Overexpression of CUG Triplet Repeat-binding Protein, CUGBP1, inMice Inhibits Myogenesis*

Received for publication, November 26, 2003, and in revised form, December 30, 2003Published, JBC Papers in Press, January 13, 2004, DOI 10.1074/jbc.M312923200

Nikolai A. Timchenko‡, Roma Patel§, Polina Iakova‡, Zong-Jin Cai§, Ling Quan§,and Lubov T. Timchenko§¶�

From the Departments of Pathology and Huffington Center on ‡Aging, §Medicine, and ¶Molecular Physiology andBiophysics, Baylor College of Medicine, Houston, Texas 77030

Accumulation of RNA CUG repeats in myotonic dys-trophy type 1 (DM1) patients leads to the induction of aCUG-binding protein, CUGBP1, which increases trans-lation of several proteins that are required for myogen-esis. In this paper, we examine the role of overexpres-sion of CUGBP1 in DM1 muscle pathology usingtransgenic mice that overexpress CUGBP1 in skeletalmuscle. Our data demonstrate that the elevation ofCUGBP1 in skeletal muscle causes overexpression ofMEF2A and p21 to levels that are significantly higherthan those in skeletal muscle of wild type animals. Asimilar induction of these proteins is observed in skele-tal muscle of DM1 patients with increased levels ofCUGBP1. Immunohistological analysis showed that theskeletal muscle from mice overexpressing CUGBP1 ischaracterized by a developmental delay, muscular dystro-phy, and myofiber-type switch: increase of slow/oxidativefibers and the reduction of fast fibers. Examination ofmolecular mechanisms by which CUGBP1 up-regulatesMEF2A shows that CUGBP1 increases translation ofMEF2A via direct interaction with GCN repeats locatedwithin MEF2A mRNA. Our data suggest that CUGBP1-mediated overexpression of MEF2A and p21 inhibitsmyogenesis and contributes to the development of mus-cle deficiency in DM1 patients.

DM11 is a multisystem disease mainly characterized by de-fects in skeletal muscle with the involvement of many tissuesand systems such as cardiac muscle, brain, eye, and endocrinesystem (1). DM1 is caused by an expansion of CTG trinucle-otide repeats within the 3�-untranslated region of the myotoninprotein kinase gene (2). In DM1 patients, the size of DNA CTGexpansion correlates with the severity of the disease. Patientswith CTG expansion containing 50–80 CTG repeats are almostasymptomatic. Individuals bearing the myotonin protein ki-nase gene with 100–500 CTG repeats develop a disease inadult life (classical adult form of DM1) that is characterized by

a progressive muscle wasting with myotonia. The most severeform of DM1, congenital disease, affects patients before or afterbirth and is associated with long CTG expansions (up to 2,000repeats). This form of disease is characterized by a delay orarrest of skeletal muscle development (3). Although there is anoverlap in range of repeats between different forms of DM1,there is a clear correlation of repeat number with severity ofphenotype and reduction of age of onset.

Investigations of molecular alterations in DM1 suggest thatthe expansion of CTG repeats causes the DM1 pathologythrough different mechanisms, mediated at both DNA andRNA levels. It has been shown that CTG repeats reduce ex-pression of myotonin protein kinase in cis (4), causing abnor-malities in cardiac muscle (5). CTG repeats also affect tran-scription of genes adjacent to myotonin protein kinase (6),leading to the development of cataracts (7, 8). A number ofrecent studies indicate that other symptoms in DM1 such asmyotonia (9, 10), delay of skeletal muscle differentiation (11,12), and a resistance to insulin (13) are mediated through anRNA-based mechanism. According to this mechanism, RNACUG repeats cause the DM1 disease via specific RNA CUG-binding proteins (14).

Two families of RNA CUG-binding proteins have been iden-tified: CUGBP (RNA-binding protein, binding to RNA CUGrepeats) (15–17) and EXP (also known as MNBL, muscle blind)proteins (18). One of the members of the CUGBP family,CUGBP1, contributes to at least three symptoms of DM1: thedelay of skeletal muscle differentiation (12), myotonia (19), andthe insulin resistance (13). Previous studies showed that theexpansion of CUG repeats in DM1 leads to a 3–5-fold elevationof CUGBP1 binding activity and protein levels (13, 19–21),perhaps by stabilizing CUGBP1 protein within RNA CUG-CUGBP1 complexes (21). CUGBP1 possesses two major biolog-ical functions: regulation of splicing and translation (12, 20–22). Translational function of CUGBP1 was investigatedmainly in cultured cells. These studies showed that CUGBP1increases translation of p21 (12) and translation of a dominantnegative isoform of transcription factor CCAAT/enhancer-bind-ing protein �, LIP (22). It has been shown that, in tissue culturemodels, CUGBP1-mediated induction of p21 is required forproper differentiation of myocytes (12). However, the role oftranslational function of CUGBP1 in vivo has not beenaddressed.

To examine the role of translational activity of CUGBP1 indevelopment of skeletal muscle deficiency in DM1 patients, wegenerated transgenic mice that overexpress CUGBP1 mainlyin skeletal muscle and to a lesser extent in the heart. We foundthat elevation of CUGBP1 causes overexpression of its trans-lational targets p21 and MEF2A (transcription factor, myocyteenhancer factor 2A) in skeletal muscle. These alterations cause

* This work is supported by National Institutes of Health GrantsAR44387 (to L. T. T.), AR49222 (to L. T. T.), GM55188, AG20752, andCA100070 (to N. A. T.), and by a grant from the Muscular DystrophyAssociation (to L. T. T.). The costs of publication of this article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

� To whom correspondence should be addressed: Baylor College ofMedicine, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-6911;Fax: 713-798-3142; E-mail: lubovt@bcm.tmc.edu.

1 The abbreviations used are: DM1, myotonic dystrophy type 1;sORF, short out of frame open reading frame; C/EBP�, CCAAT/enhancer-binding protein �; RBD, RNA-binding domain; GFP, greenfluorescent protein; H/E, hematoxylin/eosin; IF, immunofluorescence;IP, immunoprecipitation.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 13, Issue of March 26, pp. 13129–13139, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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a muscular dystrophy, an increase of slow fibers, and delay ofmuscle development.

EXPERIMENTAL PROCEDURES

UV Cross-linking—RNA transcripts encoding 123 CUG repeats, thefull-length wild type, and mutant MEF2A mRNAs lacking the CUGBP1binding site were synthesized in an in vitro transcription assay (RocheApplied Science) and incubated with 10–20 �g of purified proteins asdescribed (12). After UV cross-linking, the samples were treated withRNase A (2 units/10 �l) and separated by PAGE with 0.1% SDS. Whereindicated, MEF2A riboprobe containing the (GCA)9 repeat (CUGBP1binding site within MEF2A mRNA) or sORF riboprobe specific forC/EBP� was used.

Generation of CUGBP1 Transgenic Mice—The experiments relatedto transgenic mice complied with federal guidelines and institutionalpolicies. The full-length human CUGBP1 cDNA was amplified from aparental plasmid (17) with CUGBP1-specific primers. The sequence ofthe forward primer is as follows: 5�-TCA AAG AAA ATG AAC GGC ACCCTG-3�. The sequence of the reverse primer fused in frame with a shortsequence coding for 6 histidines is as follows: 5�-ATG GTG ATG GTGATG ATG GTA GGG CTT GCT GTC ATT CTT CGA-3�. The CUGBP1cDNA fused with the track of histidines on its C terminus was clonedinto BS vector containing chicken �-actin promoter connected withcytomegalovirus enhancer (a gift from Dr. Schneider, Baylor College ofMedicine). Transgenic construct was injected into mouse FVB inbredeggs at DNX Transgenic Sciences Co. (Princeton, NJ).

Genotyping of Transgenic Mice—Progenies were genotyped by PCRwith CUGBP1-specific primers. The sequence of forward primer is 5�-GCT GCA TTA GAA GCT CAG AAT GCT-3�. The reverse primer has asequence 5�-AGG TTT CAT CTG TAT AGG GTG ATG-3�. Two PCRproducts of different size from both wild type (540 bp) and mutantalleles (78 bp) were synthesized simultaneously. The size of PCR prod-uct from mutant allele is smaller because the transgene lacks introns.

Tissue Culture—Mouse C2C12 myoblasts and human primary myo-blast cultures were grown in 10-cm dishes in myoblast growth mediumat 60% of density. Differentiation was induced by a switch of growthmedium to fusion medium as described (12). Differentiating myoblastswere maintained for 5 days with medium changes occurring every day.

Northern Analysis—Total cellular RNA was extracted with TRI Re-agent (Molecular Research Center, Inc.) from C2C12 myoblasts andmyotubes at different time points of differentiation. RNA was hybrid-ized with CUGBP1 probe as described (15). Levels of CUGBP1 mRNA(7 � 9 kb) were calculated as a ratio to 18 S rRNA control.

Immunostaining of Mouse Tissues—Paraffin and frozen sectionswere prepared from vastus or plantaris muscles from wild type andmutant mice with different numbers of CUGBP1 copies of the matchingage and gender. Muscle sections were stained with H/E at the BaylorCollege of Medicine Histology Core. For immunoanalysis, sections wereblocked with 1% bovine serum albumin in phosphate-buffered saline for1 h and incubated for 1 h with antibodies against MEF2 (C-21; 1:1,000),myoglobin (sc-8081; 1:200) (Santa Cruz Biotechnology, Inc., SantaCruz, CA), fast myosin (MY-32; 1:100), and slow myosin heavy chain(NOQ7.54D; 1:50) (Sigma). Rhodamine-labeled anti-rabbit and anti-mouse immunoglobulins from Santa Cruz Biotechnology were used assecondary antibodies with dilution 1:200. Images were analyzed inMicroscopic Core Facilities of Aging Center and Department of Molec-ular and Cellular Biology at Baylor College of Medicine.

Isolation of Proteins and Western Blotting Analysis—Whole proteinextracts from mouse tissues were prepared with radioimmune precipi-tation buffer. Cytoplasmic, nuclear, and whole cell extracts from cul-tured cells were isolated as described (12). The primary antibodies wererabbit anti-CUGBP1, MEF2 (C-21), myoglobin (sc-8081), and MyoD(myogenic transcription factor) (C-20) and mouse p21 (H164), myogenin(CF5D), fast (MY-32) and slow myosin (NOQ7.54D), and �-tubulin(TU-02). Detection was performed using the ECL kit (Pierce). Mem-branes were stripped and reprobed with control antibodies (�-tubulin oractin), and results were quantified by densitometer analysis.

The Effect of CUGBP1 on MEF2A Levels in Cultured Cells—C2C12cells were transfected with empty pcDNA vector and with recombinantplasmid containing CUGBP1 in pcDNA vector using FuGene 6 protocol.Protein extracts were isolated in 48 h after transfection and subjectedto Western blotting with antibodies against His tag, CUGBP1, andMEF2A. CUGBP1 and MEF2A protein levels were calculated as a ratioto �-actin. To visualize the effects of CUGBP1 on MEF2A levels, C2C12cells grown in slides were transfected with GFP as control and withCUGBP1 cloned into GFP vector. Cells were fixed and subjected to IFwith antibodies against MEF2A using secondary antibodies labeled

with rhodamine. CUGBP1 (green signal) and MEF2A (red signal) wereanalyzed at �40 magnification.

Examination of MEF2A Translation in Tissue Culture—One set ofplates with C2C12 cells was co-transfected with MEF2A and CUGBP1,and another set was co-transfected with MEF2A and empty vector. Oneday after transfection, the growth medium was replaced with Met-freemedium. In 24 h, Met-deficient medium was complemented with[35S]Met, and whole cell protein extracts were prepared in 0, 1, 2, 4, and8 h after the addition of [35S]Met. Protein extracts were diluted withphosphate-buffered saline buffer and incubated with antibodies againstMEF2A (1:100) for 4 h at 4 °C, following incubation with protein A-agarose for 1 h. Immunoprecipitates were collected by centrifugation at6,000 rpm for 10 min, washed three times with 1 ml of phosphate-buffered saline, resuspended in the loading buffer, and analyzed bypolyacrylamide gel electrophoresis.

Inhibition of CUGBP1 by siRNA—Two RNA CUGBP1 primers weresynthesized in Qiagen to target the following CUGBP1 sequence: 5�-AAT TTG GCT GCA CTA GCT GCT-3�. The sequence of the first primeris r(UUUGGCUGCACUAGCUGCU)d(TT). The sequence of the secondprimer is r(AGCAGCUAGUGCAGCCAAA)d(TT). These primers wereannealed to make double-stranded siRNA-CUGBP1. Transfection ofsiRNA CUGBP1 was performed according to the Qiagen protocol.C2C12 cells were grown at 70% density. Enhancer R was mixed withsiRNA oligonucleotide and transfection reagent in a ratio of 2:1:2.5.Protein extracts were prepared from proliferating and differentiatingmyoblasts transfected with siRNA. In control plates, no siRNA wasadded.

Translation in an in Vitro Cell-free System—MEF2A plasmid wasidentified by screening of a human cardiac cDNA library with a GCAtriplet repeat probe. GCA deletion mutant was generated by usingQuikChangeTM site-directed mutagenesis kit (Stratagene). The se-quence of forward mutagenic primer is 5�-TGG CGG CGG CTG GAAGCC CGA TGG GGT CAT-3�. Rabbit reticulocyte lysate was programmedwith wild type and mutant MEF2A mRNAs, and increased amounts ofpurified CUGBP1 were added to the incubation mixture. Translationalproducts were subjected to SDS-PAGE and autoradiography.

Interaction of CUGBP1 and MEF2A mRNA in Vivo—C2C12 myo-blasts were grown in the myoblast medium until 80% density and thensubjected to differentiation in the fusion medium during 5 days. Cyto-plasmic protein extracts were collected from C2C12 differentiatingmyoblasts in cytoplasmic buffer (20 mM Tris-HCl, pH 7.5, 30 mM KCl, 5mM EDTA, and 5 mM MgCl2) containing RNase inhibitors. CUGBP1was immunoprecipitated with monoclonal antibodies 3B1. RNA wasextracted by phenol-chloroform and reverse-transcribed with AML us-ing oligo(dT) primer. PCR was performed with primers specific to mouseMEF2A mRNA. The sequence of the forward primer was 5�-GATGTT-GAGCACTACAGACCTCA-3�, and the sequence of the reverse primerwas 5�-CTCCCCTGTGGACAGTCTGAGCA-3�. The size of the PCRproduct with MEF2A primers was 743 bp.

Myogenic Conversion of Fibroblasts into Myoblasts—Mouse fibro-blasts 10T1/2 were grown onto four-chamber slides in Dulbecco’s mod-ified Eagle’s medium with 10% fetal bovine serum. Cells grown at 60%of density were transfected with plasmids expressing MyoD, wild typeand deletion mutant MEF2A, and the full-length or truncatedCUGBP1. The amount of plasmid DNA was 0.2 �g per chamber andremained constant in all experiments. When one plasmid was usedinstead of two or three, the amount of DNA was adjusted with pcDNAvector. Transfection was performed with FuGene. In 2 days after trans-fection, fibroblast growth medium was replaced with myoblast fusionmedium. Cells were grown for 5 days with medium changed every day.Cells were fixed with 4% formaldehyde for 30 min with two changes offresh formaldehyde. Slides were blocked in 1% bovine serum albumin inphosphate-buffered saline containing goat serum (dilution 1:50) for 2 h.After blocking, slides were incubated with mouse antibodies againstfast myosin (Sigma; dilution 1:50) and rabbit antibodies against MyoD(Santa Cruz Biotechnology; 1:50). Secondary anti-rabbit antibodies la-beled with rhodamine or anti-mouse antibodies labeled with fluoresceinisothiocyanate (Santa Cruz Biotechnology) were used at a dilution of1:200.

RESULTS

Generation of CUGBP1 Transgenic Mice—The full-lengthCUGBP1 coding region was placed under a modified �-actinpromoter, which is mainly active in skeletal muscle and to alesser extent in the heart (23), and fused on the C terminus toan oligonucleotide coding for 6 His amino acids. The resultingtransgenic construct was injected into inbred eggs of FVB mice,

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and pups were genotyped by PCR with CUGBP1-specific prim-ers. The expression of transgenic protein (His-CUGBP1) wasverified by Western blotting with antibodies to His tag (Fig. 1,A and B). We first examined expression of His-CUGBP1 indifferent tissues of CUGBP1 transgenic mice. In agreementwith previous publications (23), �-actin promoter fused withcytomegalovirus enhancer directs the expression of His-CUGBP1 primarily in skeletal muscle and to a lesser extent incardiac muscle (Fig. 1A). Within the sensitivity of Western blotanalysis, we did not detect His-CUGBP1 in brain, spleen, andkidney (Fig. 1A). We have generated five lines of CUGBP1transgenic animals (Table I). The analysis of His-CUGBP1 inskeletal muscle of these animals showed that the expression ofHis-CUGBP1 is increased to different levels (Fig. 1B). In addi-tion, we found that the expression of His-CUGBP1 leads to a2–3-fold elevation of the endogenous CUGBP1 levels (Fig. 1C).This estimate was done using protein extracts from vastusmuscles of 3–5 mice of each line and 17 wild type mice. Themechanisms by which the transgenic His-CUGBP1 protein in-duces protein levels of endogenous CUGBP1 are unknown. Ourdata also showed that CUGBP1 immunoreactive proteins mi-grate as several bands, perhaps because of different levels inphosphorylation (24). To examine whether these immunoreac-tive bands are isoforms of His-CUGBP1, we immunoprecipi-tated His-CUGBP1 with polyclonal antibodies to His and per-formed a two-dimensional gel electrophoresis followed byWestern blotting with monoclonal antibodies to CUGBP1. Fig.1D shows that His-CUGBP1 migrates as several spots located

between pH 6.0 and 8.0. Acidic isoforms of His-CUGBP1 rep-resent phosphorylated forms of CUGBP1, since a treatment ofHis-IPs with CIP shifts these isoforms to the alkali region ofthe strip. Given the elevation of both His-CUGBP1 and endog-enous CUGBP1, as well as phosphorylated isoforms ofCUGBP1, CUGBP1 levels were calculated as a ratio of all ofthese isoforms in transgenic animals to endo-CUGBP1 in wildtype animals. These calculations showed that an increase of

TABLE 1The loss of body weight correlates with

the levels of CUGBP1 induction

Mouse line Transgenecopies

Inductionof

CUGBP1proteina

Body weight,percentage of

wild type,newborns

%

CUGBP1–20b 5 5–6 73–77CUGBP1–29c 2 2–3 82–88CUGBP1–32d 1 3 95–100CUGBP1–36e 8 8–10 55–60CUGBP1–49f 6 4–6 72–74

a The induction of CUGBP1 levels in skeletal muscle was calculatedas a ratio of (His-CUGBP1 � endogenous CUGBP1) in transgenic miceto endogenous CUGBP1 in wild type littermates.

b Three litters (21 mice) were weighted, p � 0.001.c Four litters (28 mice) were weighted, p � 0.001.d Three litters (26 mice) were weighted, p � 0.002.e Four litters (32 mice) were weighted, p � 0.001.f Four litters (30 mice) were weighted, p � 0.002.

FIG. 1. Generation of CUGBP1 transgenic mice. A, expression of His-CUGBP1 in tissues of CUGBP1 transgenic mice. Whole cell extractswere isolated from tissues shown at the top and examined by Western blotting with antibodies to the His tag. Coomassie stain of the membraneis shown below. B, expression of His-CUGBP1 in skeletal muscle of CUGBP1-transgenic lines. Western blotting was performed with antibodies tothe His tag. The membrane was reprobed with antibodies to �-actin. The numbers at the top indicate CUGBP1 transgenic lines (see Table I). C,Western blotting of His-CUGBP1 with antibodies against CUGBP1. Western blotting with protein extracts isolated from skeletal muscle of wildtype, CTG transgenic (36), and CUGBP1 transgenic mice (line 36) was performed with antibodies against CUGBP1. The first lane was loaded withCUGBP1 purified from HeLa cells. The red arrows on the right show positions of hyperphosphorylated forms of CUGBP1. The bottom image showsCoomassie stain and the reprobe of the same membrane with antibodies to actin. A -fold increase of CUGBP1 (shown below) was calculated as aratio of (endogenous � His-CUGBP1) signals to CUGBP1 signals observed in wild type animals. D, His-CUGBP1 is hyperphosphorylated inskeletal muscle. His-CUGBP1 was immunoprecipitated from skeletal muscle of CUGBP1 transgenic mice with antibodies to His tag and examinedby two-dimensional gel electrophoresis followed by Western blotting with monoclonal antibodies to CUGBP1. The bottom image shows a similaranalysis of His-CUGBP1 after treatment with CIP. Spots 1, 2, and 3 represent hyperphosphorylated forms.

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total levels of CUGBP1 correlates with the number of trans-genic copies (see Table I). All analyzed animals showed a rel-atively narrow rate of CUGBP1 overexpression (from 2- to10-fold), perhaps due to negative effect of CUGBP1 elevation onmouse development (Fig. 2).

A Severity of Developmental Delay Correlates with Levels ofCUGBP1 Elevation—It has been previously shown that trans-genic mice overexpressing the human DM1 region with greaterthan 300 CTG repeats developed myotonia and are growth-retarded (10). Given the accumulation of CUGBP1 in thesemice, we performed gross analysis of CUGBP1 transgenic micefrom different lines. This analysis showed that mutant mice, inwhich CUGBP1 is elevated to 2–3-fold (lines 29 and 32), haveslight reduction of weight at birth but survive and developnormally. However, transgenic mice, in which CUGBP1 is 4–6-fold elevated, were growth-retarded and significantly under-weight (Fig. 2, A and B). Analysis of three or four litters fromdifferent lines confirmed that weight of newborn CUGBP1transgenic mice is �5–30% less than their wild type litter-mates (Table I) and that the reduction of weight depends on thelevels of CUGBP1 protein. Animals with 8–10-fold elevation ofCUGBP1 die in utero and are severely underdeveloped (Fig.2B). This suggests that the overexpression of CUGBP1 in vivo

might have severe impact on mouse development. Given thestrongest expression of CUGBP1 transgene in skeletal muscle(Fig. 1C), we performed cross-sections through hind limb andfemur of newborn wild type and mutant (line 36) littermatesand found that whereas the size of femur in mutant mice isunchanged, the hind limb muscles look significantly reduced(Fig. 2C). Therefore, we further focused our studies on thehistological analysis of skeletal muscle of CUGBP1 transgenicmice and on molecular pathways by which overexpression ofCUGBP1 causes the disorder in skeletal muscle.

His-CUGBP1 Precipitated from Skeletal Muscle of CUGBP1Transgenic Mice Is Able to Regulate Translation of mRNAs—Invitro studies have demonstrated that CUGBP1 is a key regu-lator of translation of p21 and C/EBP� mRNAs (12, 21, 22). Toexamine whether CUGBP1 displays this activity in vivo, intissues of transgenic mice, we first determined the RNA bind-ing activity of His-CUGBP1 isolated from these tissues and itsability to regulate translation of mRNA targets. UV cross-linkassay of skeletal muscle samples with CUG123 probe showsthat His-CUGBP1, isolated from skeletal muscle cells, is able tointeract with the RNA containing CUG repeats (Fig. 2D). Toconfirm that His-CUGBP1 binds to RNA, we immunoprecipi-tated CUGBP1 with antibodies to His tag and examined in a

FIG. 2. CUGBP1 transgenic mice. A, effect of CUGBP1 transgene on mouse weight. A picture of wild type and two transgenic mice at the ageof 5 days is shown. Heterozygous transgenic animals with 5–6-fold induction of CUGBP1 (TR1) survive but are underweight. A hemizygoustransgenic animal (TR2) with an 8-fold increase of CUGBP1 died at 7 days of age. A bar graph shows a summary of body weight for four littersof line 20 (p � 0.001) and three litters of wild type mice (p � 0.002) with a mean of 10 animals/litter weighted at 10 and 20 days of age. B, CUGBP1transgenic mice with high copies of CUGBP1 transgene die in utero. The picture of wild type and CUGBP1 transgenic mouse (at 20 days ofembryonic development) with 10-fold elevation of CUGBP1 (line 36) is shown. C, skeletal muscles of CUGBP1 transgenic mice are severelydamaged. Cross-sections through hind limbs of wild type and mutant littermates (line 36) are shown. Note that the size of femur is unchanged,but the layer of muscles is significantly reduced. D, His-CUGBP1 from skeletal muscle of CUGBP1 transgenic mice interacts with CUG repeats.Total skeletal muscle protein extracts were incubated with the CUG123 probe and analyzed by UV cross-link assay. After UV cross-link, themembrane was stained with Coomassie (below). Positions of His-CUGBP1 (line 32) and endogenous CUGBP1 are shown. The bottom (His-IP)shows UV cross-link with His immunoprecipitates from WT and CUGBP1 transgenic mice. E, His-CUGBP1 increases translation of p21 and LIPin a cell-free translation system. His-CUGBP1 was precipitated from skeletal muscle with His tag Abs and added into reticulocyte lysateprogrammed with p21 mRNA (top) or with C/EBP� mRNA. After translation, p21 or C/EBP� was immunoprecipitated and examined by gelelectrophoresis. A diagram shows the structure of C/EBP� mRNA and the position of the CUGBP1 binding site.

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UV cross-link assay. As can be seen in Fig. 2D (his-IP), His-CUGBP1 interacts with the CUG123 probe. To perform a func-tional test for translational activity of His-CUGBP1, CUGBP1was immunoprecipitated with antibodies to His tag from skel-etal muscle and added into a cell-free translation system pro-grammed with p21 or with C/EBP� mRNA. These studies dem-onstrated that His-CUGBP1 precipitated from skeletal muscleof transgenic animals induces translation of p21 mRNA as wellas translation of the dominant negative isoform of C/EBP�, LIP(Fig. 2E). Thus, these investigations showed that His-CUGBP1(expressed in tissues of CUGBP1 transgenic mice) binds toRNA and potentially might regulate translation of mRNAsin vivo.

Histological Analyses of Skeletal Muscle from CUGBP1Transgenic Mice—Histological and immunohistochemical anal-yses were performed on vastus and, where indicated, on plan-taris muscles from the five mutant lines (five mice for line 20,six mice for line 29, five mice for line 32, seven mice for line 36,five mice for line 49) and on control littermates (21 mice). Since2–3-fold elevation of CUGBP1 was observed in DM1 patientswith mild symptoms that often are not apparent for decades,the analysis of this transgenic line was performed with agedmice (1.5 years old). Mutant mice with greater increase ofCUGBP1 (4–6-fold) died between 6 and 12 months. Therefore,histology analysis on these mice was performed using musclesfrom 6–8-month-old mice. Mutant mice with the high increaseof CUGBP1 (8-fold, line 36) die after birth. H/E staining in this

case was performed on newborn pups using cross-sectionsthrough hind limb. H/E staining of transverse sections fromvastus muscle of wild type mice shows that in wild type mice,most myofibers are of similar diameter with peripheral nuclei(Fig. 3, A and B). This analysis showed that only 0.3–1% ofmyofibers in wild type vastus muscle contain internal nuclei.However, in mutant adult mice myofibers were variable in sizewith an increase of cells containing internal nuclei up to 9.5–21.1% depending on the increase of CUGBP1 (Fig. 3, A and B).Some myofibers in mutant mice contain nuclei occupying thecentral position. The presence of internal nuclei in CUGBP1mutant mice and size variability suggest that the skeletalmuscle in these mice is immature. The distance between myo-fibers was greater in CUGBP1 mutant mice with greater ele-vation of CUGBP1. We also observed the increase of slowmyofibers in CUGBP1 transgenic mice (Fig. 4C). Comparison ofmyofiber number in vastus muscles of CUGBP1 transgenicmice (lines 29 and 49) and wild type mice suggests that themyofiber number is slightly (2.5–6.3%) reduced in CUGBP1mutant mice, perhaps due to increased variability in myofibersize.

Mutant mice with the high increase of CUGBP1 (8-fold, line36) die after birth. Since congenital DM1 patients have severeunderdevelopment of muscles, we focused on the analysis ofmuscle sections through limbs of new born wild type and trans-genic littermates (Fig. 3C). Two magnified homologous areasfrom the sections showed that myofibers of CUGBP1 trans-

FIG. 3. Muscle deficiency in CUGBP1 transgenic mice. A, H/E staining of transverse and longitudinal sections of vastus muscle from aged(1.5 years) wild type and transgenic mice with 2–3-fold elevation of CUGBP1 (line 29). Myofibers from transgenic mice are variable in size andcontain internal nuclei. B, H/E staining of vastus muscle from wild type and CUGBP1 transgenic mice with a 4–6-fold increase of CUGBP1 (line49) is shown. Longitudinal sections show severe variability of myofiber size. Transverse sections indicate the presence of internal nuclei. C,H/E-stained sections through the hind limb and the femur for wild type and transgenic newborn mice (line 36) are shown. D, sections in the boxedareas show that fibers in CUGBP1 transgenic mice with 8-fold elevation of CUGBP1 are short and unfused and contain multiple nuclei. E,transverse sections of the same mutant muscle show centrally placed large nuclei in 20% of myofibers.

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genic mice are variable in length and are shorter than thosefrom wild type mice (Fig. 3D). Muscle fibers in this line arecharacterized by prominent nuclei. Analysis of transverse mus-cle sections showed that 21–33% cells in mutant mice havelarge nuclei occupying the central position (Fig. 3E). Severalmuscle spindles in mutant mice contain clusters of poorly or-ganized fibers with prominent large nuclei (data not shown).

Overexpression of His-CUGBP1 in Skeletal Muscle Leads toIncreased Expression of MEF2A and p21—We next examinedthe molecular basis for the alterations observed in skeletalmuscles of CUGBP1 transgenic mice. The presence of shortfibers in mutant mice with 8-fold elevation of CUGBP1 (Fig.3D) suggests that CUGBP1 may disrupt major regulators oflate myogenesis, such as myogenin (25) and/or MEF2 factors,which cooperate with basic helix-loop-helix myogenic factors toactivate the differentiation program (26, 27). In a parallelsearch for the possible regulators of myogenesis that might betargeted by CUGBP1, a muscle-specific library was screenedfor the transcripts containing the CUGBP1 binding site (CUGrepeats and/or GC-rich regions). We identified a member ofMEF2 family transcription factors, MEF2A, containing a GC-rich region (GCA9) in the position of nucleotide 1565. The other

members of this family also contain extensive GC-rich se-quences that might be potential binding sites for CUGBP1.Since CUGBP1 up-regulates p21 and MEF2A in cultured cells(12) (see Fig. 6), we examined whether these translationaltargets of CUGBP1 are affected in transgenic mice. As can beseen in Fig. 4A, both p21 and MEF2A are increased in skeletalmuscle of transgenic mice. This result was confirmed by immu-nostaining of skeletal muscle sections. In vastus muscle fromwild type mice, strong MEF2A signals were detected only in20% of fibers. On the contrary, strong MEF2A staining wasobserved in the majority of cells in vastus muscle fromCUGBP1 transgenic mice (line 49) (Fig. 4A).

In addition to MEF2A, we detected the induction of myoge-nin in CUGBP1 transgenic mice (Fig. 4A). The increase ofmyogenin levels might be a consequence of the MEF2A in-crease, since the MEF2 family is able to activate myogenin (28).MyoD levels were not changed in CUGBP1 transgenic mice.Given the alteration of the levels of MEF2A, myogenin, and p21in skeletal muscle of CUGBP1 transgenic mice, we examinedthese proteins in muscle of DM1 patients. Similar to CUGBP1transgenic mice, MEF2A, myogenin, and p21 are elevated inDM1 skeletal muscle (Fig. 4B). The alterations of CUGBP1,

FIG. 4. Elevation of CUGBP1 in skeletal muscle leads to the induction of MEF2A, myogenin, and p21 protein levels and toalterations of myofiber type. A, elevation of CUGBP1 in skeletal muscle increases levels of MEF2A, p21, and myogenin. Total proteins fromvastus muscles were analyzed by Western blotting with various antibodies as indicated. To verify protein loading, each membrane was reprobedwith antibodies against actin and stained with Coomassie Blue. Actin control is shown for myogenin and MEF2A filter. Right image, immuno-staining of transverse sections of vastus muscle with antibodies against MEF2A. Sections from wild type and transgenic mice with 4–6-foldelevation of CUGBP1 (line 49) at 8 months of age were analyzed under identical conditions. B, MEF2A, myogenin, and p21 levels are elevated inskeletal muscle of DM1 patients. Protein extracts were isolated from skeletal muscle of normal control and DM1 patient with large CTG expansion(1,500 repeats) and analyzed by Western blotting. Right, CUGBP1, MEF2A, and p21 levels are changed in skeletal muscle of DM1 patients butnot in muscle from patients with late onset myopathy. Total protein extracts from DM1 patients with 857 (1) or 300 (2) CTG repeats and frompatients with late-onset myopathy (M) were analyzed with antibodies against CUGBP1, p21, MEF2A, and actin as the loading control. Westernanalysis with CUGBP1 Abs detects CUGBP1 and a weak upper band corresponding to IgG. C, upper image, immunostaining of transverse musclesections for plantaris muscle from wild type (WT) and mutant mice (line 46) with antibodies against myoglobin and slow and fast myosin heavychains. Bottom image, the switch of fiber type in skeletal muscle from CUGBP1 transgenic (TR) mice. Total skeletal muscle proteins isolated fromvastus muscle of wild type and mutant mice (lines 20, 29, and 46) were examined by Western blotting with antibodies shown on the left. Eachmembrane was stripped and reprobed with antibodies to actin (not shown) or stained with Coomassie to verify protein loading.

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MEF2A, and p21 levels in DM1 muscle are not due to generaldystrophy, because the levels of these proteins were unchangedin muscle from patients with late onset myopathy (Fig. 4B).Thus, the overexpression of CUGBP1 in DM1 patients and inCUGBP1 transgenic mice affects MEF2A and p21 proteins,which play a significant role in myogenesis.

Elevation of CUGBP1, p21, and MEF2A in Skeletal Muscle IsAssociated with Alterations of Myofiber Type—At earlier stagesof the disease, DM1 patients have an increased number of slowmyofibers, which become preferential fibers at later stages ofdisease progression. Given that p21 and MEF2A are similarlyinduced in DM1 and CUGBP1 transgenic mice, we examinedwhether myofiber type is changed in skeletal muscle fromCUGBP1 transgenic mice. IF analysis of transverse musclesections with antibodies against fast myosin shows that inplantaris muscle of 8-month-old wild type mice, fast and inter-mediate fibers represent 70–85%. IF analysis of similar sec-tions from mutant littermates (line 49) showed that the num-ber of these fibers is significantly reduced (50%) (Fig. 4C, Fast).The number of slow fibers determined by IF with antibodies tofast myosin was 15–30% greater, respectively, in CUGBP1transgenic mice. The increase of slow fibers in CUGBP1 trans-genic mice was confirmed by IF with antibodies against slowmyosin (Fig. 4C, Slow). The myofiber-type switch in CUGBP1transgenic mice was also confirmed by Western analysis ofmyoglobin and fast and slow myosin. Fig. 4C shows that, inplantaris muscle from CUGBP1 transgenic mice, the levels ofslow myosin are increased, whereas the levels of fast myosinare reduced. It is known that slow fibers contain increasedlevels of myoglobin, and this is in contrast to fast fibers con-taining lower levels of myoglobin. We determined myoglobinlevels by Western blot analysis and IF assay and found thatplantaris muscle from CUGBP1 transgenic mice (line 49) haselevated levels of myoglobin, suggesting that the number ofslow fibers is greater in CUGBP1 mutant mice. These datasuggest that the elevation of CUGBP1 causes alterations infiber-type composition in skeletal muscle with an increase ofslow fibers and reduction of fast fibers similar to DM1 disease.

CUGBP1 Interacts with GCA Repeats Located withinMEF2A mRNA—The elevation of MEF2A in skeletal musclefrom CUGBP1 transgenic mice prompted us to examine molec-ular mechanisms by which CUGBP1 increases MEF2A levels.UV cross-link assay with full-length MEF2A mRNA showedthat CUGBP1 binds directly to this mRNA and that the affinityof this interaction is similar to that for RNA probe containingCUG123 (Fig. 5A). CUGBP1 binds to CAG repeats withinMEF2A mRNA, since the deletion of these repeats abolishesthe interaction. To confirm the specificity of the interaction,cold MEF2A mRNA and (CAG)10- and AU-rich RNA oligomerswere incorporated into the binding reactions. As can be seen inFig. 5A, MEF2A and CAG10 compete for the binding, whereasAU-rich oligomer does not.

We next determined which RNA-binding domains (RBDs) ofCUGBP1 bind to MEF2A mRNA. UV cross-link analysis indi-cates that full-length CUGBP1 and RBD3 bind to MEF2AmRNA, whereas truncated forms of CUGBP1 containingRBD1�2 and RBD2 do not interact with MEF2A mRNA (Fig.5B). Given the in vitro interaction of CUGBP1 with MEF2AmRNA, we asked whether this interaction takes place in vivo.For this goal, we immunoprecipitated CUGBP1 from differen-tiated C2C12 myotubes and examined the presence of MEF2AmRNA in CUGBP1 IPs using the reverse transcriptase-PCRprocedure. In these experiments, we applied primers that coverthe GCA repeat within MEF2A mRNA and produce a 743-kbDNA fragment. As can be seen, MEF2A mRNA is detectable inCUGBP1 IPs, whereas MEF2A mRNA is not observed in con-

trol precipitates with agarose only (Fig. 5C). Thus, these stud-ies revealed that CUGBP1 interacts with MEF2A mRNA invitro and in vivo.

CUGBP1 Increases Translation of MEF2A in a Cell-freeTranslation System and in Cultured Cells—We next examinedwhether the interaction of CUGBP1 with MEF2A mRNA af-fects expression of MEF2A in a cell-free translation system. Ascan be seen in Fig. 6A, CUGBP1 increases protein levels ofMEF2A in the cell-free translation system programmed withthe wild type full-length MEF2A mRNA. Deletion of GCA re-peats (CUGBP1 binding site) diminishes binding of CUGBP1 toMEF2A mRNA (Fig. 5A) and leads to a failure of CUGBP1 toinduce the translation of mutant MEF2A mRNA (Fig. 6A).Thus, we conclude that CUGBP1 increases the translation of

FIG. 5. CUGBP1 binds to MEF2A mRNA. A, CUGBP1 interactswith MEF2A mRNA via GCA repeats. Top, bacterially expressed, pu-rified CUGBP1 was incubated with RNA probes CUG123, full-lengthMEF2A mRNA, and �CAG MEF2A mRNA (shown at the top); linked tothe probes by UV; and separated by gel electrophoresis. Cold full-lengthMEF2A mRNA was added to the binding reaction with 32P-labeledMEF2A mRNA. Coomassie stain of the same membrane is shown.Bottom, cold (CAG)8 and AU-rich oligomers (100 ng each) were added tothe binding reactions with bacterially expressed CUGBP1 and MEF2AmRNA probe. B, CUGBP1 binds to ME2FA mRNA through RBD3. Top,positions of three RBDs within the CUGBP1 molecule. Full-lengthbacterially expressed CUGBP1 and truncated CUGBP1 proteins con-taining different RBDs (shown at the top) were incubated with MEF2AmRNA probe and analyzed by UV cross-link assay. C, CUGBP1 binds toMEF2A mRNA in vivo. CUGBP1 was immunoprecipitated from differ-entiated C2C12 cells (day 3), and RNA was extracted from IPs andexamined by reverse transcriptase-PCR with primers specific forMEF2A mRNA.

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MEF2A through the interaction with GCA repeats locatedwithin MEF2A mRNA.

We next examined whether CUGBP1 increases MEF2A ex-pression in cultured cells using two approaches: transienttransfection assay and a stable clone line containing antisenseCUGBP1 mRNA under Lac repressor control (21). The stableclone line contains the inducible antisense CUGBP1 mRNAunder control of the Lac repressor. Examination of MEF2Aprotein levels after the addition of isopropyl-1-thio-�-D-galac-topyranoside revealed that the inhibition of CUGBP1 causesthe reduction in the protein levels of MEF2A (Fig. 6B). Toconfirm these observations, His-CUGBP1 was transfected intoC2C12 cells, and levels of MEF2A were determined by Westernblotting. Fig. 6C shows that the levels of His-CUGBP1 ex-pressed from the transfected plasmid are significantly higherthan the levels of endogenous CUGBP1 protein. In cells over-expressing CUGBP1, protein levels of MEF2A are dramatically

induced. Densitometric analysis of multiple experiments dem-onstrated an 8–10-fold induction of MEF2A by CUGBP1 as aratio to �-actin. Immunostaining of transfected cells also re-vealed that protein levels of MEF2A are dramatically increasedin cells overexpressing GFP-CUGBP1 (Fig. 6C, right). Thiseffect is specific to GFP-CUGBP1, since GFP alone does notincrease MEF2A levels. Thus, studies in the cell-free transla-tion system and in cultured cells show that CUGBP1 increasesprotein levels of MEF2A.

Although data in a cell-free translation system suggest thatCUGBP1 increases translation of MEF2A, the CUGBP1-medi-ated increase of MEF2A levels in transfected cells could be alsodue to a stabilization of MEF2A protein. To examine this pos-sibility, we determined the half-life of MEF2A protein inC2C12 cells transfected with CUGBP1 or with empty vector asthe control. Experiments with cyclohexamide block of proteinsynthesis showed that MEF2A is a very stable protein and that

FIG. 6. CUGBP1 increases translation of MEF2A. A, GCA repeats within MEF2A mRNA are required for the CUGBP1-dependent increaseof MEF2A translation. The upper panel indicates the positions of GCA repeats within MEF2A mRNA. Full-length MEF2A mRNA (FL) and mRNAlacking GCA repeats (�GCA) were translated in a cell-free translation system in the presence of increasing amounts of CUGBP1. Positions ofproteins translated from FL MEF2A and �GCA MEF2A mRNAs are shown by arrows. B, the inhibition of CUGBP1 expression in a stable antisenseCUGBP1 clone leads to the reduction of MEF2A. Antisense CUGBP1 was induced by the addition of 10 mM isopropyl-1-thio-�-D-galactopyranoside,and proteins were isolated at 4, 12, and 24 h after isopropyl-1-thio-�-D-galactopyranoside (IPTG) addition and analyzed by Western blotting withantibodies to CUGBP1, MEF2A, and �-actin. C, overexpression of CUGBP1 in cultured cells increases protein levels of MEF2A. Left, C2C12 cellswere transfected with an empty vector (V) and with a vector expressing His-CUGBP1. Protein levels of CUGBP1 and MEF2A were examined byWestern blotting. Positions of an endogenous CUGBP1 and His-CUGBP1 are shown. MEF2A membrane was reprobed with Abs to �-actin to verifyprotein loading. Right, GFP-CUGBP1 or GFP vectors (green) were transfected into proliferating C2C12 cells. MEF2A was determined byimmunostaining with specific antibodies (red). 4�,6-Diamidino-2-phenylindole (DAPI) shows staining of nuclei on the same fields. D, CUGBP1 doesnot change the half-life of MEF2A. C2C12 cells were transfected with CUGBP1 or with empty vector, and total protein extracts were isolated atdifferent time points after the addition of cyclohexamide and analyzed by Western assay with antibodies against MEF2A. The bar graphs showdensitometric calculations of MEF2A protein levels as an average of three experiments. E, CUGBP1 increases translation of MEF2A. C2C12 cellswere transfected with CUGBP1 and with an empty vector, and [35S]Met was added into medium the next day. Protein extracts were isolated atdifferent time points after [35S]Met addition (shown at the top). MEF2A was precipitated and analyzed by gel electrophoresis. Coomassie stainingshows IgGs in the immunoprecipitates. Bar graph shows a summary of three experiments. Amounts of radioactivity in MEF2A precipitates werecounted and presented as the average of three experiments.

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CUGBP1 does not affect MEF2A stability (Fig. 6D). We thenanalyzed whether CUGBP1 increases MEF2A protein transla-tion by measuring newly synthesized radioactive MEF2A pro-tein. C2C12 cells were co-transfected with MEF2A andCUGBP1 or with empty vector, [35S]Met was added, and[35S]MEF2A was precipitated and analyzed by gel electro-phoresis. In cells transfected with CUGBP1, the incorporationof [35S]Met into MEF2A (translation) was significantly greaterthan the incorporation of [35S]Met in cells transfected with theempty vector at each time point analyzed in our studies (Fig.6E). Taken together, these investigations show that CUGBP1increases translation of MEF2A mRNA in a cell-free transla-tion system and in cultured cells.

CUGBP1 Enhances the MEF2A-dependent Myogenic Conver-sion—The analysis of MEF2A under conditions of overexpres-sion of CUGBP1 showed that CUGBP1 increases translation ofMEF2A. We next tested the hypothesis that CUGBP1 mightregulate MEF2A during a differentiation course of C2C12 myo-blasts. Northern analysis showed that the levels of two majorisoforms of CUGBP1 mRNA (7 and 9 kb) are 3–6-fold elevated

at days 3 and 5 of differentiation (Fig. 7A). Western analysisrevealed that protein levels of CUGBP1 are also elevated incytoplasm (Fig. 7B, left) and in nuclei of differentiating C2C12cells (data not shown). Analysis of p21 and MEF2A expressionin the same cell extracts shows an increase of the protein levelsof MEF2A and p21, which correlates with the induction ofCUGBP1 (Fig. 7B, left). We next examined whether CUGBP1interacts with MEF2A mRNA in differentiated myotubes.CUGBP1 was immunoprecipitated from cytoplasmic extracts,and the presence of MEF2A mRNA in CUGBP1 IPs was deter-mined by reverse transcriptase-PCR with specific primers. Fig.7B (CUGBP1 IP) shows that CUGBP1 is associated withMEF2A mRNA in differentiated myotubes. Our results in acell-free translation system and these data suggest thatCUGBP1 is required for the induction of MEF2A and p21 indifferentiating myoblasts. Therefore, we further examined therole of CUGBP1-mediated translational induction of p21 andMEF2A during differentiation of C2C12 cells using siRNA-mediated inhibition of CUGBP1. We found that the inhibitionof CUGBP1 by siRNA during differentiation leads to a signifi-

FIG. 7. CUGBP1 increases levels MEF2A during myoblast differentiation. A, CUGBP1 mRNA is increased in C2C12 cells at later stagesof differentiation. The upper panel shows CUGBP1 mRNA levels. Ethidium bromide staining of the gel is shown in the middle. The bottom panelshows the reprobe of the same filter with 18 S rRNA probe. B, protein levels of CUGBP1 and MEF2A are increased during differentiation of C2C12cells. Left, Western blotting was performed with proteins isolated from C2C12 cells at days 0, 3, and 5 after the initiation of differentiation. Themembrane was reprobed with Abs to �-actin, and MEF2A levels were calculated as ratios to �-actin. Antibodies to MEF2 (C-21; Santa CruzBiotechnology) in addition to MEF2A recognize MEF2C. CUGBP1-IP. MEF2A mRNA is associated with CUGBP1 after initiation of differentiation.CUGBP1 was precipitated from cytoplasm with monoclonal antibodies, and CUGBP1 IPs were examined by reverse transcriptase-PCR withprimers specific to MEF2A mRNA. The position of a PCR product (743 bp) is shown. Right, inhibition of CUGBP1 by siRNA reduces levels ofMEF2A and p21 in differentiating C2C12 myoblasts. C, DM1 cells fail to induce MEF2A during differentiation. Left, MEF2A Western blotting withprotein extracts isolated from control cells and from DM1 cells growing in differentiation medium for 0, 3, and 5 days is shown. The membrane wasreprobed with �-actin antibodies. Right, 4�,6-Diamidino-2-phenylindole (DAPI) staining of normal and DM1 myoblasts and myotubes is shown. D,CUGBP1 enhances MEF2A-dependent myogenic conversion. Left, fibroblasts were co-transfected with MyoD or wild type or mutant MEF2A andCUGBP1. Cells were subjected to double immunostaining with antibodies against MyoD (red) and myosin (green). Right, bar graph shows asummary of three independent experiments.

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cant reduction of MEF2A and p21 (Fig. 7B, right). Thus, thesedata demonstrate that CUGBP1 is required for the increase ofMEF2A and p21 levels during differentiation.

Given the observation that CUGBP1 increases translation ofMEF2A, we examined MEF2A levels in cultured myoblastsfrom control and from DM1 patients during differentiation. Innormal myoblasts, MEF2A levels are elevated during differen-tiation (Fig. 7C). However, in DM1 cells, the regulation ofMEF2A is altered; the levels of CUGBP1 are higher in prolif-erating myoblasts and lower in differentiating cells (Fig. 7C).Consistently with low levels of MEF2A, DM1 differentiatingmyoblasts have lower levels of myosin (data not shown) and arenot properly fused (Fig. 7C). These data suggested thatCUGBP1-mediated induction of MEF2A is required for properdifferentiation of myoblasts. To examine role of CUGBP1-me-diated induction of MEF2A in myogenesis, we applied a tissueculture model of myogenic conversion of mouse fibroblasts10T1/2 into myoblasts. It is known that although MyoD aloneinitiates myogenic conversion, the MEF2 family is required toco-activate the myogenesis (27). We next examined whetherdirect regulation of MEF2A by CUGBP1 is required for anenhancement of the myogenic conversion. Mouse fibroblastswere co-transfected with MyoD, CUGBP1, and wild typeMEF2A or with the GCA deletion mutant MEF2A. If CUGBP1was co-transfected with wild type MEF2A construct, the effi-ciency of myogenic conversion was enhanced by �50%. Thedeletion of the CUGBP1 binding site from MEF2A mRNA abol-ishes the CUGBP1-dependent enhancement of myogenesis(Fig. 7D). Thus, these studies revealed that under biologicallyrelevant concentrations and conditions, CUGBP1 enhancesmyogenic conversion through the regulation of MEF2A.

DISCUSSION

Proper myogenesis requires an orchestrated cascade of ex-pression of muscle specific genes; certain proteins should beexpressed at certain stages of muscle development and at cer-tain levels. Such programmed expression of muscle genes iscontrolled at different levels, including the regulation of tran-scription and post-transcriptional control. Protein levels ofCUGBP1 and RNA binding activity of CUGBP1 are elevated inDM1 patients by expansion of CUG RNA repeats (12, 13, 19–21). In this paper, we examine the role of CUGBP1 elevation inDM1 muscle pathology using transgenic mice. The analysis ofCUGBP1 transgenic mice shows that the overexpression ofCUGBP1 above biological levels is sufficient to disrupt myo-genesis in vivo. We found a strong correlation between levels ofCUGBP1 elevation and severity of muscle defects. Muscle inmutant mice with 2–3 fold elevation of CUGBP1 shows mildvariability in fiber size and increase of internal nuclei only at1.5–2.0 years of age. Histological muscle phenotype in thesemice is similar to that observed in DM1 patients (adult form ofDM1) during earlier changes of disease progression. In skeletalmuscle of mice with greater increase of CUGBP1 (4–6-fold, line49), a larger number of cells was variable in size with accumu-lation of internal nuclei. Comparison of two transgenic linesshows that mutant mice in line 49 develop muscle histopathol-ogy earlier in life (6–8 months). Elevation of CUGBP1 up to8-fold causes immaturity of myofibers: central nuclei, increaseof nuclei number, shortening of fibers, and reduction in myofi-ber density. These mice do not survive. Such a phenotype ispartially reminiscent of congenital DM1, since immaturity offibers and fibers with centrally positioning nuclei were found incongenital DM1. The phenotype in this line is stronger thanone in congenital DM1, perhaps because the elevation ofCUGBP1 in this line is higher than in congenital patients thatwe analyzed.

Another important similarity between muscle abnormalities

in DM1 and in CUGBP1 mice is a myofiber-type switch: in-crease of slow fibers and reduction of fast fibers. A similarchange in fiber type composition can be seen in experimentalmice affected with myotonia (29), which is a hallmark of DM1.Elevation of MEF2A in CUGBP1 transgenic mice is consistentwith recent data from Dr. Olson’s laboratory indicating thatMEF2 activity is elevated in slow fibers in mouse muscles withmyotonia (29). In these myotonic mice, �80% of fibers areoxidative or slow. The elevation of MEF2 activity in muscle ofmyotonic mice clearly indicates that hyperexcitability of myo-tonic muscle is associated with up-regulation of MEF2, result-ing in increase of slow fibers.

A number of proteins have been shown to play a key role indifferentiation and development of skeletal muscle. One ofthese proteins, p21, in collaboration with p57 is involved in theirreversible growth arrest of skeletal muscle cells (30). Ourdata from CUGBP1 transgenic mice demonstrate that, in vivo,elevation of CUGBP1 causes an increase of p21 accompanied bydelay of muscle development and dystrophic histopathology inmuscle. Since CUGBP1 increases translation of p21 in culturedcells (12), it is likely that translation of p21 is also induced inskeletal muscle of DM1 patients. On the other hand, alter-ations in p21 mRNA may also contribute to increased levels ofp21 protein. What is a possible mechanism by which increase ofp21 might delay myogenesis in DM1 and in CUGBP transgenicmouse muscles? It has been recently shown that p21 may playopposite roles in the regulating cell proliferation: 1) arrest ofproliferation by inhibiting Cdk2-cyclin E and 2) promotion ofproliferation by stimulating the assembly of active Cdk4-cyclinD1 complexes (31). Therefore, elevated levels of p21 in DM1tissues might lead to both inhibition and promotion of prolif-eration depending on the protein environment at the cell cyclestage when p21 is elevated. Since differentiation requires atightly regulated cascade of gene expression and preciseamounts of the proteins, we suggest that CUGBP1-mediatedoverexpression of p21 in DM1 tissues inhibits differentiation ofskeletal muscle.

Myogenesis is also regulated by myogenic basic helix-loop-helix transcription factors (MyoD, Myf-5, myogenin, and

FIG. 8. A hypothetical model showing the role of translationalactivity of CUGBP1 in DM1 pathology in skeletal muscle(see “Discussion”).

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MFR4) and their co-activators, MEF2 factors. An increase ofmyogenic transcription factors and their co-activators is re-quired for the activation of myogenesis. In CUGBP1 transgenicmice, however, an increase of MEF2A and myogenin is associ-ated with a delay of myogenesis. We hypothesize that such aparadox occurs because of misregulation of myogenic transcrip-tion factors. For proper myogenesis, these factors should beregulated in a very precise manner. However, forced increase ofCUGBP1 in mice might result in unscheduled activation ofmyogenic factors at the wrong time of development, delayingmyogenesis. It is interesting to note that both deletion andoverexpression of myogenin in mice leads to neo- or postnataldeath (32, 33), showing that normal development requires atight regulation of myogenin.

A similar phenomenon has also been described for MEF2A.Since MEF2A activates transcription of many muscle proteins,precise timing and levels of MEF2A expression are required toprovide distinct levels of specific proteins at the correct stage ofmuscle development. Such regulation has been earlier de-scribed for MEF2A. It has been found that the 3�-untranslatedregion of MEF2A mRNA is responsible for regulating its ownmRNA translation during differentiation (34). This regulationleads to low levels of MEF2A in proliferating myoblasts viainhibition of MEF2A translation and high levels of MEF2A indifferentiating myoblasts due to lack of repression of MEF2Atranslation. Another publication also found that proper regu-lation of MEF2 levels is required for differentiation (35). In thispaper, the authors show that too little or too much MEF2activity leads to the same result: disruption of differentiation(35). Taking into account these observations, we suggest thattoo much CUGBP1 activity in CUGBP1 transgenic micedelays myogenesis.

CUGBP1 is located in nuclei and cytoplasm and is able toregulate both RNA splicing and mRNA translation. In thispaper, we focused our studies on the translational function ofCUGBP1. Based on our observations, we suggest a hypotheti-cal pathway by which the cytoplasmic portion of CUGBP1contributes to DM1 pathology in skeletal muscle (Fig. 8). Wehypothesize that in normal differentiating cells, CUGBP1causes an increase of MEF2A and p21 at the correct time. InDM1 patients, basal overexpression of CUGBP1 caused byCUG repeats leads to overall overexpression of p21 and MEF2Athat results in inhibition of myogenesis.

Acknowledgments—We are very grateful to J. Lopez, A. Cortez, andD. Nopierawa for the excellent assistance, J. Gordon and D. Moncktonfor providing mouse tissues from CUG transgenic mice, and H. Vogel forthe advice with histology experiments. We thank A. Welm for thediscussion of experimental data and for critical review of the paper.

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CUGBP1 as a Translational Regulator of Myogenesis 13139

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T. TimchenkoNikolai A. Timchenko, Roma Patel, Polina Iakova, Zong-Jin Cai, Ling Quan and Lubov

MyogenesisOverexpression of CUG Triplet Repeat-binding Protein, CUGBP1, in Mice Inhibits

doi: 10.1074/jbc.M312923200 originally published online January 13, 20042004, 279:13129-13139.J. Biol. Chem. 

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