Human Molecular Genetics, 2001, Vol. 10, No. 11 1129-1139
© 2001 Oxford University Press
The product of an oculopharyngeal muscular dystrophy gene, poly(A)-binding protein 2, interacts with SKIP and stimulates muscle-specific gene expression
1Department of Neuromuscular Research, National Institute of Neuroscience, NCNP, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan and 2Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0031, Japan
Received 29 November 2000; Revised and Accepted 23 March 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Oculopharyngeal muscular dystrophy (OPMD) is caused by short expansions of the GCG trinucleotide repeat encoding the polyalanine tract of the poly(A)-binding protein 2 (PABP2). PABP2 binds to the growing poly(A) tail, stimulating its extension during the polyadenylation process, and limits the length of the newly synthesized poly(A) tail. Whereas PABP2 is expressed ubiquitously, the clinical and pathological features of OPMD patients are restricted to the skeletal muscle. To elucidate the possible role of PABP2 in skeletal muscle, we established the stable C2 cell lines expressing human PABP2. These stable cell lines showed morphologically enhanced myotube formation accompanied by an increased expression of myogenic factors, MyoD and myogenin. In nuclear run-on assay, the transcription rate of the MyoD gene was significantly increased by PABP2 transfection. We found the N-terminal region of PABP2 was responsible for the up-regulation of these myogenic factors. Furthermore, Ski-interacting protein (SKIP) was isolated as a binding protein for PABP2 using the yeast two-hybrid system. The interaction of PABP2 and SKIP was confirmed by glutathione S-transferase-pulldown assay and immunoprecipitation. Confocal laser scanning showed PABP2 was co-localized with SKIP in nuclear speckles. The reporter assays showed that PABP2 co-operated with SKIP to synergistically activate E-box-mediated transcription through MyoD. Moreover, both PABP2 and SKIP were directly associated with MyoD to form a single complex. These findings suggest that PABP2 and SKIP directly control the expression of muscle-specific genes at the transcription level.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disease with autosomal dominant inheritance with progressive swallowing difficulties (dysphagia), eyelid drooping (ptosis) and proximal limb weakness after the age of 50. Unique intranuclear inclusions in the skeletal muscle are a pathological hallmark of OPMD. A recent genetic study identified the responsible mutation as a GCG repeat expansion encoding a polyalanine tract at the N-terminus of the poly(A)-binding protein 2 (PABP2) which causes OPMD (1).
PABP2 is an abundant nuclear protein which binds poly(A) with high affinity and specificity. In vitro analyses showed that PABP2 stimulated poly(A) polymerase (PAP) in conjunction with cleavage and polyadenylation-specific factor (CPSF) to increase the processivity of the polyadenylation reaction (2–4). Although the polyadenylation reaction is directly catalyzed by PAP, this enzyme by itself is almost inactive due to a low affinity with RNA (5). Processive and efficient poly(A) tail synthesis requires both CPSF and PABP2 at the 3'-end processing complex (2,4). PABP2 is also involved in the mechanism that limits the length of the newly synthesized poly(A) tail (4). In vitro reconstitution of the poly(A) tail synthesis resulted in a rapid and processive synthesis of the poly(A) tail to approximately 200–250 residues which corresponded to the length of the nascent poly(A) tail in mammals. The Poly(A) length restriction mechanism is probably associated with the stoichiometric binding of multiple PABP2 to the nascent poly(A) tail, CPSF and PAP (4). A recent study using electron microscopy provided evidence to support this stoichiometric binding (6).
Despite ubiquitous expression patterns of PABP2, the clinical and pathological phenotypes are restricted to the skeletal muscle, relatively specific to the levator palpebrea superioris and pharyngeal muscle. Characterizing the functional roles of PABP2 in skeletal muscle may contribute to elucidating the possible pathogenic mechanism in OPMD. To gain insight into the possible roles of PABP2 in skeletal muscle, we established stable C2 cell lines expressing human PABP2. These stable cell lines acquired the ability of enhanced myogenic differentiation compared with their parent cell line. We demonstrate how PABP2 affects myogenic differentiation and elucidate its functional role.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Exogenous expression of human PABP2 promotes myogenic differentiation in C2 cells
The mouse skeletal muscle cell line, C2, provides a good model for the study of myoblasts and their myogenic differentiation (7). Exposure of proliferating myoblasts to media lacking mitogen induces withdrawal from the proliferative cell cycles, and cells fuse to form multinucleated myotubes.
To examine the possible role of PABP2 in skeletal muscle, C2 cells were transfected with full-length human PABP2 cDNA under the control of the cytomegalovirus promoter. The human PABP2 was Flag-epitope tagged at the C-terminus (Flag-PABP2) to distinguish it from the endogenous (mouse) PABP2. G418-resistant clones were selected and screened by examining human PABP2 expression using RT–PCR and western blot analyses. Twenty-eight G418-resistant clones expressing human PABP2 were obtained. Clones S1 and S7, which showed similar morphology to their parent cells during the growth phase were chosen for further characterization. We determined the expression rate of transfected human PABP2 relative to endogenous PABP2 using quantitative RT–PCR. The mRNA expression levels of exogenous (human) PABP2 in clones S1 and S7 were 14.6 and 28.9% of their endogenous PABP2 levels, respectively (data not shown). However, these clones showed 3-fold greater mRNA expression of the endogenous PABP2 with respect to their parent cells (data not shown).
Parental C2 cells and the stable clones were cultured to confluence, and then induced to fuse into myotubes as described in Materials and Methods. S1 and S7 showed accelerated morphological differentiation compared with the parental C2 (Fig. 1). In cultures approaching confluence, myotubes began to be observed in S7 cultured in growth medium [Fig. 1, GM (day 0)]. Two days after transferring to differentiation medium, large myotubes were observed in the S1 and S7 clones, but not in parental C2 [Fig. 1, DM (day 2)]. In parental C2, myotubes were observed 3 days after culture in differentiation medium [Fig. 1, DM (day 3)]. Thus, exogenous expression of human PABP2 in C2 cells leads to enhanced morphological differentiation.
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The protein expression of the exogenous PABP2 in the stable cell lines was characterized by immunofluorescence and western blotting using anti-Flag polyclonal antibody. Flag-PABP2 was detected more prominently in the nuclei of myotubes than myoblasts by immunofluorescence (Fig. 1). No staining could be seen in parental C2 (Fig. 1). Specific bands for Flag-PABP2 (
50 kDa) were detected in S1 and S7 by western blot analysis (Fig. 2A). Consistent with the result of immunofluorescence analysis, expression of Flag-PABP2 was increased in differentiated myotubes relative to myoblasts in western blot analysis (Fig. 2A). The bands of 75 kDa that the anti-Flag polyclonal antibody detected in all lanes were unknown but probably non-specific because they are present in the C2 lanes as well (Fig. 2A).
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The mRNAs for MyoD and myogenin were up-regulated in the stable cells expressing exogenous PABP2
Myogenesis is a result of a multiple transcription process of muscle-specific genes by the myogenic factors (basic helix–loop–helix transcription factors). Previous studies have demonstrated that expressions of myogenic factors, MyoD and myogenin, are induced as an early event in myogenic differentiation in vitro (8), and that their actions as transcriptional regulators are required for terminal myoblast differentiation in vivo (9–11).
We examined the mRNA expression levels of myogenic factors, such as MyoD and myogenin, in the stable clones using quantitative RT–PCR. At the confluence stage in the growth medium, S1 and S7 expressed 4.2- and 8-fold greater MyoD mRNA compared with parental C2, respectively (Fig. 2B). The protein level of MyoD was also higher in S1 and S7 than in parental C2 at this stage (Fig. 2A, GM). The maximal differences of MyoD expression among C2, S1 and S7 were observed after 1 day of culture in the differentiation medium. At this stage, S1 and S7 showed 6.4- and 12-fold higher expression levels of MyoD mRNA compared with their parent cells, respectively (Fig. 2B, DM1). The expression level of myogenin mRNA during the growth stage and confluence phase was extremely low in each clone (Fig. 2B, Gr and Co). After culture for 1 day in the differentiation medium, the expression levels of myogenin mRNA in S1 and S7 were 25- and 37-fold higher than in parental C2, respectively (Fig. 2B, DM1). The myogenin protein was also up-regulated during the differentiation (Fig. 2A). One of the muscle-specific mRNAs, myosin light chain 1a (MLC1a), was highly expressed in S1 and S7 compared with parental C2 during the differentiation (Fig. 2B). The expression levels of Myf-5 mRNA were not significantly changed between the stable clones and parental C2 (Fig. 2B).
Overexpression of PABP2 results in an increase in the transcription rate of the MyoD gene
To clarify the up-regulation mechanism of MyoD, we performed nuclear run-on assay to measure the transcription rate of the MyoD gene from the C2 cells transiently transfected with PABP2 expression plasmid. The transcription rate of the MyoD gene relative to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene showed a 2.5-fold increase in the PABP2 transfection (Fig. 3A). In agreement with the result of the nuclear run-on assay, northern blot analysis showed that the mRNA expression of MyoD was increased by the transfection of PABP2 expression plasmid (Fig. 3B). Thus, MyoD mRNA is specifically up-regulated by PABP2 transfection.
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The N-terminal region of PABP2 (PABP2-N) is responsible for the up-regulation of MyoD and myogenin mRNAs
PABP2 contains an acidic N-terminus, highly conserved RNP-type RNA-binding domain known as RNA recognition motifs (RRMs) and a basic C-terminus (12). To determine which region of PABP2 is involved in the up-regulation of MyoD and myogenin, we transiently transfected the expression plasmids encoding either the full-length or truncated PABP2 (Fig. 4A) in C2 cells and measured the expression levels of MyoD and myogenin mRNAs using quantitative RT–PCR. The proteins expressed in each transfected sample were confirmed by western blot analysis (Fig. 4B). At 24 h after transfection, cells had almost reached confluence and no marked change was observed in the measured mRNA levels in each transfected sample (Fig. 4C). At 48 and 72 h after transfection, the mRNA levels of MyoD and myogenin showed a 2-fold increase in the cells transfected with full-length PABP2 (PABP2-FL) and PABP2-N expression plasmids relative to the mock control (Fig. 4C). However, the cells transfected with the RNA-binding domain of PABP2 (RRMs) showed no notable increase in the amount of the target mRNAs compared with the mock controls (Fig. 4C). In addition, MLC1a expression was activated 2-fold by transient expression of PABP2-FL and PABP2-N relative to the mock control (Fig. 4C).
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PABP2 interacts with a nuclear protein Ski-interacting protein (SKIP)
To identify proteins that interact with the N-terminal region of PABP2 (amino acids 1–145), we performed yeast two-hybrid screening. The yeast-expressing N-terminal region of PABP2 was transformed with a human skeletal muscle cDNA library which consisted of clones carrying cDNA fused to the GAL4 activation domain. Finally, several positive clones were obtained from 3 x 106 leucine and tryptophan auxotrophic transformants. The restriction enzyme digestion and sequence analysis resulted in the identification of four kinds of independent cDNAs encoding SKIP (13), DnaJ-like heat shock protein 40 (14), human SWI-SNF component BRG1 (15) and an unknown protein containing zinc-finger motifs. Since SKIP was found in eight overlapping clones including a full-length open reading frame, it was used for additional studies.
To verify the interaction between PABP2 and SKIP, a glutathione S-transferase (GST)-pulldown assay was carried out. For the GST-pulldown assay, GST-fusion proteins were purified from Escherichia coli (BL21) harboring expression plasmids encoding PABP2-FL (GST-PABP2-FL), its N-terminal region (GST-PABP2-N) and C-terminal region which contained the RNA recognition motifs (GST-RRMs). The purified fusion proteins were incubated with cell lysates from COS-7 cells transiently transfected with the expression plasmid for SKIP fused to the Myc-epitope tag (Myc-SKIP). The GST-pulldown assay revealed that Myc-SKIP was specifically bound to GST-PABP2-FL and GST-PABP2-N, but not to GST-RRMs or GST alone (Fig. 5A). Furthermore, the interaction between PABP2 and SKIP was also confirmed by immunoprecipitation. For the immunoprecipitation assay, COS-7 cells were transiently co-transfected with the expression plasmids encoding Myc-SKIP and Flag-PABP2. The protein complex was immunoprecipitated from the cell lysates using anti-Flag polyclonal antibody recognizing Flag-PABP2. Immunoprecipitates were analyzed by western blotting using anti-Myc monoclonal antibody. As shown in Figure 5B, Myc-SKIP was specifically co-immunoprecipitated with Flag-PABP2 from the co-transfected COS-7 lysate. As controls, Myc-SKIP was not detected in the immune complex from the COS-7 lysates transfected with the expression plasmids encoding either Myc-SKIP or Flag-PABP2 (Fig. 5B). These results clearly indicate that PABP2 interacts with SKIP in mammalian cells.
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SKIP is co-localized with PABP2 in nuclear speckles
Intranuclear localization of PABP2 and SKIP in HeLa cells was examined under a confocal microscope. A previous study reported that PABP2 was localized in the nuclear speckles of HeLa cells (16). The splicing factor, SC-35, was used for the marker of the nuclear speckles (16,17). As shown in Figure 6A, transiently expressed Flag-PABP2 was co-localized in nuclear speckles with SC-35. This is thought to be a similar pattern to that for endogenous PABP2. In HeLa cells co-transfected with the Flag-PABP2 and Myc-SKIP expression plasmids these two proteins were strongly co-localized in nuclear speckles (Fig. 6A). No staining was detected in non-transfected cells (data not shown). This finding also supports the suggestion that these two proteins interact with each other in the nuclear speckles.
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At 48 h after transfection
5% of transfected HeLa cells showed some large aggregates with strong staining by anti-Flag antibody in the nucleus, indicating that they contain a high concentration of Flag-PABP2 (Fig. 6B). These aggregates appeared round in shape with variable size, and SKIP and SC-35 were strongly associated with them (Fig. 6B). These presumably appear to be the result of overexpression of PABP2. Although it is not known whether these aggregates are similar to inclusions observed in OPMD muscle, we found that the nuclear distributions of SC-35 and SKIP were changed by the aggregate formation.
PABP2 and SKIP co-operate E-box-mediated transcription in the presence of MyoD
Myogenic factors control the expression of muscle-specific genes by binding to a specific DNA sequence (CANNTG), called E-box, in their promoter region. To evaluate the effect of PABP2 and SKIP on E-box-mediated transcription, luciferase reporter assays were performed using C3H10T1/2 cells. C3H10T1/2 is a mouse non-muscle fibroblastic cell line. Exogenous expression of myogenic factors in C3H10T1/2 cells is sufficient to convert them into a muscle-specific lineage (18,19). As a model system for muscle-specific transcription, we used the MyoD-responsive reporter plasmid (4RE-tk-luc), which contains a firefly luciferase gene controlled by four tandem E-boxes from muscle creatine kinase enhancer upstream of the thymidine kinase basal promoter (20). C3H10T1/2 cells were transiently co-transfected with the reporter plasmid and the expression plasmids encoding PABP2, SKIP and MyoD. This reporter plasmid was activated to 7-fold greater luciferase activity by MyoD expression (Fig. 7). In the absence of MyoD, the expression of PABP2 and SKIP failed to activate the reporter over the basal levels (Fig. 7A). In the presence of MyoD, the effect of SKIP and PABP2 on MyoD-dependent transactivation was characterized at various amounts of expression plasmids. MyoD-responsive luciferase activity was greatly activated by PABP2 transfection in a dose-dependent manner (Fig. 7A). The cells transfected with SKIP expression plasmid showed a similar result, but more efficient luciferase transactivation was observed compared with cells transfected with PABP2 expression plasmid of the same dose (Fig. 7A). The transfections of PABP2-FL or PABP2-N activated reporter activity in the presence of MyoD (Fig. 7B).
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To evaluate the synergistic effect of PABP2 and SKIP on the reporter gene, the cells were co-transfected with expression plasmids encoding the truncated PABP2 and SKIP. We determined the amount of PABP2 and SKIP expression plasmids for transfection as a 2-fold transactivation on the reporter in the presence of MyoD. This is consistent with the result of quantitative RT–PCR from C2 cells as described previously (Fig. 4C). In the presence of MyoD, the transfection of PABP2 synergistically stimulated the luciferase activity with its binding partner, SKIP (Fig. 7B). We observed an approximately 6-fold synergistic transactivation (Fig. 7B). Moreover, a similar transactivation in the cells transfected with PABP2-N was also observed (Fig. 7B). In contrast, few synergistic activities were observed in the cell co-transfected with the expression plasmid for RRMs and SKIP (Fig. 7B).
We next investigated whether PABP2 and SKIP exist in a single complex with MyoD because the transactivation for MyoD-responsive reporter might be caused by protein–protein interaction. Immunoprecipitation assay was performed using the anti-MyoD polyclonal antibody in C3H10T1/2 cells transiently co-transfected with the indicated expression plasmids. Western blot analysis showed that PABP2 or SKIP as well as both PABP2 and SKIP could be associated with MyoD (Fig. 8). These associations with MyoD were specific because omission of the anti-MyoD antibody or MyoD protein did not result in the co-precipitation of SKIP and PABP2 (Fig. 8).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In the present study, we demonstrate the possible involvement of PABP2 in the expression of muscle-specific genes and skeletal myogenesis. We established stable C2 cell lines expressing human PABP2. A cell line constitutively expressing human PABP2 showed accelerated morphological differentiation accompanied by enhanced mRNA expression of MyoD as well as the differentiation markers, myogenin and MLC1a. Although the cytomegalovirus promoter tends to induce very high levels of expression of transgenes, our stable cell lines showed only 14.6 and 28.9% of the exogenous PABP2 expression. On the other hand, the entire mRNA expression levels of PABP2 were approximately 3.5-fold higher than their parent cells for unknown reasons. In addition, the exogenous PABP2 protein was easily detected by western blotting during the myogenic differentiation. Therefore, the enhanced myotube formation of the stable cell lines may be due to the overexpression of PABP2.
The poly(A) length of mRNA has functional implications for its turnover, translation and export. In general, regulation of polyadenylation is distinct from the nuclear reaction and may depend on several specific factors and a cis-acting sequence element (21). PABP2 acts to limit the overall length of newly synthesized poly(A) tail to 200–250 residues (4). PABP2 can shuttle between the nucleus to the cytoplasm suggesting its involvement in mRNA export (22). Chen et al. (23) reported that the influenza virus NS1A protein inhibited PABP2 function during 3'-end processing which is responsible for the block in the nuclear export of cellular mRNAs (23). Therefore, the enhanced differentiation observed in the stable cell lines might be caused by specifically promoted mRNA export of MyoD due to the PABP2 overexpression. However, we wonder why mRNA of MyoD is specifically exported. One of the possible explanations is overexpressed PABP2 which presumably affects the transcription of the MyoD gene. In the present study, the result of nuclear run-on assays indicates that high levels of the MyoD transcription occur in the nucleus depending on the transfection of PABP2 expression plasmid. In addition, nuclear run-on assay as well as Northern blot analysis showed no notable change of the expression level of GAPDH mRNA by the PABP2 transfection. These findings suggest that MyoD mRNA is specifically regulated at the transcription level by the PABP2 transfection.
We also demonstrated that PABP2-N played a crucial role in the up-regulation of MyoD mRNA. One muscle-specific mRNA, MLC1a, was increased by the tranfections of expression plasmids encoding PABP2-N as well as PABP2-FL, suggesting that the N-terminal region of PABP2 contributed to activating muscle-specific gene expression via the up-regulation of myogenic factors. In addition, we identified that PABP2 interacted with SKIP. SKIP was originally identified as a protein that interacts with both the cellular and viral forms of the oncoprotein Ski (13). SKIP is highly homologous to Bx42, a Drosophila melanogaster nuclear protein involved in ecdysone-stimulated gene expression (24). A recent study demonstrated that NcoA-62 functions as a co-activator protein involved in vitamin D-mediated transcription (25). SKIP has a nucleotide sequence virtually identical to the cDNA termed NcoA-62 (25). This protein also shows a transactivation activity as a co-activator for the retinoic acid-, estrogen- and glucorticoid receptor-mediated transcription pathways (25). Several lines of evidence have been reported suggesting that skeletal myogenesis and muscle-specific transcription are positively regulated by hormone-related factors including the receptors of retinoic acid (26–29) and insulin-like growth factors (30–32), and by several oncoproteins (33–36). These are directly or indirectly related to the transcriptional machinery mediated by myogenic factors. Through the reporter assay, we showed that transient transfection of PABP2 results in transactivation for E-box-mediated transcription in the presence of MyoD. SKIP co-operated with PABP2 to stimulate the E-box-mediated transcription in the presence of MyoD. Moreover, we identified that SKIP and PABP2 were associated in a single complex with MyoD. This finding suggests that the co-operation by PABP2 and SKIP to stimulate MyoD-dependent transcription may depend on their ability to form such a complex and provides evidence that PABP2 may function as a potential co-factor of transcription or its related protein in the skeletal muscle. This is a function of PABP2 distinguishable from that involved in the polyadenylation process.
Our immunofluorescence study also demonstrated the co-localization of PABP2 and SKIP in nuclear speckles. The nuclear speckles are highly enriched in pre-mRNA splicing factors (37–40) including the 3'-end-processing factors and poly(A) RNA (16,41). These correspond to the interchromatin granule clusters and perichromatin fibrils imaged by electron microscopy (42). Several recent findings suggest that most splicing and 3'-end processing occurs co-transcriptionally on perichromatin fibrils, and interchromatin granule clusters represent reservoirs, from which pre-mRNA processing factors are recruited to the nascent transcript (40,43,44). The nuclear distribution of PABP2 shows a unique pattern compared with major 3'-end processing factors including cleavage stimulation factor (CstF), CPSF and PAP. A previous immunofluorescence study has shown that PABP2 is concentrated in nuclear speckles (16); however, other components of the 3'-end processing machinery (CPSF, CstF, PAP) do not concentrate in speckles (45). Although these components are found in transcription sites, they show different nuclear distributions from PABP2 under physiological condition (45). These differences in nuclear distributions indicate that PABP2 may differ functionally from other 3'-end processing components. We showed that the exogenous PABP2 in the stable cell lines was accumulated in the nuclei during myogenic differentiation. It appears that the muscle-specific gene expression during myogenesis requires the accumulation of PABP2.
In the skeletal muscle from OPMD patients, unique intranuclear filamentous inclusions are found in 2.5% of nuclei by electron microscopy (46,47). It appears that increased repeat expansion causes abnormal aggregation of PABP2 and the formation of the filamentous nuclear inclusions in skeletal muscle (48–52). These observations raise the possibility that the nuclear filament inclusions formed by the GCG repeat expansion of PABP2 could exert a gain of toxic function to accelerate cell death. We transfected PABP2 containing expanded repeats of GCG (nine GCG repeats) and compared the frequency of aggregate formation with normal PABP2 (six GCG repeats). However, significant differences in frequency and distribution were not observed (data not shown). Nevertheless, we found that 5% of PABP2 transfected HeLa cells contained large aggregates that altered the spatial distribution of nuclear speckles. Similar aggregates were observed with a high frequency in COS-1 cells transfected with ataxin-1 containing an expansion of the polyglutamine tract, which is responsible for spinocerebellar ataxia type 1 (SCA1) (53). The aggregates observed in the COS-1 cells transfected with expanded ataxin-1 altered the distribution of the nuclear matrix-associate structures and suggested their involvement in the pathogenesis of SCA1 (53). PABP2 is also known as a nuclear matrix-associated protein (54) and localized in the nuclear speckles. The nuclear speckles play an important role in splicing and processing of pre-mRNA, and their altered distribution may be associated with OPMD pathogenesis. Further studies should be needed to clarify the pathophysiological mechanism of OPMD whose muscles contain inclusions with accumulation of PABP2 (52).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Plasmids
The coding region of PABP2 was amplified from the first strand cDNA for human skeletal muscle by PCR. The cDNA fragments encoding the N- and C-terminus of PABP2 were amplified separately using the following primers: N-terminal fragment, 5'-GATGGCGGCGGCGGCGGCGGCG-3' (sense) and 5'-GCTTCTCTACCTCGTTCTGATAGCT-3' (antisense); C-terminal fragment, 5'-CCCGGAGCTGGAAGCTATCAA-3' (sense) and 5'-TTACGTAAGGGGAATACCATGATGT-3' (antisense). For Flag-epitope (DYKDDDDK) tagging, the antisense sequence against the Flag-epitope was added to the 5'-end of the C-terminus antisense primer which was used for PCR amplification. The amplified product was subcloned into pGEM-T Easy vector (Promega) and sequenced. Basically, these subclones were used to generate the expression constructs of PABP2 for mammalian cells, yeasts and E.coli. To construct the PABP2 expression plasmid for mammalian cells, the EcoRI-XhoI fragment of the N-terminus and the XhoI-NotI fragment of the C-terminus were fused into EcoRI-NotI sites of pCI-neo (Promega) to generate pCI-PABP2-FL. The expression plasmid for the truncated form of PABP2, pCI-RRMs (amino acids 127–306), was derived from pCI-PABP2-FL by XhoI digestion and self-ligation. The EcoRI-XhoI fragment of the N-terminus was subcloned into EcoRI-SalI sites to generate pCI-PABP2-N (amino acids 1–145). The expression plasmids for GST-fusion proteins were constructed in the same manner as described above using the pGEX-6P-2 vector (Amersham Pharmacia Biotech). Full-length SKIP cDNA was isolated from a human skeletal muscle cDNA library (Clontech) using yeast two-hybrid screening. The SKIP cDNA insert was excised from the yeast plasmid, pACT2 (Clontech), and then subcloned into EcoRI-XhoI sites of pMyc-CMV (Clontech) to generate pCMV-Myc-SKIP. MyoD cDNA was amplified by RT–PCR from C2 cells and subcloned into pRc/RSV (Invitrogen) to generate pRSV-MyoD. The luciferase reporter plasmid (4RE-tk-luc) was generated as described previously (20).
Cell culture and stable transfectants
C3H10T1/2, HeLa and COS-7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma) supplemented with 10% fetal bovine serum. All cultures were grown at 37°C in a humidified atmosphere containing 5% CO2.
C2 cells were cultured in growth medium (DMEM containing 15% fetal bovine serum) at low cell-density to sustain a proliferative state. To induce differentiation, cells were plated at a density of 104 cells/cm2 into collagen coated culture plates (IWAKI) in growth medium. After 2 days incubation the cells were almost confluent and the growth medium was changed to a differentiation medium (DMEM containing 5% horse serum) and then cells were cultured for 3 days.
To establish the C2-derived stable transfectants, C2 cells were plated at 5 x 105 cells in 60 mm dishes for 24 h prior to transfection. The cells were transfected with an expression plasmid for C-terminally Flag-epitope tagged PABP2 cDNA using lipofectamine reagent (Gibco BRL Life Technologies). Following incubation for 24 h, cells were selected in the presence of 800 µg/ml G418 (Gibco BRL Life Technologies) in growth medium. The resistant clones were pooled 7 days after incubation and replated in 96-well culture plates at an extremely low density to avoid cross-contamination. After an additional 7 days incubation, G418-resistant clones were subcultured and maintained in growth medium containing 400 µg/ml G418. The expression of Flag-tagged PABP2 was confirmed by RT–PCR and western blot analysis from each individual clone.
Quantitative RT–PCR
Quantitative RT–PCR was performed by the method of Kinoshita et al. (55) with slight modification. Total RNAs were isolated from the growth and differentiation phases of C2 cells using TRIzol reagent (Gibco BRL Life Technologies). First-strand cDNA was synthesized from 2 µg of total RNA by reverse transcription at 37°C for 1 h. The reaction mixture was diluted in water to 100 µl, and used for PCR amplification using primers that are specific to MyoD, myogenin, Myf-5 and MLC1a. The length of the PCR products was in the 250–400 bp range. The fluorescent reporter dye, NED, was covalently linked to the 5'-end of the sense primers. ß-actin was used as the internal standard of the cDNA content. The sense primer of ß-actin was labeled with HEX amidite. Amplification was first performed with ß-actin primers to normalize the amount of cDNA present in each preparation, and the normalized values were used to adjust for the amount of templates used in the PCR reaction with the gene-specific primers. The PCR reaction was performed in a final volume of 25 µl containing 10 pmol of each primer, 0.2 mM dNTP and 1.2 U of Tag DNA polymerase (Promega) under the following conditions; denaturation at 95°C for 30 s, primer annealing at 56°C for 30 s, and primer extension at 72°C for 40 s. Appropriate cycling numbers for each primer pair were pre-determined to ensure that PCRs were in a linear concentration range. One microliter of the amplified products was loaded for capillary electrophoresis using an ABI PRISM 310 genetic analyzer (Perkin-Elmer Applied Biosystem) and quantified using GeneScan Analysis software (Perkin-Elmer Applied Biosystem).
Nuclear run-on assay and northern blot analysis
For the nuclear run-on experiment, nuclei from the control and PABP2 transfected cells were isolated using a previously reported procedure (56). The nuclei were resuspended in 200 ml of transcription buffer (10 mM Tris pH 8.0, 150 mM KCl, 3 mM MgCl2, 20% glycerol, 2 mM DTT) and subjected to in vitro transcription by adding 1 mM each of ATP, GTP, CTP and 200 µCi of [
-32P]UTP (Amersham Pharmacia Biotech) to label nascent transcripts for 30 min at 30°C. Following DNase I and proteinase K treatment, radiolabeled RNA was extracted using TRIzol reagent. The cDNA fragment (5 µg of MyoD, 2 µg of GAPDH) and 5 µg of linearized pUC18 plasmid DNA were denatured with NaOH, slot-blotted on positively charged nylon membrane (Hybond N+, Amersham Pharmacia Biotech), and then hybridized overnight at 68°C with 1 x 107 c.p.m. of labeled transcripts in ExpressHyb hybridization solution (Clontech). After hybridization, filters were stringently washed and exposed to an imaging plate for quantification of radioactivity using an image analyzer, BAS-2500 (FUJIFIRM)
For northern blot analysis, 10 µg of each total RNA was size fractionated on 1.2% formaldehyde-agarose gel and transferred to positively charged nylon membrane with an alkaline condition. The filters were hybridized for 2 h at 68°C with 32P-radiolabeled MyoD or GAPDH cDNA fragments in ExpressHyb hybridization solution (Clontech).
Yeast two-hybrid screening
The cDNA fragment encoding PABP2-N (amino acids 1–145) was subcloned into a yeast two-hybrid vector, pAS2-1 (Clontech) containing the GAL4 DNA-binding domain, which was used as bait. Yeast strain AH109 (Clontech) was transformed using the lithium acetate transformation method (57). Yeast two-hybrid screening was performed by transforming a human skeletal muscle cDNA library (Clontech) fused to the activation domain into the AH109 expressing bait protein. Approximately 3 x 106 transformants were plated on selection medium lacking leucine, tryptophan, histidine and adenine, and then cultured at 30°C for 5 days. To reduce background transformants, primary screened yeast colonies were collected and replated on selection medium containing 15 mM 3-amino-1, 2, 4-triazole (Sigma). The positive clones were confirmed by a ß-galactosidase filter-lift assay.
Immunofluorescence analysis
Immunofluorescence analysis was performed on C2 myoblasts and myotubes grown on collagen coated culture plates. Phosphate-buffered saline (PBS) was used to wash cells extensively at room temperature before fixation and after each step of the described procedures. Cells were fixed and permeabilized in 3% paraformaldehyde, 0.3% Triton X-100 in PBS for 5 min, followed by incubation with 3% paraformaldehyde for 15 min. The fixed cells were blocked for 1 h using 5% normal goat serum in PBS, and then incubated for 1 h at room temperature with 1:500 rabbit anti-Flag polyclonal antibody (ZYMED Laboratories) recognizing Flag-PABP2. Subsequently, cells were incubated with 1:100 FITC-conjugated goat anti-rabbit antibody (Tago) for 1 h. Samples were photographed using an Olympus AX 70 microscope (Olympus Optical).
To determine intranuclear localization, HeLa cells were cultured on coverslips and transfected with the expression plasmid for Flag-PABP2 and/or Myc-SKIP. Cells were fixed at 48 h after transfection and incubated with 1:500 rabbit anti-Flag polyclonal antibody and/or mouse anti-Myc monoclonal antibody (Clontech). The secondary antibodies, 1:100 FITC-conjugated goat anti-rabbit antibody and rhodamine-conjugated anti-mouse antibody (Tago) were used. Samples were observed under a confocal laser-scanning microscope (Leica). For localization of the SC-35 protein, a monoclonal antibody against SC-35 was used as the culture supernatant at a 1:100 dilution.
Luciferase assay
C3H10T1/2 cells were transiently co-transfected with 0.6 µg of reporter plasmid (4RE-tk-luc), 0.3 µg of MyoD construct (pRSV-MyoD), 0.02 µg of pRL-CMV (Promega) and various amounts of each expression construct per well in 6-well plates. The CMV promoter-driven sea-pansy luciferase plasmid, pRL-CMV, was used as an internal control to normalize firefly luciferase activity. The total amount of plasmid DNA was adjusted to 2 µg by addition of pUC18 plasmid DNA. The growth medium was changed to a differentiation medium 24 h after transfection, and 48 h later the cells were harvested for luciferase assay. The luciferase activity was assayed using a dual-luciferase assay system (Promega) according to the manufacturer’s instruction with a TD-20/20 luminometer (Turner Designs).
Preparation of cell lysate and western blot
Cell lysates were prepared from C2, COS-7 and C3H10T1/2 cells for western blot analysis and protein interaction assay. After washing with PBS, pelleted cells were incubated in five volumes of buffer (10 mM Tris pH 8.0, 10 mM NaCl, 2 mM MgCl2) supplemented with a mixture of protease inhibitor (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin and 5 µg/ml aprotinin) for 10 min on ice. The detergent, Igepal CA-630 (Sigma) was added to 0.5% and incubation was allowed to continue for 10 min. Cells were pelleted by centrifugation, and resuspended in extraction buffer (20 mM HEPES pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 50 mM NaF, 0.2 mM EDTA, 0.5 mM DTT, 20% glycerol) supplemented with the mixture of protease inhibitors, and incubated for 1 h with slow rotation at 4°C. The lysate was centrifuged at 15 000 g for 20 min and the supernatant was recovered.
The cell lysates were analyzed by SDS–PAGE and transferred to polyvinylidene difluoride filter using a trans-blot electrophoretic transfer cell (Bio-Rad). Filters were blocked for non-specific binding in Tris-buffered saline containing 0.1% Tween-20 with 5% powdered skimmed milk for 1 h and then incubated with primary antibody for 1 h at room temperature. The following primary antibodies were used; 1:500 anti-Flag polyclonal antibody (ZYMED), 1:500 anti-MyoD polyclonal antibody (C-20; Santa Cruz Biotechnology) and anti-myogenin polyclonal antibody (M-255; Santa Cruz Biotechnology). The filters were incubated with 1:2000 horseradish peroxidase-conjugated secondary antibody (Tago) for 1 h. Immunoreactive bands were visualized using an enhanced chemiluminescence system (Amersham Pharmacia Biotech).
GST-pulldown assay and immunoprecipitation
Approximately 200 µg of protein from cell lysate was diluted in the same volume of buffer (50 mM Tris pH 7.5, 0.2% Igepal CA-630) for the GST-pulldown assay and immunoprecipitation. For the GST-pulldown assay, the diluted cell lysate was incubated with 1 µg of purified GST-fusion protein and 30 µl of glutathione-Sepharose 4B (Amersham Pharmacia Biotech) for 2 h at 4°C with gentle rocking. The resins were washed five times with 1 ml of ice-cold wash buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% Igepal CA-630) and boiled in 20 µl of SDS–PAGE sample buffer for 5 min. Eluted proteins were analyzed by western blot using an anti-Myc monoclonal antibody (Clontech).
For immunoprecipitation the diluted cell lysate was incubated with 2 µg of anti-Flag polyclonal antibody or anti-MyoD polyclonal antibody (M-318; Santa Cruz Biotechnology) and 30 µl of protein G–Sepharose for 2 h at 4°C with slow rotation. Washing conditions and elution of associated proteins were identical to those of the GST-pulldown assay. To detect associated proteins by western blotting, the following antibodies were used; 1:1000 anti-Flag M2 monoclonal antibody (Sigma) for Flag-PABP2, 1:1000 anti-Myc monoclonal antibody for Myc-SKIP, 1:100 anti-MyoD monoclonal antibody (Novocastra).
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ACKNOWLEDGEMENTS |
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The authors thank Dr C. Akazawa for helpful advice and encouragement. This work was supported by Research Grant and Grants-in-Aid for Scientific Research for Center of Excellence (COE) from the Ministry of Health and Welfare, Japan and from the Core Research for Evolutional Science and Technology, Japan Science and Technology. Y.-J.K. was supported by a scholarship from the Kambayashi scholarship foundation.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +81 42 341 2711; Fax: +81 42 346 1742; Email: hayasi_y@ncnp.go.jp
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REFERENCES |
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