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Biochimica et Biophysica Acta 1779 (2008) 74–80www.elsevier.com/locate/bbagrm

Promoter paper

Identification of two E-boxes that negatively modulate the activity of MyoDon the alpha-sarcoglycan core promoter

Paul Delgado-Olguín a,b, J. Manuel Hernández-Hernández a, Fabio Salamanca a,Félix Recillas-Targa b, Ramón M. Coral-Vázquez a,⁎

a Unidad de Investigación Médica en Genética Humana, Hospital de Pediatría, Centro Médico Nacional Siglo XXI-IMSS,Av. Cuauhtémoc No. 330, Col. Doctores, Delegación Cuauhtémoc, México D.F. 06725, México

b Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad de México, México

Received 14 May 2007; received in revised form 22 September 2007; accepted 24 September 2007Available online 3 December 2007

Abstract

The α-SG promoter is composed of a plethora of cis-regulatory elements, whose individual contribution to α-SG gene expression modulationremains unknown. We have identified a negative regulatory element in the α-SG distal promoter including two conserved E-boxes (E1 and E2),which interact with MyoD. We found that E1 and E2 negatively modulate the transactivation potential of MyoD on the α-SG core promoter.Moreover, such negative effect is mainly mediated by E2, which is surrounded by conserved nucleotides conferring MyoD binding capacity. Ourresults suggest that modulation of MyoD activity by E1, and particularly E2, contributes to the negative regulation of α-SG gene expressionduring myogenic differentiation.© 2007 Elsevier B.V. All rights reserved.

Keywords: α-sarcoglycan promoter; MyoD transactivation; Core promoter

Alpha sarcoglycan (α-SG), a protein whose gene is expressedsolely in striated muscle [1], forms part of the sarcoglycan (SG)subcomplex, composed of transmembrane proteins sarcospan,and α-, β-, γ-, and δ-SGs, which form part of the Dystrophinglycoprotein complex (DGC) [2–4]. Mutations in the SG genesare responsible for limb girdle muscular dystrophies type 2D,2E, 2C and 2F, respectively [5–8], while mutations in thedystrophin gene lead to Duchenne and Becker musculardystrophy [9]. Other components of the SG subcomplex, ɛ-and ζ-SGs, have been discovered [10,11], although theirdeficiency has not been directly associated with musculardystrophy. The DGC confers a structural link between laminin-α2in the extracellular matrix and F-actin in cytoskeleton [12], andis thought to protect muscle cells from contraction-induceddamage [13].

The analysis of SGs' knock out mouse models have broadenedour knowledge about the roles of SGs in muscular dystrophy,and have highlighted the importance of controlling their gene

⁎ Corresponding author. Tel.: +52 55 56276941; fax: +52 55 55885174.E-mail address: rmcoralv@prodigy.net.mx (R.M. Coral-Vázquez).

1874-9399/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.bbagrm.2007.09.002

expression level in potential therapeutic approaches [14,15]. Inthis regard, deficiency and over-expression of γ-SG causemuscular dystrophy in mice [16,17], suggesting that SGs geneexpression is highly regulated. In support of this idea, γ- and α-SGs are activated during myogenic differentiation [18,19] andtheir promoters are composed of multiple regulatory elementsdirecting differential activities [18–20]. However, the mechan-isms governing SG gene expression remain poorly understoodand there is very little data regarding the individual contributionof such sequences to controlled expression during myogenicdifferentiation.

The α-SG promoter presents basal activity in myoblasts andis upregulated during myogenic differentiation [18,19] through aprocess probably involvingNFI transcription factors, which playpositive and negative regulatory functions [19]. In addition, theα-SG promoter is targeted by MyoD [20], which controlsmuscle-specific gene expression [21] through cis regulatorysequences known as E-boxes [22]. MyoD is the foundingmember of the family of myogenic regulatory factors (MRFs),also including Myf5, Myogenin and MRF4, which inducemyogenesis when transfected in a variety of non-muscle cells

Fig. 1. A α-SG promoter regulatory element negatively modulates the activityof MyoD. Constructs F5 and F6, spanning the indicated nucleotides with respectto the transcription start site (upper panel), were transiently transfected in C2C12cells in the absence (−) or presence (+) of a MyoD cDNA. Luciferase activity wasmeasured in myoblasts and myotubes. The data in this and followingexperiments, expressed as fold activation, represent the mean±S.D. of twoindependent experiments performed in triplicate.

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[21,23]. Gene expression analysis in MyoD transduced fi-broblasts revealed subsets of up and downregulated genes [24],suggesting that the MyoD transcriptional output is modulated ina promoter-specific way. Recent evidence showed thatMyoD, inconjunction with basal transcription factors, interact with a DNAregion located in the α-SG distal promoter in the chromatincontext of C2C12 myoblasts, and concomitantly with the distaland proximal promoter in myotubes [20]. Interestingly, deletionof such a distal promoter region, which includes two E-boxes,caused increasedα-SG promoter activity, opening the possibilityof MRFs involvement in negative α-SG gene regulation [20].

In order to further our understanding of the mechanismsgoverning α-SG gene expression during myogenic differenti-ation, we investigated the function of the potential suppressorelement located in the α-SG distal promoter and whether MyoDplays a role in its transcriptional influence. We found that the α-SG promoter region spanning from −1710 to −1892 negativelymodulates the transactivation potential of MyoD on the α-SGpromoter during myogenic differentiation. Overexpressionassays revealed that such negative effect on MyoD relies atleast in part on two conserved E-boxes, named E-box1 (E1) andE-box2 (E2), which bind MyoD in vitro. Further analysisshowed that the negative influence of MyoD activity on α-SGcore promoter is mediated by conserved nucleotides near the E2core sequence, which are indispensable for MyoD interaction.These results suggest that modulation of MyoD activity by E1and E2 could be a conserved event for suppression of α-SGgene expression during myogenic differentiation.

We previously identified a putative negative regulatoryelement in the α-SG distal promoter spanning nucleotides from−1710 to −1892, relative to the transcription start point, thatinclude E1 and E2, around which, MyoD and basal transcriptionfactors locate during C2C12 cells myogenic differentiation [20].These facts raised the possible involvement of MyoD or otherMRFs in α-SG promoter negative regulation. To address if theα-SG distal promoter region including nucleotides from −1710to −1892 influences the transactivation potential of MyoD onpromoter activity, we transfected constructs F5 and F6, whichexpress luciferase under control of two α-SG promoter frag-ments [20], in the absence or presence of aMyoD-encoding cDNAinto C2C12 cells (Fig. 1). F5 and F6 constructs include nu-cleotides spanning from +4 to −1892 and from +4 to −1710,respectively [20]. Cell maintenance, differentiation, transfec-tion, as well as luciferase activity assays in myoblasts andmyotubes were performed as previously described [19]. In-tegrity of the constructs employed in this work was confirmedby sequencing with a dye terminator cycle reaction kit (ABIPRISM 310, Applied Biosystems, Foster City, CA, USA).Luciferase activity data in this and further experiments are rep-resented as mean±standard deviation (S.D.). Deletion of thefragment comprising nucleotides from −1710 to −1892 inconstruct F6 resulted in 1.5- and 2.5-fold activity increment, ascompared with F5, in myoblasts and myotubes, respectively(Fig. 1; lanes 1 and 3) in agreement with a previous study [20].When co-transfected with MyoD, construct F5 was induced by2.5- and 2-fold in myoblasts and myotubes, respectively (Fig. 1;lane 2). In contrast, construct F6, which lacks nucleotides

−1710 to −1892 was 4- and 32-fold trans-activated by MyoD inmyoblasts and myotubes, respectively (Fig. 1; lane 4). Theseresults confirm the negative role of the α-SG distal promoterregion including nucleotides from −1710 to −1892; moreover itsuggests that negative modulation of MyoD transactivationpotential by such a distal regulatory element could contribute toα-SG gene repression.

Alignment of α-SG promoter sequences from human andmouse revealed that E1 and E2, located in the putative repressorelement at the α-SG distal promoter, are conserved in bothspecies (Fig. 2A). In contrast to E1, which has central variablenucleotides that differ between the analyzed sequences, E2 isfully conserved (Fig. 2A). This observation in conjunction withthe previous result (Fig. 1) opened the possible involvement ofE1 and E2, particularly E2, in α-SG gene expression negativeregulation. We investigated whether E1 and E2 influence the α-SG core promoter during myogenic differentiation (Fig. 2B);and if they play a role in the negative effect exerted by the α-SGdistal promoter region on MyoD activity (Fig. 3). To test theinfluence of E1 and E2 on α-SG gene expression, we transfectedconstructs α-SGCP [20] and E1-E2CP, which contain the α-SG

Fig. 2. E1 and E2 negatively influence the α-SG core promoter and interact with MyoD. (A) Alignment of the sequences corresponding to E-box1 (E1) and E-box2(E2) from mouse and human showed partial and full conservation of E1 and E2, respectively. ⁎ indicates nucleotide identity. – indicates gaps. (B) Constructs, α-SGCPor E1–E2 CP, containing the α-SG core promoter alone, or fused to E1 and E2, respectively, were transiently transfected in C2C12 cells. Luciferase activity, expressedas fold reduction, was assessed inmyoblasts andmyotubes. (C) EMSAswere performed employing as a probe oligonucleotides including the E1 and E2 sequence (A), or anSp1 binding site. The reactions were prepared with myoblast (MB) or myotube (MT) nuclear extracts (NE) in the presence (+) or absence (−) of anti-MyoD or -GFPrecognizing antibodies (α-MyoD and α-GFP). The identified complexes are indicated with numbers at the right of each panel. Arrows indicate the complexesdiminished by the presence of anti-MyoD antibody. Self unlabeled oligonucleotides were employed as competitor (Comp). The right triangle indicates that increasingamounts of competitor were employed. ⁎ indicates unspecific complexes. (D) EMSAusing in vitro synthesizedMyoD–E12 (TNTMyoD–E12) in the presence of the E1–E2probe. Incubation of extracts (E) with the probe caused formation of a complex (arrow), which was competed with unlabeled oligonucleotides (self) and not with themutated version of E2 (E2mut) or an Sp1 binding site. ⁎ indicates the unspecific complex revealed in the presence of unprogrammed extract (lane 1).

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core promoter, spanning nucleotides from +4 to −76, and aDNA sequence including E1 and E2 upstream the α-SG corepromoter, respectively, into C2C12 cells (Fig. 2B). E1-E2CPconstruct was generated by insertion of annealed complemen-tary oligonucleotides, containing E1 and E2 sequences(Fig. 2A), upstream the α-SG core promoter in construct α-SGCP. E1 and E2 caused α-SG core promoter activity down-regulation by 2.5- and 1.8-fold in myoblasts and myotubes,respectively (Fig. 2B), suggesting that in fact E1 and E2 couldplay a role α-SG gene repression.

Previous chromatin immunoprecipitation experiments re-vealed that MyoD and basal transcription factors interact with aDNA region potentially including E1 and E2 in the C2C12cell's chromatin context [20]; however, if MyoD directly in-

teracts with such regulatory elements remained uncertain. Toaddress this issue we employed annealed [γ-32P] ATP labeledoligonucleotides including E1 and E2 in electrophoretic mo-bility shift assays (EMSA), in presence of C2C12 myoblast andmyotube nuclear extracts (Active Motif) (Fig. 2C), aspreviously described [19]. The sequence of the oligonucleotidesemployed as a probe, as well as their position relative to the α-SGtranscription start site, is shown in Fig. 2A. Protein interactionwith E1 and E2 probe in myoblast and myotube nuclear extractsfavored the formation of three and six complexes, respectively(Fig. 2C; lanes 2 and 8), suggesting that E1 and E2 interact withproteins whose encoding genes are differentially regulated orthat protein–protein interaction between complexes' memberschange during myogenic differentiation. Several examples have

Fig. 3. E1 and E2 negatively modulate the activity of MyoD on the α-SG corepromoter. (A) Constructs α-SGCP or E1–E2 CP, containing the α-SG corepromoter alone, or fused to E1 and E2, respectively, were transfected alone (–)or co-transfected with increasing amounts of MyoD, or 1 μg of myogenin cDNAsin C2C12 cells. Luciferase activity was assessed in myoblasts and myotubes.(B) Constructs α-SGCP, E1–E2 CP, described in panel A, as well as constructsE1MUT CP, E2MUT CP and E1–2MUT CP, including mutations in E1, E2 orboth, respectively, were co-transfected with a MyoD cDNA. Luciferase activitywas assessed in myoblasts and myotubes.

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been reported showing differentiation-dependent switching ofMyoD regulatory partners. For example, MyoD interacts withp300/CBP and PCAF histone acetyltransferase complexes, andHDAC1 histone deacetylase in C2C12 myoblasts and myo-tubes, respectively [25]. To find out if MyoD forms part of anyof the identified complexes interacting with E1 and E2, weadded an anti-MyoD antibody (M-318 Santacruz Biotechnol-ogy) before incubation of the probe with myoblast or myotubenuclear extracts (Fig. 2C; lanes 4, 5 and 12). Addition of suchantibody abolished complexes 1 and 3 in myoblasts and myo-

tubes, respectively (Fig. 2C; lanes 4, 5 and 12). In contrast, thepresence of an anti-GFP antibody had no effect on the com-plexes (Fig. 2C; lanes 6 and 13). Self competition withan unlabeled probe abolished the complexes (Fig. 2C; lanes 3 and9–11). Incubation of myoblast or myotube nuclear extracts withan Sp1 binding probe revealed defined complexes, supportingthe integrity of the extracts employed (Fig. 2C; lanes 14–17). Tostrengthen these results, we addressed the capacity of forcedMyoD–E12 heterodimers to interact with E1 and E2 (Fig. 2D).E12 and E47 transcription factors, which confer DNA bindingspecificity [26], are necessary for MRFs' function in vivo [27].We employed the TnT Quick Coupled Transcription/TranslationSystem (Promega) to synthesizeMyoD–E12 heterodimers from aPCR template amplified from pFB-FL-MyoD/E47 vector [28].Incubation of the E1–E2 probe with TNT extract resulted inthe formation of a retarded complex (Fig. 2D; lane 2), which wasself competed with an unlabeled probe (Fig. 2D; lanes 3 and 4);while preincubation with the mutant version of E2 or unlabeledSp1 binding oligonucleotides, did not affect the DNA–proteininteractions (Fig. 2D; lanes 5 and 6). These results suggest thatthe α-SG distal promoter elements E1 and E2 interact withprotein complexes including MyoD in C2C12 myoblasts andmyotubes, opening the possibility of their involvement innegative modulation of MyoD activity on the α-SG corepromoter during myogenic differentiation.

Next, we tested if indeed E1 and E2 influence the activity ofMyoD on the α-SG core promoter (Fig. 3). To this end, we co-transfected constructs α-SGCP or E1–E2CP, containing the α-SG core promoter alone or fused to E1 and E2, respectively, withincreasing amounts of a myc-tagged MyoD-encoding plasmid,into C2C12 cells (Fig. 3A). We also transfected a myogenin-encoding cDNA, which did not affect the activity of the α-SGcore promoter (Fig. 3A). MyoD transactivated the α-SG corepromoter in myoblasts and myotubes by 33- and 38-fold,respectively (Fig. 3A); whereas in the presence of E1 and E2,such promoter was transactivated byMyoD to a maximum of 13-and 22-fold in myoblasts and myotubes, respectively (Fig. 3A),suggesting that E1 and E2 negatively modulate the transactiva-tion potential of MyoD on the α-SG core promoter. To supportthis hypothesis, we generated constructs E1MUT CP, E2MUTCP and E1–E2MUT CP, which contain mutations disrupting E1,E2 and both, respectively. Mutant constructs were generatedwith the Quickchange mutagenesis kit (Stratagene) according tomanufacturer's instructions. Primer sequences employed inthese and further experiments are available upon request. Thoseconstructs were co-transfected with MyoD in C2C12 cells.Consistent with our previous experiment (Fig. 2B), E1 and E2caused a decrease in the activity of MyoD on the α-SG corepromoter in myoblasts and myotubes (Fig. 3B). Ablation of E1relieved the negative effect on the activity of MyoD on the α-SGcore promoter in myoblasts, but not in myotubes (Fig. 3B).In contrast, disruption of E2 relieved the negative influenceon MyoD in myoblasts and myotubes; moreover it seemedto cause an increment in the promoter response to this myogenicregulatory factor (Fig. 3B). These data confirm a negativeregulatory role of E1 and E2 on α-SG promoter activity; more-over they suggest differential contribution of such elements in

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modulation of MyoD activity on the α-SG core promoter duringmyogenic differentiation. In conjunction with the fact that E2 isfully conserved in mouse and human (Figs. 2A and 4A), theseresults suggest that the negative influence of E2 over MyoDcould play a relevant role on α-SG gene expression regulation.

Core and flanking sequences, besides the central variablenucleotides, determine the specificity and affinity of the inter-action of bHLH transcription factors with E-boxes [29]. Align-ment of E2 and the surrounding sequence frommouse and humanrevealed conserved nucleotides (Fig. 4A), which could be im-portant for E2-mediated negative modulation of the transactiva-tion potential of MyoD on the α-SG promoter. To address thishypothesis, we first asked whether E2 is able to interact withforced MyoD-E12 heterodimers (Fig. 4B). To this end, weperformed EMSA employing as a probe annealed oligonucleo-tides corresponding to E2 sequence. Incubation of E2 probe within vitro synthesized MyoD–E12 favored the formation of aretarded complex, which was self competed with an unlabeledprobe (Fig. 4B; lanes 11 and 12) and not with annealed oligo-nucleotides including the E1 sequence (Fig. 4B; lanes 4 and 5), orthe Sp1 recognition site, employed as negative control (Fig. 4B;

Fig. 4. E2 conserved surrounding nucleotides are critical for MyoD activity modulatihuman are boxed. Mutant versions of the mouse E2 tested in EMSA are shown (mut1,E2 sequence and in vitro synthesized MyoD–E12 (TNT MyoD–E12). Oligonucleosubstitutions indicated in A, and an Sp1 binding site, were employed as competitotriangle indicates that increasing amounts of competitor were used. ⁎ indicates the unsp(C) EMSA employing E2 and mut3 (A) (m3), as probes, and in vitro synthesized Minteractions were competed with E2 and E1 unlabeled oligonucleotides (self and E1probe. Arrows in B and C indicate specific retarded complexes. (D) Constructs, mut1mut2 and mut3, (A) respectively, as well as a mutated E1. These constructs, as well ascells. Luciferase activity was measured in myotubes.

lanes 6, 23 and 24). This finding indicates that E2 but not E1interacts with MyoD–E12 heterodimer in vitro; nonetheless, E1mutation relieved the negative modulation of MyoD activity onthe α-SG core promoter in myoblasts (Fig. 3B) indicatingits functionality. Next we addressed if the conserved nucleotidessurrounding E2 are involved in MyoD–E12 binding (Fig. 4B). Tothis end, we performed EMSA employing E2 as a probe, and ascompetitors, annealed oligonucleotides containing the E2sequence with substitutions in its conserved surrounding nu-cleotides (Fig. 4A and 4B). We found that the mutated unlabeledoligonucleotides competed DNA–protein interactions to a sim-ilar extent as the intact self competitor (Fig. 4B; compare lanes11 and 12 with 15–16, and 19–22), suggesting that the sub-stituted nucleotides are not involved in MyoD–E12 recognitionof E2. However, incubation of mut2 competitor rendered a moreclearly defined complex (Fig. 4B; lanes 17 and 18), suggestingthat the E2 central nucleotides confer MyoD binding affinity.These results clearly demonstrate that E2 is able to interactwith MyoD–E12 heterodimers, supporting its involvement innegative modulation of MyoD activity on the α-SG promoter.Next, we tested if the conserved E2 surrounding nucleotides are

on. (A) Conserved nucleotides in and around E2 core sequence from mouse andmut2, mut3 and mut4). (B) EMSA assays employing oligonucleotides includingtides containing E1 sequence, a disrupted E2 sequence (E2mut) or E2 with thers. E2 unlabeled oligunucleotides were employed as self competitor. The rightecific complex revealed in the presence of unprogrammed extract (lanes 2 and 9).yoD and Myogenin (TNT MyoD and TNT Myog, respectively). DNA–protein, respectively). Lane 1 corresponds to unprogrammed extract in presence of E2CP, mut2 CP, and mut3 CP contain the nucleotide substitutions indicated as mut1,α-SGCP and E1–E2 CP, were co-transfected with a MyoD cDNA (+) in C2C12

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involved in the recognition of MyoD homodimers, which bindDNAwith less specificity as compared with the MyoD–E12/E47heterodimer [30]. Ablation of the GGCC nucleotides 5′ of E2 didnot affect MyoD interaction with E2, (data not shown) whereasablation of the central CC nucleotides affected the interaction ofMyoD in a similar way as MyoD–E12 (Fig. 4B) (data notshown). In contrast, substitution of the CCC nucleotides 3′ of E2abolished the interaction of E2 with MyoD and Myogeninhomodimers (Fig. 4C; lanes 6 and 11). The complex formed byinteraction of E2 with MyoD and myogenin (Fig. 4C; lanes 3 and8) was self competed with unlabeled oligonucleotides (Fig. 4C;lanes 4 and 9) and was not affected by preincubation with E1competitor (Fig. 4C; lanes 5 and 10), employed as negativecontrol. These results suggest that the CCC nucleotides conferMyoD binding capacity and could modulate the specificity ofMRFs interactions with E2.

Finally, we addressed the functionality of the conserved E2surrounding nucleotides in the negative effect on MyoD activityon the α-SG promoter (Fig. 4D). To reach this goal we generatedthree constructs in which we introduced the same nucleotidesubstitutions as those tested in the EMSA (Fig. 4A). Weemployed as template construct E1MUT CP (Fig. 3B), in whichE1 is disrupted, thereby providing a good means to test thefunctionality of E2 conserved central and flanking nucleotideswithout potential interference from E1 activity. The MyoDresponsiveness of the generated constructs: mut1 CP, mut2 CP,mut3 CP, containing theα-SG core promoter fused to a disruptedE1, and E2 harboring substitutions of the central and flankingconserved nucleotides (Fig. 4A), was analyzed in C2C12myotubes, in which ablation of E1 did not affect the activity ofMyoD on the α-SG core promoter (Fig. 3B). Substitution ofGGCC nucleotides located 5′ of E2, as well as the central CCnucleotides in constructs mut1 CP and mut2 CP, respectively,relieved the negative effect onMyoD activity over theα-SG corepromoter (Fig. 4D). MyoD activated the α-SG core promoter inthese constructs to a similar extent as the core promoter alone(Fig. 4D). In contrast, substitution of the CCC nucleotides 3′from E2 (Fig. 4A) in construct mut3 CP, caused a significantincrement in the α-SG core promoter responsiveness to MyoD(Fig. 4D). These results further support a role of E2 in negativeα-SG gene expression regulation by modulating MyoD activitythrough its central and flanking conserved nucleotides. More-over, the results suggest that E-boxes, which are thought to beessentially cis-regulatory activators, finely modulate the MyoD-responsiveness of muscle promoters.

In this work we identified E1 and E2, which negativelyregulate the α-SG gene promoter during myogenic differenti-ation potentially by modulating MyoD transactivation potential.In agreement with this proposal, MyoD in conjunction withbasal transcription factors interact with a genomic region in-cluding E1 and E2 but not with the α-SG core promoter inC2C12 myoblasts, where the promoter is inactive [20]. MyoDand basal transcription factors contact both genomic regions inmyotubes, where α-SG gene is fully expressed [20], althoughwe found that at this stage, E1 and E2 still negatively modulatethe activity of MyoD on the α-SG promoter. In this scenario, it ispossible that E2, which is flanked by conserved nucleotides that

appear to confer MyoD binding specificity, mediate MyoDpreferential association with E1 and E2 over the core promoter inmyoblasts and myotubes. These facts suggest squelching ofMyoD and basal transcription factors as a potential way of actionof E1 and E2 in negative modulation of MyoD activity on the α-SG promoter during myogenic differentiation. However thispossibility seems unlikely because our data show that E1 and E2exert a negative effect on the activity of MyoD when clonedimmediately upstream of the α-SG core promoter. Alternatively,inaccessible chromatin structure could prevent MyoD binding tothe α-SG core promoter in myoblasts. MyoD has been linked togene repression in myoblasts as well as in MyoD-transduceddifferentiating cells [24]. Different co-repressors, like HDACs[31], and histone methyltransferases like Suv39h1 inhibit myo-genesis by linking negative activities to MyoD [32]. On the otherhand, interaction of MyoD with co-activators like SWI/SNFcomponents favors myogenic differentiation [33]. These factsraise the possible interaction of MyoD-associated repressor oractivator complexes specifically with E1 and E2 or the α-SGcore promoter, respectively. In this regard, it is possible that inthe α-SG promoter chromatin context, the MyoD-containingcomplex associated with E1 and E2 incorporates negative ac-tivities from nearby interacting transcriptional repressors. Forexample, there is evidence suggesting that nuclear factor I-C(NFI-C), which negatively regulates the α-SG promoter, binds toa sequence located downstream from E1 and E2 [19]. In ad-dition, such NFI binding site is adjacent to a consensus sequencefor recognition by Sp1, which also plays a negative role in α-SGpromoter activity (unpublished results). These facts open theinteresting possibility of the involvement of NFI and Sp1 inmodulation of MyoD-mediated transcriptional activation.

Our results indicate that the conserved E2 surroundingnucleotides are critical for the negative modulation of MyoD onthe α-SG core promoter; despite that they seem to be dispensablefor recognition by MyoD–E12, which is the physiologicallyactive heterodimer [27]. In contrast, the conserved CCC nucle-otides 5′ from E2 appear to mediate interaction with MyoDhomodimers, which present modest E-boxes binding affinity[30]; however, ablation of such nucleotides caused an increase inthe α-SG promoter responsiveness to MyoD. A possible ex-planation could be that substitution of such nucleotides would beunfavorable for MyoD homodimers binding, while permittingMyoD-E12/E47 heterodimers-mediated transactivation. Theseresults open the possibility that MyoD homodimers formationconstitute a means to negatively modulate transactivation po-tential through interaction with key nucleotides surrounding E-boxes, which seems interesting given the limited informationabout the biological function of MyoD homodimers.

We conclude that α-SG gene expression is tightly regulatedby the action of several distal regulatory elements including E1and E2, which exert a negative influence on the trans-activationpotential of MyoD on the core promoter. In addition, E2 couldnegatively affect the activity of MyoD when such factor in-teracts with conserved surrounding nucleotides, suggestingthat E-boxes, besides acting as transcriptional enhancers, fine-ly modulate the muscle-specific promoter responsiveness toMRFs.

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Acknowledgements

We thank to Georgina Guerrero Avendaño for technicalassistance. We thank to Benoit G, Bruneau for his help with ex-periments and critical reading of the manuscript, Jeffrey F.Dilworth (Ottawa Health Research Institute) and Robin Meech(The Scripps Research Institute) for providing pFB-FL-MyoD/E47 and pCDNA-MyoD vectors, respectively. P.D.O was sup-ported by the Consejo Nacional de Ciencia y Tecnología(CONACyT) (Grant 158524), the Dirección General de Estudiosde Posgrado, UNAM, and the IMSS fellowship award. R.C.V. issupported by the Association Française contre les Myopathies(Grant MNM 2000 Groupe 1). F.R.T. is supported by CONACyT(Grants 33863-N and 42653-Q), PAPIIT-DGAPA (IN203200 andIN209403), The Third World Academy of Sciences (TWAS,Grant 01-055RG/BIO/LA) and Fundación Miguel Alemán.

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