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Address correspondence to Mani S. Mahadevan, University of Virginia, P.O. Box 800904, Charlottesville, VA 22908. Tel.: (434) 243-4816. Fax: (434) 924-1545. E-mail: mahadevan{at}virginia.edu
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Abstract |
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Key Words: myotonic dystrophy; trinucleotide repeats; differentiation; MyoD; muscle development
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Introduction |
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The DM1 mutation, which accounts for 98% of DM cases, is a triplet repeat (CTG) expansion on human chromosome 19 located in the 3' untranslated region (3'-UTR) of a protein kinase gene (dystrophia myotonica protein kinase [DMPK]) (Brook et al., 1992; Fu et al., 1992; Mahadevan et al., 1992). It is not known exactly how this mutation causes disease, but several mechanisms have been proposed (for review see Tapscott and Thornton, 2001). These include altered DMPK levels and/or isoforms, misexpression of neighboring genes due to chromatin reorganization at the DM1 locus, and a trans-dominant mutant DMPK mRNA. The DM2 mutation was recently identified as a CCTG expansion in the first intron of the zinc finger protein 9 (ZNF9) gene on human chromosome 3 (Liquori et al., 2001). The similarity of the nonprotein coding repeat expansion in both mutant ZNF9 and DMPK transcripts, plus the absence of genes analogous to DMPK or its neighbors at the DM2 locus, suggest that dominant effects mediated by CUG/CCUG expansioncontaining RNA molecules play the predominant role in causing DM.
How the mutant RNA causes disease is under intense investigation. RNA FISH experiments show that both mutant DMPK and ZNF9 transcripts localize to distinct foci in the nuclei of DM patient cells (Taneja et al., 1995; Davis et al., 1997; Mankodi et al., 2001). It is thought that interactions between these transcripts and RNA binding proteins result in the aberrant expression of other genes. Several proteins bind the expanded DMPK 3'-UTR RNA in vitro (Timchenko et al., 1996; Lu et al., 1999; Miller et al., 2000; Tian et al., 2000; Tiscornia and Mahadevan, 2000), and one family of factors, MBNL, MBLL, and MBXL, colocalizes with RNA foci in both DM1 and DM2 patient cells (Fardaei et al., 2001, 2002; Mankodi et al., 2001). RNAs containing expanded CUG tracts have been shown to skew the alternative splicing of unrelated transcripts (Philips et al., 1998), including the insulin receptor mRNA, which likely explains insulin resistance seen in patients (Savkur et al., 2001). Perhaps the most compelling evidence implicating the mutant RNA in DM pathogenesis comes from transgenic mice that express heterologous transcripts containing 250 CUG repeats and develop myotonia and myopathy similar to what is seen in DM patients (Mankodi et al., 2000).
We have studied the effects of the mutant DMPK 3'-UTR RNA in a cell culture model. To establish this model, the human DMPK 3'-UTR sequence containing either a pathogenic (CUG)200 repeat tract or a wild-type (CUG)5 tract was fused to a reporter gene (lacZ or GFP) and expressed in C2C12 mouse myoblasts (Amack et al., 1999; Amack and Mahadevan, 2001). C2C12 are muscle precursor cells originally isolated from injured adult mouse muscle that have proven an excellent system to study myogenic differentiation (Yaffe and Saxel, 1977; Blau et al., 1983). When cultured in differentiation media lacking growth factors, C2C12 cells up-regulate muscle-specific genes, exit the cell cycle, and fuse into multinucleated myotubes. We have shown that transcripts containing the mutant DMPK 3'-UTR aggregate into nuclear foci and block C2C12 differentiation (Amack et al., 1999). This differentiation defect may represent muscle development abnormalities found in congenital DM patients (Harvey et al., 1976; Farkas-Bargeton et al., 1988) and/or point to defects in muscle regeneration, which could contribute to muscle wasting in adult patients. Recently, the differentiation defect was confirmed in cultured myogenic satellite cells taken from DM1 patients (Furling et al., 2001; Timchenko et al., 2001).
Here, we have used our cell culture model to investigate the molecular mechanisms that underlie how the mutant DMPK 3'-UTR RNA disrupts myoblast differentiation. The sequence of events in the differentiation pathway has been well characterized using C2C12 cells (Andres and Walsh, 1996; Fig. 1 A). MyoD and Myf5 are myogenic transcription factors expressed in committed myoblasts capable of inducing expression of myogenin and other genes required for differentiation. After myogenin expression, cells withdraw from the cell cycle and fuse into multinucleated myotubes. Previous RNA analyses using our cell culture system showed that the programmed up-regulation of myogenin and p21 is severely blunted in cells expressing the mutant DMPK 3'-UTR (Amack and Mahadevan, 2001). This suggested that the mutant 3'-UTR RNA interferes with the earliest stages of differentiation, perhaps by impeding the initiation of differentiation-specific gene expression. In this report, we show that MyoD protein levels are compromised in cells expressing mutant DMPK 3'-UTR transcripts at time points thought to be critical for the initiation of C2C12 differentiation.
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Results |
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GFP+mut 3'-UTR pool cells infected with the myogenin retrovirus formed myotubes after 3 d in differentiation media, whereas cells infected with a control retrovirus expressing LacZ and uninfected cells failed to form myotubes (Fig. 1 B). Infection with the LacZ retrovirus demonstrated that myotube formation in these cells requires myogenin and is not simply a result of retroviral infection. The myotubes seen in GFP+mut 3'-UTR pool cultures infected with the myogenin retrovirus were similar to those seen in infected control C2C12 and GFP+wt 3'-UTR pool cells, which were larger than myotubes seen in uninfected C2C12 and GFP+wt 3'-UTR pool populations (Fig. 1 B). RNA FISH analysis showed that myotubes in the GFP+mut 3'-UTR pool were foci positive (unpublished data), indicating that expression of the mutant DMPK 3'-UTR RNA had not been lost. Exogenous myogenin expression in infected cells was confirmed by Western blot analysis (Fig. 1 C), and LacZ expression from the control retrovirus was detected by X-gal staining (unpublished data). These results show that myogenin is sufficient to correct the differentiation defect in cells expressing the mutant 3'-UTR, and that mechanisms downstream of myogenin required for myoblast differentiation in these cells are intact and functional.
MyoD protein levels are reduced in myoblasts expressing the mutant DMPK 3'-UTR
Our next step was to investigate the effects of the mutant DMPK 3'-UTR RNA on myogenic factors that act before myogenin induction. However, the initiation of myogenin expression and myoblast differentiation is governed by a complex regulatory network (for review see Puri and Sartorelli, 2000). Determining a priori which molecule(s) may be affected by the mutant RNA is daunting. Fortunately, we were presented a clue by an exceptional GFP+mut 3'-UTR clone (termed mut 3'-UTR-res) that expresses the mutant DMPK 3'-UTR RNA, but remains capable of differentiating into myotubes (Fig. 2 A). Any number of alterations in genetic background could account for this resistance, but further characterization revealed that steady-state MyoD mRNA levels were 2.6-fold higher than those in control C2C12 populations (Fig. 2 B). MyoD protein levels are also elevated in mut 3'-UTR-res cells, but only by 1.2-fold, as compared with control C2C12 cells (Fig. 2 C). This hinted to us that if increased MyoD is indeed responsible for "protecting" these myoblasts, then perhaps it is MyoD that is compromised by the mutant DMPK RNA.
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MyoD levels are further reduced under differentiation conditions
Although MyoD protein levels are lower in proliferating myoblasts that express mutant DMPK 3'-UTR transcripts, this difference may not have deleterious consequences in proliferating cells. The transcriptional activity of MyoD and Myf5 is held in check until cells are stimulated to enter the differentiation program by the removal of growth factors (Wei and Paterson, 2001). Therefore, we characterized the pattern of MyoD and Myf5 expression in GFP+mut 3'-UTR cells under differentiation conditions. In control C2C12 cells, MyoD levels decreased by an average of 28% relative to the starting levels in proliferating cells after 24 h in differentiation media, and then remained between 5060% of the starting amount over the next 48 h (Fig. 4 A). A similar pattern was seen in GFP+wt 3'-UTR pool cells, in which MyoD levels decreased by 36% after 24 h. In the GFP+mut 3'-UTR pool, however, the average initial drop in MyoD after 24 h was significantly greater at 72% (Fig. 4 A). MyoD remained low at 48 h in these cells, but then rebounded by 72 h to levels similar to those in C2C12 cells (Fig. 4 A). The sharp drop in MyoD in GFP+mut 3'-UTR cells, coupled with already low starting levels, results in nearly undetectable levels of MyoD after 24 h in differentiation media.
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Activation of E-boxmediated gene expression is blunted in GFP+mut 3'-UTR cells
The blunted up-regulation of genes such as myogenin and p21 in GFP+mut 3'-UTR cells (Amack and Mahadevan, 2001) suggests defects in early differentiation-specific transcription. MyoD and Myf5 regulate transcription at DNA sequences (CANNTG) called E-boxes in promoter regions of muscle-specific genes (Lassar et al., 1989). Do diminished MyoD levels adversely affect E-boxdirected gene expression, or can Myf5, which is unaffected, compensate for the loss of MyoD? We measured E-box activation using a reporter construct from which expression of a luciferase gene is driven by a thymidine kinase (TK) promoter containing three tandem E-boxes (pE-boxLuc). To study the fold activation of E-box expression over several days, cells were cotransfected with a luciferase reporter gene and a puromycin resistance marker that was used to select for stable transfectants. We determined the amount of luciferase activation that was E-box mediated by subtracting the contribution made by the TK promoter sequences, as measured using a control plasmid lacking the E-boxes (pTKLuc). In transfected control C2C12 and GFP+wt 3'-UTR pool cells, an average of three experiments showed that E-boxluciferase expression was activated 1.7-fold after 24 h in differentiation media and approximately twofold after 72 h (Fig. 4 C). In contrast, no activation was detected at any time point in transfected GFP+mut 3'-UTR pool cells (Fig. 4 C). This suggested that the consequence of reduced MyoD levels after 24 h in differentiation media may be blunted E-box expression, and indicates that Myf5 cannot compensate for the lack of MyoD.
MyoD protein levels and E-box activation are compromised within the first 6 h under differentiation conditions
Because each of the MRFs can transactivate E-boxcontaining promoters, we cannot separate out the individual contributions of MyoD, Myf5, and myogenin (MRF4 is expressed only in mature C2C12 myotubes). It is possible that the difference observed in E-box activation is due to unequal myogenin expression, which first starts to appear after 24 h in differentiation media in control C2C12 and GFP+wt 3'-UTR cells, but not in GFP+mut 3'-UTR cells (see Fig. 7 C, uninfected cells). To determine whether blunted E-box expression correlates directly with reduced MyoD levels, we analyzed luciferase expression in transfected cultures before myogenin expression. After 6 h in differentiation media, E-boxluciferase expression was up-regulated 1.5-fold in C2C12 and GFP+wt 3'-UTR pool cells, but no up-regualtion was seen in the GFP+mut 3'-UTR pool (Fig. 5 A). This is a time point at which no myogenin was detected by Western blotting (unpublished data). Analysis of MyoD at these time points showed that the reduction of MyoD protein levels in GFP+mut 3'-UTR pool cells occurs within 6 h of withdrawing growth factors (Fig. 5 B). Thus, the lack of E-boxluciferase expression in these cells correlates with reduced MyoD levels, and, at least initially, is not due to differential expression of myogenin. This suggests that the reduction of MyoD results in blunted E-boxmediated gene activation.
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MyoD reduction is posttranscriptional
To determine whether the reduction of MyoD protein is due to transcriptional or posttranscriptional defects, we analyzed steady-state MyoD RNA levels during the first 12 h in differentiation media. In control C2C12 cells, MyoD RNA levels dropped to 52% of the starting levels in proliferating cells during the first 6 h under differentiation conditions (Fig. 6). Similar decreases were observed in GFP+wt 3'-UTR pool (46%) and GFP+mut 3'-UTR pool cells (44%) (Fig. 6). Even after 12 h there were no significant differences in MyoD RNA levels among the three cell populations (Fig. 6). Similar results were obtained when comparing MyoD RNA levels in the lacZ+20059, lacZ+20059Rev, and Cre-8 clones (unpublished data). This demonstrates that reduced MyoD protein levels in GFP+mut 3'-UTR cells within the first 6 h in differentiation media are not due to decreased MyoD transcription or unstable MyoD mRNA.
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Discussion |
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MyoD is compromised by mutant DMPK 3'-UTR RNA
Genetic studies using knock-out mice have placed MyoD and Myf5 upstream of myogenin (for review see Rudnicki and Jaenisch, 1995). This made MyoD and Myf5 candidate targets of the mutant DMPK RNA, but there are several other factors and signaling cascades that help regulate the initiation of the differentiation pathway (Puri and Sartorelli, 2000). Fortunately, analysis of C2C12 myoblasts resistant to the effects of the mutant DMPK 3'-UTR RNA demonstrated that MyoD is overexpressed in these cells, providing us with a hint that MyoD levels may be linked to the RNA-mediated block of differentiation. Although MyoD mRNA levels are not affected by mutant DMPK 3'-UTR transcripts in our cell culture system (Amack and Mahadevan, 2001), we investigated whether MyoD protein levels were altered in proliferating or differentiating myoblasts. We have identified two separate effects on MyoD protein levels in cells expressing the mutant 3'-UTR. First, MyoD levels in proliferating GFP+mut 3'-UTR cells were approximately three times lower than in control cells (Fig. 3 A). Second, MyoD levels were reduced an additional 70% in these cells during the first 24 h in differentiation media, as compared with only an
30% decrease in control C2C12 and GFP+wt 3'-UTR cells (Fig. 4 A; Fig. 5 B). No significant differences were found in Myf5 protein levels at either time point, indicating that these negative effects have some specificity for MyoD, and are not due to a general effect on protein levels. MyoD levels are also reduced in cells expressing a (CUG)200 DMPK 3'-UTR fused to a lacZ transcript (lacZ+20059 cells). Significantly, this affect is abrogated when expression of the RNA is silenced (Fig. 5 C). Taken together, these results demonstrate that the presence of DMPK 3'-UTR transcripts containing 200 CUG repeats, but not five CUG repeats, compromises MyoD protein levels.
The loss of MyoD function has been studied using MyoD-/- mice. Cultured MyoD-/- myoblasts show differentiation defects and blunted up-regulation of differentiation-specific genes (Sabourin et al., 1999; Cornelison et al., 2000), a phenotype strikingly similar to what we observe in cells expressing the mutant 3'-UTR. This is also reminiscent of the phenotype described for DM1 patient myoblasts in culture (Furling et al., 2001; Timchenko et al., 2001). Importantly, DM1 patient cells show reduced MyoD protein levels as compared with control cells (Timchenko et al., 2001), suggesting that the molecular defects that reduce MyoD levels in GFP+mut 3'-UTR cells may also compromise MyoD in patient cells. Exogenous MyoD is capable of rescuing the defects seen in MyoD-/- cells (Sabourin et al., 1999) and overriding the negative effects of the mutant DMPK 3'-UTR RNA on differentiation (Fig. 7). This is consistent with the idea that spontaneous overexpression of MyoD provides the "resistance" in mut 3'-UTR-res cells (Fig. 2). Because the differentiation defect can be rescued by elevating MyoD levels, or by adding exogenous myogenin, which acts downstream of MyoD, we conclude that compromised MyoD levels are responsible for inhibiting myoblast differentiation.
Loss of MyoD function in GFP+mut 3'-UTR cells is consistent with the blunted induction of differentiation-specific genes such as myogenin and p21. The expression of these genes is regulated in part through E-boxes (Yee and Rigby, 1993; Halevy et al., 1995), providing evidence of a defect in MyoD and/or Myf5 activity. Consistent with the lack of activation of endogenous genes containing E-boxes, we found that E-boxluciferase expression was not induced in GFP+mut 3'-UTR pool cells under differentiation conditions (Fig. 4 C; Fig. 5 A). During the first 24 h in differentiation media, the lack of E-box activation correlated with the sharp reduction of MyoD protein levels. Elevating MyoD levels in these cells restores the induction of myogenin (Fig. 7 C) and E-boxluciferase (unpublished data). This suggests that the amount of MyoD in GFP+mut 3'-UTR pool cells is not sufficient to initiate and/or sustain E-boxmediated gene expression, which explains why myogenin, p21, and other differentiation-specific genes are not properly expressed.
Why can't the normal levels of Myf5 up-regulate E-box genes in these cells? Consistent with our results that Myf5 cannot compensate for the lack of MyoD, myoblasts derived from adult MyoD-/- mice show dampened up-regulation of differentiation-specific genes despite a >10-fold up-regulation of Myf5 (Sabourin et al., 1999). This indicates that MyoD has essential functions that Myf5 is unable to perform. Additional studies have shown that during differentiation of both C2C12 and primary mouse myoblasts, MyoD is found exclusively in differentiating cells and Myf5 is expressed only in the cells that do not differentiate (Kitzmann et al., 1998; Lindon et al., 1998). These observations argue that proper MyoD expression and activity are absolutely required for myoblast differentiation.
How do mutant DMPK 3'-UTR transcripts compromise MyoD?
It is thought that mutant DMPK RNA disrupts cellular processes by interacting aberrantly with RNA binding proteins. The aggregation of DMPK transcripts in nuclear foci could either activate or sequester proteins, leading to a variety of downstream consequences. Characterization of one such protein, CUGBP1, has shown that RNAs containing expanded CUG repeats affect CUGBP1 function in both mRNA splicing and translation (Philips et al., 1998; Timchenko et al., 1999). Defects in any one of several processes could account for the reduction of MyoD protein levels in cells expressing the mutant DMPK 3'-UTR (Fig. 8). We first tested MyoD expression at the RNA level and found that transcription and mRNA stability are not affected in the GFP+mut 3'-UTR pool during the first 12 h in differentiation media (Fig. 6). An intriguing possibility downstream of transcription was that MyoD transcripts get "caught" in the nuclear RNA foci and are not transported to the cytoplasm for translation. However, nuclear and cytoplasmic fractionation of RNA from GFP+mut 3'-UTR cells showed no evidence that MyoD mRNA was retained in the nucleus (unpublished data). RNA splicing and/or processing defects could reduce MyoD protein levels, but gross changes in transcript length were not apparent on Northern blots. Any splicing alterations would have to be subtle and could not change the size or stability of the transcript. Other possibilities include mislocalization of MyoD RNA in the cytoplasm, poor translation efficiency, and decreased MyoD protein stability (Fig. 8). Our cell culture model will provide an excellent system to test each of these possibilities and further characterize the trans effect on MyoD protein levels.
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In conclusion, we have identified MyoD as a target of a trans effect mediated by mutant DMPK 3'-UTR RNA. The reduction of MyoD protein levels provides a molecular explanation for why cells expressing mutant 3'-UTR transcripts fail to differentiate. Future projects, some of which are already underway, are designed to determine how the mutant DMPK RNA compromises MyoD. In addition, as demonstrated by the successful rescue of the differentiation defect by retroviral expression of myogenin and MyoD, our cell culture model may be useful in developing strategies to override the effects of the mutant DMPK RNA. Understanding how mutant DMPK RNA causes deleterious effects at the molecular level may provide insight into how muscle defects arise in DM patients and how to correct them.
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Materials and methods |
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Retroviral infection
For transduction with retroviral vectors, cells were plated at low density (2 x 105 cells) in 10-cm dishes. The next day, cells were exposed to 4 ml of media harvested from confluent cells producing the retrovirus, supplemented with 8 µg/ml polybrene (Sigma-Aldrich), for 3 h. 24 h later, the cells were exposed to a second infection. Mock infections (referred to as uninfected cells) were conducted in parallel using regular growth media plus polybrene. For differentiation studies, infected and uninfected cells were switched into differentiation media 1 d after the second infection. The following retroviruses were used: LMDSN expressed MyoD (Weintraub et al., 1989); and JR-gal expressed LacZ (Wang et al., 1991). Media containing the myogenin retrovirus was a gift from R. Ilaria (University of Texas Southwestern, Dallas, TX).
Immunostaining and RNA FISH
To detect myosin heavy chain (MHC) expression, cells cultured in differentiation media for 3 d were fixed in 4% paraformaldehyde for 15 min at room temperature and stored in 70% ethanol at 4°C. Cells rehydrated in PBS were incubated with MY32 primary antibody (Sigma-Aldrich) diluted in PBS + 1% BSA for 1 h at 37°C. After washing, cells were incubated with a Texas redconjugated secondary antibody (Jackson ImmunoResearch Laboratories) for 1 h at 37°C. Nuclei were stained with DAPI (Sigma-Aldrich).
RNA FISH experiments were performed as previously described (Amack et al., 1999). In brief, fixed cells were incubated with a CY3-conjugated (CAG)10 oligonucleotide probe (Operon) diluted to 0.1 µg/ml in RNA hybridization buffer for 2 h at 37°C. After three washes in PBS, nuclei were stained with DAPI.
Fluorescent signals were visualized using an Olympus IX 50 microscope with epifluorescence. Pictures were taken with a SPOT II digital camera (Diagnostic Instruments), and images were assembled using Adobe Photoshop® and Canvas (Deneba Systems) software.
Western blot analysis
Cells were lysed in 50 mM Hepes, pH 7.6, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 0.5 mM DTT, and 0.1 mM EDTA supplemented a with protease inhibitor cocktail (Roche). 50 µg of total protein was fractionated by SDS-PAGE and transferred onto a nitrocellulose membrane (Schleicher & Schuell). Membranes were blocked overnight in PBS containing 10% skimmed milk, and then incubated for 13 h with primary antibodies diluted in PBS containing 1% BSA. The following primary antibodies were used: MyoD (BD Biosciences), Myf5 (Santa Cruz Biotechnology, Inc.), myogenin (Santa Cruz Biotechnology, Inc.), and dynein (Santa Cruz Biotechnology, Inc.). After incubation with the primary antibody, membranes were washed in PBS containing 0.3% Tween-20, and then incubated with a secondary antibody conjugated to HRP (Santa Cruz Biotechnology, Inc.) for 1 h. After several washes, proteins were detected using ECL reagents. Films were scanned and protein bands were quantified using ImageQuant software (Molecular Dynamics). The Mann-Whitney test was used for all statistical analyses.
Luciferase assays
The reporter plasmid pE-boxLuc was generated by subcloning a HinDIII-XhoI fragment from p3R-CAT (Weintraub et al., 1990), which contains three tandem "right" E-boxes from the muscle-specific creatine kinase enhancer inserted 5' of TK promoter sequences, upstream of the luciferase gene in pTKLuc (a gift from E. Olson, University of Texas Southwestern). pTKLuc serves as a control construct that contains the TK promoter and luciferase, but lacks E-boxes.
Control C2C12, GFP+wt 3'-UTR pool, and GFP+mut 3'-UTR pool cells were transfected as previously described (Amack and Mahadevan, 2001) with pE-boxLuc and a plasmid conferring puromycin resistance, and stable transfectants were selected in media containing 4 µg/ml puromycin. After selection, puromycin-resistant colonies (an average of 270 per transfection) were trypsinized, combined, and analyzed as a collection of independent transfection events. Visualization of GFP and RNA FISH experiments demonstrated that transfected GFP+wt 3'-UTR and GFP+mut 3'-UTR cells retained expression of the 3'-UTR transcripts (not depicted). These cells were grown in selective media containing both G418 and puromycin. In parallel, each population of cells was separately transfected with the control pTKLuc construct.
To measure luciferase, samples were prepared using the luciferase assay system (Promega), and relative light units were determined by a MLX luminometer (Dynex Technologies). Raw relative light units were normalized to protein concentration, which was measured using the Bio-Rad Laboratories protein assay. The amount of luciferase produced in proliferating cells, which varied among the different stably transfected cell lines, was set equal to one, and changes in luciferase expression during differentiation were analyzed relative to these starting levels. To determine the fold change in luciferase expression that was due strictly to the E-boxes, we subtracted changes that were due to the TK promoter sequences, as observed in cells transfected with the control pTKLuc construct. The fold induction of E-boxmediated luciferase expression was analyzed in three independent differentiation experiments.
RNA analysis
Total RNA was prepared by lysing cells in 4 M guanidinium thiocyanate, 20 mM sodium acetate, pH 5.4, and 0.5% sarkosyl and then isolating RNA by ultracentrifugation through a 5.7 CsCl density gradient. Northern and RNA slot blots were generated and analyzed as previously described (Amack and Mahadevan, 2001). Results were quantified using ImageQuant software (Molecular Dynamics).
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Footnotes |
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* Abbreviations used in this paper: DM, myotonic dystrophy; DMPK, dystrophia myotonica protein kinase; MHC, myosin heavy chain; MRF, myogenic regulatory factor; TK, thymidine kinase; 3'-UTR, 3' untranslated region; ZNF9, zinc finger protein 9.
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Acknowledgments |
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This work was supported by grants from the Muscular Dystrophy Association and the National Institutes of Health grant R01 AR45992-01. This is paper no. 3602 from the University of Wisconsin-Madison Laboratory of Genetics.
Submitted: 4 June 2002
Revised: 9 September 2002
Accepted: 30 September 2002
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References |
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