Subcellular Localization of the Steroid Receptor Coactivators (SRCs) and MEF2 in Muscle and Rhabdomyosarcoma Cells

Shen Liang Chen, S.-C. Mary Wang, Brett Hosking and George E. O. Muscat

University of Queensland Centre for Molecular and Cellular Biology Institute for Molecular Bioscience St. Lucia, 4072 Queensland, Australia


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Skeletal muscle differentiation and the activation of muscle-specific gene expression are dependent on the concerted action of the MyoD family and the MADS protein, MEF2, which function in a cooperative manner. The steroid receptor coactivator SRC-2/GRIP-1/TIF-2, is necessary for skeletal muscle differentiation, and functions as a cofactor for the transcription factor, MEF2. SRC-2 belongs to the SRC family of transcriptional coactivators/cofactors that also includes SRC-1 and SRC-3/RAC-3/ACTR/AIB-1. In this study we demonstrate that SRC-2 is essentially localized in the nucleus of proliferating myoblasts; however, weak (but notable) expression is observed in the cytoplasm. Differentiation induces a predominant localization of SRC-2 to the nucleus; furthermore, the nuclear staining is progressively more localized to dot-like structures or nuclear bodies. MEF2 is primarily expressed in the nucleus, although we observed a mosaic or variegated expression pattern in myoblasts; however, in myotubes all nuclei express MEF2. GRIP-1 and MEF2 are coexpressed in the nucleus during skeletal muscle differentiation, consistent with the direct interaction of these proteins. Rhabdomyosarcoma (RMS) cells derived from malignant skeletal muscle tumors have been proposed to be deficient in cofactors. Alveolar RMS cells very weakly express the steroid receptor coactivator, SRC-2, in a diffuse nucleocytoplasmic staining pattern. MEF2 and the cofactors, SRC-1 and SRC-3 are abundantly expressed in alveolar and embryonal RMS cells; however, the staining is not localized to the nucleus. Furthermore, the subcellular localization and transcriptional activity of MEF2C and a MEF2-dependent reporter are compromised in alveolar RMS cells. In contrast, embryonal RMS cells express SRC-2 in the nucleus, and MEF2 shuttles from the cytoplasm to the nucleus after serum withdrawal. In conclusion, this study suggests that the steroid receptor coactivator SRC-2 and MEF2 are localized to the nucleus during the differentiation process. In contrast, RMS cells display aberrant transcription factor SRC localization and expression, which may underlie certain features of the RMS phenotype.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Understanding the processes that control muscle differentiation, decay, hypertrophy, and tumors provides the foundation for therapeutic intervention into muscle-related diseases. These processes are adaptive responses to physical, injury-related and pathological stimuli. Muscular atrophy, cachexia, myopathies, and rhabdomyosarcomas (RMSs) reflect failures in the balance between proliferation and differentiation, and/or regeneration and wasting. Muscle wasting is observed in 20–80% of cancer patients and also in people with chronic diseases (1, 2, 3). RMS, a malignant tumor of skeletal muscle, is the most common soft tissue sarcoma in children under the age of 15, accounting for 5–8% of all cases of childhood cancer (4, 5). It is the third most common extracranial solid tumor after neuroblastoma and Wilm’s Tumor. It is commonly believed that RMSs originate from transformed muscle precursor cells; however, the appearance of RMSs in regions that contain few (if any) progenitors suggests another mechanism is involved in this process (6).

Great strides have been made in improving our understanding of muscle differentiation (myogenesis), hypertrophy, regeneration, and decay through the exploitation of the mouse muscle cell line, C2C12 [a subclone of C2 cells (7) deposited into ATCC (Manassas, VA)]. In this in vitro culture system, proliferating C2 myoblasts fuse into postmitotic multinucleated myotubes that acquire a muscle-specific phenotype. This cellular morphogenesis is accompanied by muscle-specific gene expression. RMSs are classified into two broad types, i.e. embryonal and alveolar, that have characteristic clinical, pathological, and histological features (8). Embryonal RMSs occur in young children and account for 60% of cases. The tumor is characterized by malignant spindle and round cells that contain skeletal muscle cross-striations and are located in specific sites, including the head/neck region, genitourinary tract, and orbit. In contrast, alveolar RMSs occur during adolescence as primary tumors of the extremities or trunk. This histological variant is characterized by the presence of fibrovascular septa that form alveolar like spaces filled with monomorphous malignant cells (5, 6, 8).

A group of basic helix-loop-helix (bHLH) proteins encoded by the myoD gene family (myoD, myf-5, myogenin, and MRF-4) and a second class of transcription factors, the myocyte enhancer factor-2 family (MEF2A-D), are required for proper muscle differentiation. The bHLH proteins and MEF2 transcription factors function in a cooperative manner to control the mutually exclusive events of division and differentiation (9, 10, 11).

Transcription is also regulated by the structural conformation of chromatin, a complex made up of DNA, histones, and other proteins. Chromatin structure is regulated by cofactors such as histone acetyltransferases (HATs) and deacetylases (HDACs). Histone acetylation and deacetylation affect accessibility of DNA to the transcriptional machinery, leading to transcriptional activation or repression, respectively. Both histone hyperacetylation and hypoacetylation have been associated with the neoplastic process, underscoring the need for improving our understanding of this process (12).

The cofactors PCAF and p300 that have HAT activity have been demonstrated to function as critical coactivators for the muscle-specific bHLH protein, MyoD, during myogenic commitment. However, skeletal muscle differentiation and the activation of muscle-specific gene expression are dependent on the concerted action of another bHLH factor, myogenin, and the MADS protein, MEF2, that function in a cooperative manner.

The cofactors GRIP-1 (glucocorticoid receptor interacting protein 1) and CBP/p300 have HAT activity and function as coactivators for MEF2C during myogenesis. (Refs. 13, 14, 15 and references therein). Class II HDACs (HDAC–4 and –5) interact with the MEF2 proteins, and inhibit MEF2-dependent transactivation (16) and myoblast differentiation (17). The transcriptional activity of class II HDACs is controlled by compartmentalization (16) and 14–3-3-mediated subcellular localization (18). HDACs-4 and -5 are expressed constitutively in myoblasts and myotubes during C2C12 myogenesis, presenting a dilemma in terms of transcriptional control and the differentiation process (17). However, recently it has been demonstrated that myogenesis is controlled via differentiation-dependent nucleocytoplasmic trafficking of HDACs.

GRIP-1 belongs to the structurally related but genetically distinct SRC family (reviewed in Ref. 19) has three members variously denoted as SRC-1/N-CoA-1 (20, 21, 22), SRC-2/GRIP-1/TIF-2/N-CoA-2 (23, 24), and SRC-3/ACTR/pCIP/RAC-3/AIB1/TRAM1(22, 25, 26, 27, 28).

Many recent studies have implicated and confirmed the link between alterations in chromatin structure and differentiation, disease, and cancer. Chromatin remodeling by histone acetylation is generally associated with cell cycle arrest and differentiation, whereas deacetylation promotes cellular proliferation and growth. Many lines of evidence suggest a strong link between chromatin structure, normal and aberrant growth, and tumorigenesis. Recent studies implicate alterations in chromatin structure by histone hyperacetylation/deacetylation as playing an important role in either the genesis or suppression of cancer. Whether histone hyperacetylation or deacetylation is involved appears to depend on the specific target gene (reviewed in Refs. 12, 15, 29, 30) and references therein). For example, studies suggest that factors with HAT activity, e.g the SRC/p160 factors, are involved in transcriptional activation and exert antiproliferative effects. For example, translocations of SRC-2/TIF-2/GRIP-1 [i.e. coactivators with HAT activity] to genes implicated in chromatin structure and function have been documented in several leukemias: monocytic leukemia zinc finger protein gene (MOZ)-mediated monocytic leukemia; mixed lineage leukemia gene (MLL)-mediated acute lymphoblastic leukemias (31, 32, 33). Amplification and/or overexpression of AIB-1 is implicated in the onset, development, and prognosis of breast cancer (26).

Recently, we have observed that muscle differentiation is regulated by MEF2C and GRIP-1 (13). The mechanism involves direct interactions between MEF2 and GRIP-1. However, the subcellular localization and nucleocytoplasmic trafficking of the HAT complexes in the process of mammalian muscle differentiation and/or in skeletal muscle tumors have not been investigated. RMS cells have impaired myogenic factor activity, and it has been suggested that they lack some critical cofactors (34). In this study we have observed that RMS cells derived from skeletal muscle tumors harbor defects in the subcellular localization, expression, and activity of key transcription factors and SRC cofactors that control essential elements of the muscle-specific differentiation program.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SRC-2/GRIP-1 Is Expressed in the Nucleus during Skeletal Muscle Differentiation
Nuclear receptor-mediated activation of transcription is associated with cofactors collectively denoted, as the steroid receptor coactivators (SRCs). These SRC cofactors interact with p300/CBP, recruit PCAF to a complex that synergistically activates transcription and remodels the chromatin (15). PCAF, p300, and SRC-2/TIF-2/GRIP-1 have also been demonstrated to function as critical coactivators for MyoD and MEF2 (13, 14). We have previously observed that the mRNA encoding SRC-2 is expressed in proliferating myoblasts and postmitotic differentiated myotubes and that the protein levels increase during differentiation (13). The SRC-1 mRNA is rarely expressed and SRC-3/pCIP/AIB-1/ACTR/RAC-3 mRNA was not detectable during the differentiation of mouse muscle cells in culture (13). From this point we will refer to all the SRC coactivators as SRC-1, -2, and -3 to establish the nomenclature usage in this manuscript.

We used immunofluorescence to investigate the subcellular localization and expression of the SRCs and MEF2 proteins during skeletal myogenesis in culture. To detect and analyze the subcellular localization pattern of SRC-2 we conducted immunofluorescence staining using monoclonal anti-SRC-2 antibody. SRC-2 was endogenously expressed in all low and high confluency proliferating C2C12 myoblasts grown in high serum (DMEM supplemented with 20% FCS) (Fig. 1Go, A and B). The majority of staining was restricted to the cell nucleus, with weak (but significant) staining observed in the cytoplasm (Fig. 1Go, A and B). The staining pattern in the nucleoplasm was extranucleolar and appeared in prominent nuclear dots in a punctate pattern characteristic of nuclear and splicing bodies.



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Figure 1. SRC-2 Progressively Localizes to the Nucleus of Muscle Cells during Differentiation

A and B, SRC-2 (green) expression in increasingly confluent C2C12 proliferating myoblasts grown in growth medium (DMEM supplemented with 20% FCS). C, GRIP-1 in confluent myoblasts after 24 h serum withdrawal (cultured in differentiation medium, DMEM supplemented with 2% HS). D, Myotubes after 72 h serum withdrawal stained with SRC-2. MEF2 localizes to the nucleus of muscle cells. E, MEF2 (red) expression in C2C12 proliferating myoblasts grown in growth medium (DMEM supplemented with 20% FCS) and G, Myotubes after 72 h serum withdrawal stained with MEF2 (red). F and H show the DAPI-stained myoblast and myotube nuclei. C2 cells were grown on cover slides held in six-well dishes. SRC-2 immunostaining (green) of fixed cells was performed with the monoclonal antibody against GRIP-1 (T73620, 1:200 dilution, Transduction Laboratories, Inc.); cells were washed and incubated with the BODIFY-conjugated goat antimouse IgG secondary antibody (1:2,000). MEF2 immunostaining of fixed cells was performed with the rabbit polyclonal antibody against MEF2 (C-21, sc313, 1:200 dilution, Santa Cruz Biotechnology, Inc.); cells were washed and incubated with the secondary antibody Texas red conjugated goat antirabbit IgG (1:2,000).

 
Interestingly, within 24 h after the induction of the differentiation program by serum withdrawal [DMEM supplemented with 2% horse serum (HS)] the staining becomes predominantly localized to the nucleus (Fig. 1CGo). Furthermore, the nuclear staining is progressively more localized to the dot-like structures or nuclear bodies that appear more prominently in a differentiation-dependent manner.

We then conducted further immunofluorescence staining of cells after 72–96 h of serum withdrawal. At this stage the cells form postmitotic multinucleated cells (myotubes), where the nuclei line up end to end. This demonstrated that SRC-2 was endogenously expressed in differentiated postmitotic muscle cells and also localized to the nucleus in prominent dot-like structures. (Fig. 1DGo). We also conducted immunofluorescent analysis of SRC-1 and -3 using antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Affinity BioReagents, Inc. (Golden, CO) but the level of staining was low to insignificant and consistent with the Northern analysis (data not shown).

MEF2 is Expressed in the Nucleus during Skeletal Muscle Differentiation: MEF2 Is Expressed in a Variegated/Mosaic Pattern in Proliferating Myoblasts
We among others have previously demonstrated that when proliferating C2C12 myoblasts are induced to biochemically and morphologically differentiate into postmitotic multinucleated myotubes, that the transition is associated with an increase in the levels of MEF2 proteins during myogenesis.

To detect and analyze the subcellular localization pattern of MEF2, we conducted immunofluorescence staining using polyclonal anti-MEF2 antibody. MEF2 was endogenously expressed in the nucleus of proliferating C2C12 myoblasts grown in high serum (Fig. 1EGo). We observed consistently that in a population of proliferating myoblasts (which express low levels of MEF2), that only 1–5% of the cells (nuclei) express MEF2. This can be seen by comparing Fig. 1EGo to the 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei in Fig. 1FGo. Importantly, although the staining of the protein is restricted to the nucleus, the expression pattern in proliferating myoblasts is mosaic/variegated (35, 36), i.e. it is either active or completely inactive in individual cells, which may suggests this transcription factor maybe regulated in a binary fashion (35, 36). In contrast, MEF2 was expressed in more than 80% of the nuclei in differentiated postmitotic myotube cells (Fig. 1Go, G and H).

It should be noted that the MEF2 antibody used in these analyses was made against an epitope at the C terminus of MEF2A and has significant cross-reactivity to MEF2C, but not MEF2D or MEF2B. Previous MEF2 RNA and protein analysis using the cell culture model of skeletal myogenesis suggests MEF2D is expressed in the nuclei of proliferating myoblasts grown in high serum (whereas MEF2A and C are entirely absent) and myotubes (37). In contrast, MEF2A expression is induced only after serum withdrawal (38). Furthermore, MEF2C is expressed only in well differentiated postmitotic myotubes after more than72 h of serum withdrawal (39, 40). Our analysis in the context of these previous investigations suggests that early MEF2 expression is probably MEF2A and, during the progressive increase in MEF2 positive nuclei (coupled to the differentiation program) that MEF2A and subsequently MEF2C are expressed. This pattern of expression, i.e. the progressive increase in the proportion of nuclei expressing MEF2 from mosaic/variegated to total expression in all nuclei has been described as a characteristic of binary regulation (35, 36). The future availability of MEF2A and C-specific antibodies will help to determine whether this suggestion is correct.

GRIP-1 and MEF2 Are Colocalized in the Nuclei of Differentiated Muscle Cells
We had previously demonstrated that the steroid receptor coactivator, GRIP-1, coactivates MEF2C-mediated transcription, and that the mechanism involves direct interaction between MEF2C and SRC-2 (13). This was demonstrated by a number of biochemical interaction assays and two-hybrid analysis. In this study we examined the expression of SRC-2 and MEF2 in muscle cells by double staining myotubes with antibodies directed against SRC-2 and MEF2. Conventional and confocal two-color immunofluorescence microscopy confirmed that the SRC-2 (Fig. 2Go, A and D) and MEF 2 (Fig. 2Go, B and E) proteins were endogenously expressed in differentiated muscle cells. The colocalization of SRC-2 and MEF2 in differentiated muscle cell nuclei was confirmed by merging these two images, which showed considerable colocalization (Fig. 2Go, C and F, yellow) of the two proteins in the nucleus.



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Figure 2. Myotubes after 72 h Serum Withdrawal Stained with SRC-2 (Green, A and D) and MEF2 (Red, B and E), Respectively

Merged (yellow) SRC-2 and MEF2 images (C and F). Immunofluorescence was examined by conventional (panels A–C) and confocal microscopy (D–F). C2 cells were grown on cover slides held in six-well dishes. SRC-2 immunostaining (green) of fixed cells was performed with the monoclonal antibody against GRIP-1 (T73620, 1:200 dilution, Transduction Laboratories, Inc.), and cells were washed and incubated with the BODIFY-conjugated goat antimouse IgG secondary antibody (1:2,000). MEF2 immunostaining of fixed cells was performed with the rabbit polyclonal antibody against MEF2 (C-21, sc313, 1:200 dilution, Santa Cruz Biotechnology, Inc.), and cells were washed and incubated with the secondary antibody Texas red-conjugated goat antirabbit IgG (1:2,000).

 
Analysis of SRC Expression in Muscle and RMS Cells
To gain insight into which of the p160 cofactor(s), SRC-1, SRC-2/GRIP-1/TIF-2 and SRC-3/RAC-3/ACTR/pCIP/AIB-1 are expressed in alveolar RMS Rb-2061 cells, we initially investigated the expression pattern of the mRNAs encoding the SRC family in RMS cells relative to mouse myoblast C2C12 cells [the expression pattern in C2C12 cell has been described previously (13)]. Proliferating C2C12 myoblasts were induced to biochemically and morphologically differentiate into postmitotic multinucleated myotubes by serum withdrawal in culture over a 4- to 72-h period. The transition from a nonmusical phenotype to a contractile phenotype is associated with the activation/expression of 1) the myoD gene family (myoD, myogenin, myf-5, and MRF-4], 2) the MADs box protein, MEF2C, and -3) a structurally diverse group of genes that encode a functional sarcomere responsible for contraction. Concomitant with these events is terminal cell cycle exit, characterized by the repression of cyclin D1 and the activation of the cell cycle inhibitor, p21.

PolyA+ RNA from C2C12 cells was isolated from proliferating myoblasts, confluent myoblasts, and postmitotic myotubes after 4, 8, 24, and 72 h of serum withdrawal and examined by Northern blot analysis. Similarly, RNA was isolated from alveolar RMS Rb-2061 cells grown in high serum after 72 h of serum withdrawal.

We have previously demonstrated in C2 cells (13) that SRC-2/GRIP-1 mRNA was constitutively expressed in proliferating myoblasts as the cells exited the cell cycle and fused to form postmitotic differentiated multinucleated myotubes that had acquired a muscle-specific phenotype (Fig. 3AGo). The hybridization signal corresponded to the expected transcript size of approximately 7.5 kb. It was necessary to use polyadenylated RNA since the level of expression was quite low relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or myogenin, and exposure times required for the detection of SRC-2 were approximately 10-fold longer than that of GAPDH (Fig. 3AGo). The SRC-1 mRNA was very weakly expressed, however, and we could not detect the mRNA transcript that encoded SRC-3. In alveolar RMS Rb-2061 cells, SRC-2 mRNA was not detectable by Northern analysis; however, the mRNAs encoding SRC-1 and SRC-3 were abundantly expressed relative to GAPDH.



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Figure 3. GRIP-1 Is Expressed in Muscle Cells Whereas SRC-1 and AIB-1 Are Abundantly Expressed in RMS Cells

A, Poly A+ RNA was isolated from proliferating C2 myoblasts (PMB, ~50% confluent), confluent myoblasts (CMB, 100% confluent) cultured in growth medium, and from developing myotubes 4, 8, 24, and 72 h after serum withdrawal (i.e. propagated in DM, differentiation medium); and alveolar RMS cells (Rb 2061) cultured in growth medium, and after 72 h of serum withdrawal. After blotting, RNA was probed with 32P-radiolabeled cDNA encoding GAPDH, MyoD, myogenin,, CyclinD1, p21, and SRC-1, -2, and -3. GM, Growth medium (DMEM containing 20% FCS); DM, differentiation medium (DMEM containing 2% HS). B, Western analysis of GRIP-1 expression in C2 and RMS cells cultured in growth and differentiation medium. Nuclear extract was isolated from cells before and after differentiation and subjected to Western analysis with the T73620 monoclonal antibody to human TIF-2 (Transduction Laboratories, Inc.) that cross-reacts with GRIP-1 (mouse).

 
Nuclear extract was isolated from proliferating C2C12 myoblasts grown in high serum and C2C12 myotubes after 3 days of serum withdrawal. Similarly, samples were isolated from RMS cells grown in high serum and after 3 days of serum withdrawal. Western analysis with the T73620 monoclonal antibody to human SRC-2 (Transduction Laboratories, Inc., Lexington, KY) that cross-reacts with the mouse species demonstrated that the levels of the SRC-2 protein increased during myogenesis (Fig. 3BGo). SRC-2 levels in RMS cells were low. The identity of the signal was confirmed with the use of an SRC-2-positive control (T73620, Transduction Laboratories, Inc.) from Jurkat cells (data not shown).

Differential Expression and Subcellular Localization of SRC-2 in Alveolar (Rb-2061) and Embryonal (A-204) RMS Cells
We used immunofluorescence to investigate the subcellular localization and expression of the steroid receptor coactivator, SRC-2, in alveolar RMS Rb-2061 cells. To detect and analyze the subcellular localization pattern of SRC-2, we conducted immunofluorescence staining using monoclonal anti-SRC-2 antibody. SRC-2 was very weakly expressed after 3 days of serum withdrawal in alveolar RMS Rb-2061 cells (Fig. 4AGo). In contrast to C2C12 cells, the staining pattern did not appear in prominent nuclear dots characteristic of nuclear and splicing bodies. The staining was rather diffuse, and observed in the nucleus and cytoplasm. Merging the SRC-2 image (green) with DAPI image (blue) clearly highlighted the expression of SRC-2 in the cytoplasm (Fig. 4BGo).



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Figure 4. Differential Subcellular Localization of SRC-2 in Alveolar and Embryonal RMS Cells

A, SRC-2 expression in alveolar RMS cells (Rb-2061/RMS13) grown in differentiation medium. B, DAPI (blue)-stained nucleus merged with GRIP-1 staining highlighting cytoplasmic staining. C and D, SRC-2 staining, and merged GRIP-1/DAPI images after ectopic GRIP-1 expression. E and F, SRC-2 expression and DAPI staining in embryonal RMS cells (Rb-204) grown in differentiation medium. GRIP-1 immunostaining (in green) of fixed cells was performed with the monoclonal antibody against SRC-2 (T73620, 1:200 dilution, Transduction Laboratories, Inc.); cells were washed and incubated with the BODIFY-conjugated goat antimouse IgG secondary antibody (1:2,000).

 
Transfection of GRIP-1 into alveolar RMS Rb-2061 cells, followed by immunofluorescence with SRC-2 antibodies, indicated increased SRC-2 expression in these cells (Fig. 4CGo). We could not discriminate transfected from endogenous SRC2; however, the elevated expression was diffuse and was observed in both the nucleus and cytoplasm (Fig. 4DGo).

We then used immunofluorescence to investigate the subcellular localization and expression of the steroid receptor coactivator, SRC-2, in embryonal RMS A-204 cells. To detect and analyze the subcellular localization pattern of SRC-2, we conducted immunofluorescence staining using monoclonal anti-SRC-2 antibody. SRC-2 was strongly expressed in proliferating embryonal RMS A-204 cells (Fig. 4EGo). In contrast to alveolar RMS Rb-2061, the staining pattern in embryonal A-204 cells appeared in prominent nuclear dots characteristic of nuclear and splicing bodies. SRC-2 was predominantly expressed in the nucleus. Comparing the SRC-2 image (green) (Fig. 4EGo) with the DAPI image (blue) (Fig. 4FGo) clearly highlighted the specific nature of SRC-2 staining, in the nucleus of embryonal RMS A-204 cells.

SRC-1 and SRC-3 Are Abundantly Expressed in Embryonal and Alveolar RMS Cells: Subcellular Localization of p160 Factors Is Defective
We used immunofluorescence to investigate the subcellular localization and expression of the steroid receptor coactivators, SRC-1 and –3, in the alveolar (Rb-2061) and embryonal (A-204) RMS cells after 72 h of serum withdrawal. To detect and analyze the subcellular localization pattern of SRC-1 and -3, we conducted immunofluorescence staining. SRC-1 in alveolar RMS Rb-2061 (Fig. 5Go, A and B) and embryonal RMS A-204 cells (Fig. 5Go, C and D) were expressed in RMS cells. The staining was rather diffuse and was observed in cytoplasm and nucleus. The staining pattern did not appear in prominent nuclear dots characteristic of nuclear and splicing bodies. Merging the SRC-1 image (green) with the DAPI image (blue) clearly highlighted the significant SRC-1 staining in the cytoplasmic expression (Fig. 5Go, B and D). Similarly, significant expression of SRC-3 is highlighted in alveolar RMS Rb-2061 (Fig. 5Go, E and F) and embryonal RMS A-204 cells. (Fig. 5Go, G and H). These data suggest SRC -1 and -3 are aberrantly localized in alveolar and embryonal RMS cells, in contrast to the nuclear-specific pattern of SRC-2 staining in embryonal RMS A-204 cells (Fig. 4EGo).



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Figure 5. Aberrant Localization of SRC-1 and SRC-3 in RMS Cells

A and C, SRC-1 expression in alveolar and embryonal RMS cells grown in differentiation medium. B and D shows the DAPI (blue)-stained nucleus merged with SRC-1 staining highlighting cytoplasmic staining. E and G, SRC-3 staining in alveolar and embryonal RMS cells grown in differentiation medium and merged SRC-3/DAPI images (F and H). SRC-1 immunostaining of fixed cells was performed with the antibody against SRC-1(M-20, sc6098, 1:200 dilution, Santa Cruz Biotechnology, Inc.); cells were washed and incubated with the secondary antibody FITC-conjugated rabbit anti-goat IgG (1:2000). SRC-3 immunostaining of fixed cells was performed with the antibody against RAC-3 (C-20, sc7216, 1:200 dilution, Santa Cruz Biotechnology, Inc.); cells were washed and incubated with the FITC-conjugated rabbit antigoat IgG secondary antibody (1:2,000).

 
MEF2 Is Expressed in Alveolar and Embryonal RMS Cells, but Accumulates in the Cytoplasm of Alveolar Rb-2061 Cells
To detect and analyze the subcellular localization pattern of MEF2 in alveolar and embryonal RMS cells, we conducted immunofluorescence staining using polyclonal anti-MEF2 antibody. MEF2 was endogenously expressed in alveolar RMS cells cultured in growth medium (RPMI supplemented with 10% FCS) and after serum withdrawal (differentiation medium, RPMI supplemented with 2% HS) (Fig. 6Go, panels B and E, respectively). In contrast to the mosaic/variegated expression pattern observed in proliferating C2 cells, MEF2 was expressed in all RMS cells in high and low serum conditions. However, the staining pattern was not exclusively localized to the nucleus, with many cells displaying predominantly cytoplasmic staining (Fig. 6Go, B and E). Some cells displayed an accumulation of MEF2 in the cytoplasm after serum withdrawal (Fig. 6EGo, yellow arrows). Serum withdrawal leads to increased expression of MEF2 (see also Fig. 6Go, M and N), but does not induce nuclear localization (Fig. 6Go, E and F).



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Figure 6. Defective Subcellular Localization and Cytoplasmic Accumlation of MEF2 in Alveolar RMS Cells

A, MyoD expression in alveolar RMS (Rb 2061) cells cultured in growth medium. B, MEF2 expression in RMS (Rb 2061) cells cultured in growth medium, immunostained with MEF2 (red). C, Merged MEF2 (red) and MyoD (green) images highlighting cytoplasmic MEF2 expression. D, MyoD expression in RMS Rb-2061 cells cultured in differentiation medium, immunostained with MyoD (green). E, MEF2 expression in RMS Rb-2061 cells cultured in differentiation medium. F, Merged MEF2 (red) and MyoD (green) images highlighting the cytoplasmic accumulation of MEF2 in alveolar RMS Rb-2061 cells. G, SRC-2 expression in embryonal RMS A-204 cells cultured in growth medium. H, MEF2 expression in embryonal RMS cells cultured in growth medium, immunostained with MEF2 (red). I, Merged MEF2 (red) and SRC-2 (green) images highlighting cytoplasmic MEF2 expression. J, SRC-2 expression in embryonal RMS cultured in differentiation medium. K, MEF2 expression in embryonal RMS cells cultured in differentiation medium. L, Merged MEF2 (red) and SRC-2 (green) images highlighting the translocation of MEF2 into the nucleus of embryonal RMS cells after serum withdrawal. Western analysis of total lysate (M) and nuclear extracts (N) from C2 and alveolar RMS Rb-2061 cells cultured in growth and differentiation medium with the MEF2A antibody that cross-reacts with MEF2C. PMB and CMB, Proliferating and confluent myoblasts, respectively. GM, Growth medium (DMEM containing 20% FCS); DM, differentiation medium (DMEM containing 2% HS). O, IVT denotes in vitro transcribed and translated MEF2 isoforms used as markers of specific MEF2 (A and C) isoforms. P, Western analysis of nuclear extracts from C2 and alveolar and embryonal RMS cells cultured in growth and differentiation medium with the MEF2A antibody that cross-reacts with MEF2C.

 
MEF2 functions in a cooperative manner with the muscle-specific basic helix loop helix proteins encoded by the myoD gene family through the formation of a complex (9) that also includes the cofactors p300 and SRC-2 (13, 14). The formation of this complex mediated by direct protein-protein interaction is critical to the activation and progression of the differentiation program. Hence, we proceeded to investigate the subcellular localization of the MEF2 partner, and master regulatory protein, MyoD, in the alveolar RMS Rb-2061 cells by double immunostaining. The diffuse staining pattern and cytoplasmic expression of MEF2 were further highlighted by double staining cells with antibodies directed against MyoD and MEF2. Immunostaining with MyoD clearly showed that MyoD (Fig. 6Go, A and D, green) was localized to the nucleus of all cells grown in high serum (Fig. 6AGo). Serum withdrawal induced the punctate nuclear dot staining pattern in the nucleus (Fig. 6DGo). Two-color immunofluorescence and merging (Fig. 6Go, C and F, yellow) highlighted the nuclear expression of MyoD and the predominant cytoplasmic localization of MEF2 in the cytoplasm. The expression of MEF2 in the cytoplasm is clearly displayed in red, in contrast to the green or yellow nuclei in Fig. 6Go, C and F.

We then analyzed the subcellular localization pattern of MEF2 in embryonal RMS cells. Immunofluorescence staining using polyclonal anti-MEF2 antibody demonstrated MEF2 was endogenously expressed in alveolar RMS cells growth medium (RPMI supplemented with 10% FCS) and after serum withdrawal (differentiation medium, RPMI supplemented with 2% HS) (Fig. 6Go, panels H and K, respectively). The staining pattern of MEF2 in alveolar and embryonal RMS cells grown in high serum was similar. In contrast to the accumulation of MEF2 in the cytoplasm after serum withdrawal in Rb 2061 cells (Fig. 6EGo, yellow arrows), MEF2 accumulated in the nucleus after serum withdrawal in the embryonal RMS A-204 cells (Fig. 6KGo). This suggested the trafficking of MEF2 into the nucleus was regulated by serum withdrawal and perhaps occurred in a density-dependent manner.

Embryonal RMS A-204 cells express SRC-2 in the nucleus; furthermore, serum withdrawal leads to a predominant localization of MEF2 in the nucleus. MEF2 functions through the formation of a complex that also includes the cofactor SRC-2. The formation of this complex mediated by direct protein-protein interaction is critical to the activation and progression of the differentiation program (13). Hence, we proceeded to investigate the subcellular localization of the MEF2 cofactor SRC-2 in the RMS A-204 cells by double immunostaining after serum withdrawal. The expression of MEF2 and SRC-2 in embryonal A-204 cells was further highlighted by double staining cells with antibodies directed against SRC-2 and MEF2. Immunostaining clearly showed that SRC-2 (Fig. 6Go, G and J, green) was localized to the nucleus of all cells cultured in growth and differentiation medium. Two-color immunofluorescence and merging (Fig. 6Go, I and L, yellow) highlighted the nuclear expression of SRC-2 and the predominant localization of MEF2 from the cytoplasm to the nucleus after serum withdrawal.

We investigated this differential expression and localization of MEF2 by Western analysis of total lysates and nuclear extracts from C2 cells and alveolar RMS Rb 2061 cells. We observed that MEF2 proteins were expressed and induced in both C2C12 cells and RMS cells after serum withdrawal (Fig. 6MGo). However, RMS cells abundantly expressed MEF2A and the faster migrating MEF2C species (~50 kDa). Total expression of MEF2 in both cell types after serum withdrawal, as measured by immunostaining and Western analysis, was similar. Curiously, Western analysis of nuclear extracts (Fig. 6NGo) from C2 and alveolar RMS Rb-2061 cells indicated that significant amounts of the faster migrating MEF2C species from the RMS cells was not shuttled into the nucleus after serum withdrawal. This observation was consistent with the immunofluorescent analysis. In summary, this indicates that MyoD is exclusively localized to the nucleus; however, the partner protein, MEF2, is predominantly localized to the cytoplasm. This confirmed that the trafficking of MEF2C into the nucleus of alveolar RMS Rb-2061 cells is impaired.

Similarly, Western analysis of the embryonal RMS A-204 cells demonstrates that these cells accumulate large amounts of MEF2C in the nucleus after serum withdrawal (Fig. 6PGo), in contrast to the alveolar cells. This correlated with the expression of SRC-2 in the nuclei of these cells detected by immunostaining.

The Transcriptional Activity of the Myogenic Factors Is Impaired in Alveolar RMS Rb-2061 Cells: MEF2 Function Is Significantly Compromised in Alveolar RMS Rb-2061 Cells
We compared the relative activity of MEF2C, MyoD, and myogenin in muscle and RMS cells using the GAL4 hybrid assay. We observed that MEF2C, MyoD, and myogenin induced transcription by approximately 25-, 50-, and 120-fold (relative to GAL0, arbitrarily set to 1) in C2 muscle cells. In comparison, in alveolar RMS Rb-2061 cells, MEF2C, MyoD, and myogenin induced transcription by approximately 4-, 22-, and 45-fold. This suggested that in RMS cells, the transcriptional activity of MEF2C is significantly impaired (>5-fold). MyoD and myogenin activity is reduced about 2-fold (Fig. 7AGo).



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Figure 7. The Activity of MEF2 Is Impaired in Rhabdomyosarcoma Cells

A, GAL4 hybrid analysis in C2C12 and embryonal RMS Rb-2061 cells with GAL4-MEF2C, -MyoD, and -myogenin in the presence and absence of GRIP-1. GAL0 is arbitrarily set to 1. B, GAL4 hybrid analysis in C2C12 and embryonal RMS Rb-2061 cells with GAL4-TBP, and -VP16 as controls for the transcriptional activity of nonmyogenic transactivators in C2C12 and RMS cells. C, Activity of a MEF2-dependent reporter comprised of 3 x mef-2 sites linked to a basal E1b-LUC reporter and the basal E1b promoter in C2C12 and RMS cells. The C2C12 and RMS cells were passaged into 12-well plates and transfected at 60–80% confluence with 1,000 ng reporter and 330 ng of the specified GAL-chimera by liposome-mediated transfection for 6–12 h. Cells were harvested 48 h after transfection for the assay of luciferase.

 
Interestingly, cotransfection of the cofactor, SRC-2, increased MEF2C-, MyoD-, and myogenin-mediated transcription to approximately 10-, 29-, and 53-fold (relative to GAL0, arbitrarily set to 1). This demonstrated that MEF2C activity is preferentially increased by SRC-2 expression, whereas MyoD or myogenin activity is relatively unaffected (Fig. 7AGo). We confirmed that this effect was not due to a generalized repression of transcription in RMS cells by transfection of GAL-TBP (TATA box binding protein) and the chimeric GAL4VP16 trans-activator (Fig. 7BGo) into C2C12 and Rb-2061 cells. TBP activity was similar in both cells; furthermore, GALVP16 activity was increased 2-fold in RMS Rb-2061 cells.

To further substantiate these results we tested the activity of a MEF2-dependent reporter with three tandem copies of the MEF2 binding sites upstream of a basal E1b promoter in C2C12 and RMS Rb-2061 cells cultured in differentiation medium (Fig. 7CGo). These experiments clearly demonstrated again that the activity of a MEF2-dependent reporter is reduced by 4-fold in alveolar RMS Rb-2061 cells. In conclusion, our studies demonstrate that the activity and localization of MEF2C in alveolar RMS cells are impaired.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our investigations provide evidence for the predominant localization of SRC-2 to the nucleus in a differentiation-dependent manner. Furthermore, the nuclear staining is increasingly localized to aggregates of distinct foci. The cytoplasmic-nuclear shuttling of the coactivators provides a novel mechanism for the control of cell differentiation. MEF2 expression in proliferating (undifferentiated) myoblasts is variegated, and induction of the differentiation program results in the activation of MEF2 expression in all nuclei in the postmitotic myotube cell.

Muscle cells abundantly express SRC-2 (and weakly express SRC-1). Alveolar RMS-Rb 2061 cells derived from malignant skeletal muscle tumors very weakly express the steroid receptor coactivator, SRC-2, and more abundantly express SRC-1 and SRC-3. In contrast, the embryonal A-204 cells express SRC-2; however, they also express SRC-1 and SRC-3. Furthermore, SRC-1 and -3 are inappropriately localized in cells derived from alveolar and embryonal RMS. This observation correlates with a number of other studies on steroid receptor cofactor expression, cancer development, and progression. For example, normal brain tissue is positive for SRC-2(GRIP-1/TIF-2) and SRC-1; however, meningiomas (associated with breast cancer) abundantly express SRC-3 and SRC-1 (41). SRC-3 (AIB-1) was originally identified in a search for genes whose expression and copy number were elevated in human breast cancers (26). SRC-3 amplification and increased expression correlate with steroid receptor-positive breast and ovarian tumors (42). SRC-3 amplification in association with other coamplifications (e.g. ERBB2, MYC, CCND1, or FGFR1) suggest poor prognosis and worsened outcome (43, 44, 45). Pancreatic carcinoma and gastric tumors are also linked to SRC-3 amplification/overexpression and chromosomal aberration (46, 47). Our study suggests that elevated SRC expression may also be a marker for RMSs.

Aberrant subcellular localization of SRCs, and of critical transcription factors in RMSs, rather than over/mis-expression, suggests that inappropriate/dysfunctional trafficking of essential transcription factors and cofactors is associated with the RMS phenotype. However, we cannot rule out increased export or reduced degradation of cytoplasmic MEF2. Interestingly, inappropriate localization of SRC-2 and dysfunctional trafficking of MEF2 in alveolar cells relative to embryonal RMS cells may correlate with the poor prognosis for individuals with alveolar RMS.

Class II HDACs (HDAC-4 and -5) interact with the MEF2 proteins and inhibit MEF2-dependent transactivation and myoblast differentiation (17). The transcriptional activity of class II HDACs is controlled by compartmentalization (16, 17) and 14–3-3 protein- mediated subcellular localization (18). HDACs-4 and -5 are expressed constitutively in myoblasts and myotubes during C2C12 myogenesis, presenting a dilemma in terms of transcriptional control and the differentiation process (16). However, recently it has been demonstrated that myogenesis is controlled via differentiation-dependent nuclear export of HDACs. The shuttling of HDACs and the nucleocytoplasmic trafficking of these transcriptional repressors are regulated by phosphorylation. Calcium-calmodulin-dependent protein kinase stimulates myogenesis, blocks HDAC-mediated inhibition of MEF2, and induces export of the class II HDACs from the nucleus. Export of these repressors from the nucleus is mediated by the 14–3-3 proteins in a phosphorylation-dependent manner.

These results raise the scenario that kinase signaling regulates trafficking of the SRCs into the nucleus. The mitogen-activated protein kinases (MAPKs), p38 and ERK5, stimulate MEF2 activity by direct phosphorylation of the C-terminal activation domain (46). Analogously, SRC-3 has been demonstrated to be a phosphoprotein targeted by MAPK (47). Furthermore, MAPK activation of SRC-3 facilitates the recruitment of other cofactors and associated HAT activity (47). These observations in the context of defective SRC cofactor localization in RMS cells (this work) suggest that protein kinase signaling mediates the active shuttling of the SRCs into the nucleus. This hypothesis is strongly supported by several observations: 1) myogenesis is dependent on p38 MAPK activation; 2) RMS cells are deficient in p38 MAPK; 3) MAPK kinase 6 expression in RMS cells stimulates MEF2 activity; and 4) ectopic expression of an activated MAPK kinase 6, which induces p38 MAPK, restored MyoD and MEF2 function and led to terminal differentiation.

Our current studies are directed at examining whether MAPK activation stimulates the localization of MEF2 and the SRC cofactors into the nucleus. Our preliminary data suggest O-tetradecanoylphorbol-13-acetate treatment completely restores MEF2C activity in alveolar RMS cells (data not shown). Time will tell whether drug intervention targeted at protein kinases, and nucleocytoplasmic trafficking of SRC cofactors is a plausible therapeutic strategy.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Extract Preparation
To isolate nuclear protein, cells were resuspended in 0.5–2 ml of TM buffer (10 mM Tris at pH 7.4, 2 mM MgCl, 5 mM dithiothreitol) freshly supplemented with protease inhibitors and incubated on ice for 5–10 min. Then the cells were spun down at 2,000 rpm for 10 min at 4 C. The supernatant was discarded and the cells were frozen and thawed twice to break the cell membranes. The broken cells were resuspended in 0.5 ml TM buffer and incubated on ice for 10 min followed by spinning at 2,000 rpm at 4 C for 10 min. The supernatant was aspirated for analysis of cytosolic protein, and 0.5 ml nuclear extract buffer (20 mM Tris, 400 mM KCl, 2 mM EDTA, 2 mM MgCl, 1 mM dithiothreitol, 10% glycerol) freshly supplemented with protease inhibitors was added to resuspend the cells and then incubated on ice for 60–90 min. The cells were vigorously vortexed every 10 min during this incubation to maximize the extraction efficiency. Cell debris were cleared by centrifugation at 15,000 rpm at 4 C for 20 min, and the supernatant was dialyzed against PBS at 4 C to reduce the salt concentration. After dialysis the protein concentrations were determined by the Bradford dye method.

For isolating total proteins, cells were lysed in lysis buffer from LUC-Lite luciferase assay kit (Packard Instruments, Meriden, CT) and cleared by centrifugation. Proteins were dialyzed against PBS to reduce the detergent’s concentration, and the protein concentration were determined by DECA protein assay kit (Pierce Chemical Co., Rockford, IL).

Immunoblotting
Aliquots of 30 µg nuclear protein or 50 µg total protein were used for each sample. The proteins were blotted onto nitrocellulose membrane at 160–220 mA overnight in Towbin buffer (25 mM Tris at pH 8.3, 200 mM glycine, 20% methanol, 0.4% SDS) after being resolved on 7% or 10% SDS-PAGE gels. The blots were blocked in blocking solution (5% skim milk, 0.05% Tween 20 in PBS) at 37 C for 1 h and then incubated with 0.1 µg/ml MEF2 or TIF2 antibodies diluted in blocking solution at room temperature for at least 2 h or at 4 C overnight. Blots were washed several times in PBST (0.05% Tween in PBS) after the primary antibody incubation and then incubated with secondary antibody diluted in blocking solution at room temperature for 1 h. After the secondary antibody incubation the blots were washed in PBST for at least 20 min with several changes of PBST and then viewed with chemiluminescence (ECL; Amersham Pharmacia Biotech, Arlington Heights, IL).

Northern Analysis
Total RNA from different differentiation stages of C2C12 and RMS cells was prepared by using an acid guanidinium thiocyanate-based method (50). Briefly, cells were harvested in PBS containing 5 mM EDTA and then lysed in solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol.). Cell lysates were kept at -20 C before RNA was extracted. To extract total RNA, water-saturated phenol was added to the lysate and then vortexed. Chloroform was added and vortexed vigorously for 30 sec before centrifugation at 4 C to separate the aqueous phase from the organic phase. RNA in the aqueous phase was precipitated by isopropanol precipitation and was resuspended in diethyl pyrocarbonate (DEPC)-treated water.

Total RNA was further purified using the Oligotex mRNA Mini Kit (QIAGEN, Chatsworth, CA), and an aliquot of 5 µg poly (A)+ RNA was used for each sample. RNA was denatured at 70 C for 10 min in RNA sample buffer (50% formamide, 2 M formaldehyde, 1x MOPS, 50 µg/ml ethidium bromide), and quickly chilled on ice before being run on a 1% agarose-2 M formaldehyde denaturing gel in 1x 3-[N-morpholino]propanesulfonic acid (MOPS) buffer (20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA). The fractionated RNA was blotted onto nylon membranes (Hybond N; Amersham Pharmacia Biotech) overnight with a vacuum blotter (LKB, Rockville, MD) to ensure that the high molecular weight RNA was transferred completely. The nylon membranes were UV cross-linked and baked at 80 C after blotting. The nylon membranes were prehybridized in 5x SSPE, 5x Denhardts solution, 0.1% SDS, 100 µg/ml herring sperm DNA, 5% glycine overnight at 42 C before the addition of specific probes. Specific probes were labeled by a random priming method using specific cDNAs as templates and were further purified with NICK column (Pharmacia Biotech, Piscataway, NJ). Prehybridized blots were hybridized with approximately 107 cpm of specific probe for another 24–48 h in prehybridization buffer at 42 C. After hybridization the blots were washed in 1x or 0.5x SSC for 30–60 min at 65 C and were visualized by autoradiography.

The SRC-1 probe encompasses amino acids 1,138–1,441 and 180 bp of the 3'- untranslated region, the GRIP-1 probe spans amino acids 545–1,157, and the RAC-3 probe contains amino acids 507–937. The MyoD, myogenin, p21, and cyclin D1 probes were EcoRI digestion fragments isolated from pEMSV-myoD, pEMSV-myogenin, pCMW35, and pGEX-3X-CYL1 plasmids, respectively. GAPDH cDNA was amplified by RT-PCR and gel isolated for use as a template for random priming. cDNA probes were radioactively labeled by random priming. DNA fragments (50–100 ng) were boiled with 20 ng of random primers (pdN6; Pharmacia Biotech) at 100 C for 10 min and quickly chilled on ice for another 10 min. Denatured DNA was then incubated overnight with 5–10 µl of [{alpha}-p32]-dCTP, 62.5 µM of dATP, dGTP, dTTP, and 5 U of Klenow enzyme (New England Biolabs, Inc., Beverly, MA) in 1x EcoPol buffer (New England Biolabs, Inc.). Probes were purified using NICK columns (Pharmacia Biotech) according to the manufacturer’s instructions.

Fluorescence Immunohistochemistry
For fluorescence immunohistochemistry analysis, C2C12 and RMS cells were grown on cover slides held in six-well dishes. Cell were washed in PBS once and then fixed in 100% methanol at -20 C for 5 min. After fixation cells were washed once in PBS and then quenched in PBS containing 50 mM NH4Cl to avoid the deleterious effect of the methanol on the antibodies. Cells were then blocked in blocking solution (0.2% fish skin gelatin and 0.2% BSA diluted in PBS) at room temperature for 10 min before being incubated with primary antibody diluted 1:200 in blocking solution at room temperature for 30 min. Then cells were washed with PBS four times before being incubated with Bodify-, Texas red-, or fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (Molecular Probes, Inc., Eugene, OR) diluted 1:2,000 in blocking solution at room temperature for 30 min and then washed four times with PBS again. To visualize the nuclei, cells were incubated with DAPI (1:2,000 dilution in PBS) at room temperature for 10 min after the secondary antibody incubation and were then washed thoroughly with PBS.

Transient Transfection and GAL4 Hybrid Analysis
C2C12, and RMS cell line SJRH30/RMS13 were passaged into 12-well plates and transfected at 60–80% confluence with 1,000 ng of reporter, G5E1b-LUC, and 330 ng of GAL-MEF2C, GAL-MyoD, or GAL-myogenin in the presence and absence of GRIP-1 SJRH30) by liposome (Dotap and Dosper, Roche Molecular Biochemicals, Indianapolis, IN)-mediated method for 6–12 h. Cells were harvested 48 h after transfection for assay of luciferase activity. Each experiment represented at least two sets of independent triplicates to overcome the variability inherent in transfection experiments.


    ACKNOWLEDGMENTS
 
We thank Drs. Michael Stallcup and J. Don Chen for the generous gift of plasmids.


    FOOTNOTES
 
Address requests for reprints to: Dr. George E. O. Muscat, X-Ceptor Therapeutics, Inc., 4757 Nexus Centre Drive, Suite 200, San Diego, California 92121. E-mail: G.Muscat{at}imb.uq.edu.au

This investigation was supported by the National Health and Medical Research Council (NHMRC) of Australia. G.E.O.M. is an NHMRC Principal Research Fellow. The Centre for Molecular and Cellular Biology, IMB, is part of the Special Research Centre for Functional and Applied Genomics that is supported by the Australia Research Council (ARC).

Received for publication October 26, 2000. Revision received February 19, 2001. Accepted for publication February 20, 2001.


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