certain abdominal genes to limit the domains of engrailed expression in parasegmental pattern in Drosophila (16,53). It has a conserved domain RHD, with an ATP binding box located within, which is essential for DNA binding and heterodimerization. Thus, Nished, based on its sequence similarity to these functional domains, appears to belong to a family of transcriptional inhibitors. Activation of Nished binding activity in skeletal muscle may be an important prerequisite for suppression of cardiac MLC2 gene expression in skeletal muscle. It is not yet known whether Nished is one of the two CSSBPs observed in skeletal nuclear extracts. The generation of Nished-specific antibodies, presently in progress in our laboratory, may help in elucidation of its role in repression mechanism(s) underlying the tissue-specific expression of cardiac MLC2.