Repositioning of Muscle-specific Genes Relative to the Periphery of SC-35 Domains during Skeletal Myogenesis

Cell Culture and In Vitro Myogenic Differentiation

Normal human fibroblast cells (Detroit 551 or WI-38; American Type Culture Collection, Manassas, VA) were used as nonmyogenic cell controls, and were cultured as described previously (Xing et al., 1995). Flow-sorted undifferentiated normal human myoblast cells (strain 50Mb-1), isolated from the vastus lateralis muscle of a 10-year-old male, were kindly provided by Dr. Helen Blau (Stanford University, Palo Alto, CA) (Webster et al., 1988; Smith et al., 1999). Proliferative myoblast cells were cultured in growth media (GM) comprised of Ham's F-10 media supplemented with 20% fetal bovine serum (Hyclone Laboratories, Logan, UT), 1% (vol/vol) chick embryo extract (60-Å ultrafiltrate), with penicillin and streptomycin, and seeded onto glass coverslips (Corning Glassworks, Corning, NY). In vitro myogenic differentiation was induced by culturing myoblasts in GM to ∼70% confluence, and then switching to low serum differentiation media (DM; DMEM-low glucose supplemented with 2% horse serum (Hyclone Laboratories), 1 mM insulin, 1 mM dexamethasone (Sigma-Aldrich), 100 U/ml penicillin and 100 μg/ml streptomycin). Multinucleated myofibers typically were first noted on day 3, and differentiation was typically complete by day 7. Cell processing for in situ hybridization, involving detergent permeabilization and paraformaldehyde fixation, was as described previously (Xing et al., 1995).

A second cell differentiation model utilized mouse NIH3T3-derived cells (B22 cells) that had been induced to form myoblasts using a MyoD-expressing retrovirus, as previously described (Novitch et al., 1996). Cultures at ∼50% confluency were infected for 30 h with one-third of the supernatant from a confluent 100-mm plate of BOSC23 cells generating MyoD retrovirus and polybrene (4 μg/ml) diluted into fresh media. Mock-infected plates were exposed to media and polybrene only. Differentiation was induced by growth in low serum medium for 36 h or more (Novitch et al., 1996).

The B22 cell line expresses dominant-negative BRG1 protein under the control of the tet promoter (de La Serna et al., 2000). For analysis of normal cells, B22 cells were grown in tetracycline, which represses expression of the mutant protein. To prevent myogenic gene expression, cells were grown without tetracycline, which allows expression of mutant BRG1. The dominant-negative BRG1 is ATPase deficient and forms dominant-negative SWI/SNF complexes that interfere with MyoD-induced differentiation (de La Serna et al., 2001a)

Hybridization Probes and Immunofluorescence Reagents

Human β-cardiac myosin heavy chain (MyHC) was detected using a 12.3-kb clone (p8-1A) from L. Leinwand (University of Colorado, Boulder, CO) (Liew et al., 1990) encompassing the intergenic spacer and extending partly into the 5′ region of the closely linked α-gene. The closely linked α- and β-genes are highly homologous, but only β is expressed in cultured muscle (Yamauchi-Takihara et al., 1989). We used a 30-kb cosmid clone containing the entire MyoD gene (cHMD-13) and 5′-upstream sequences (Goldhamer et al., 1992) from C. Emerson (University of Pennsylvania Medical Center, Philadelphia, PA). Human myogenin (myf-4) was detected using a 5.8-kb plasmid clone containing the entire human gene (Braun et al., 1989), supplied by H. Arnold (Technische Universität Braunschweig, Braunschweig, Germany). Mouse myogenin (Edmondson et al., 1992) was detected with a 16-kb genomic probe from D. Edmondson and E. Olson (University of Texas, M.D. Anderson Cancer Center, Houston, TX). Nonmyogenic sequences used were described previously (Xing et al., 1995). Whole chromosome 14 was detected using a digoxigenin-labeled Coatasome total chromosome probe (Oncor, Gaithersburg, MD).

A mouse monoclonal antibody against the SR (arginine/serine) spliceosome assembly protein SC-35 (Fu and Maniatis, 1990) was obtained from X.-D. Fu (University of California, San Diego, San Diego, CA) or Sigma-Aldrich (St. Louis, MO). SRM300, a 300-kDa SR-related nuclear matrix antigen used to detect SC-35 domains in mouse cells was provided by Dr. B.J. Blencowe (University of Toronto, Toronto, ON, Canada). Mouse anti-myogenin was purchased from BD PharMingen (San Diego, CA) and rabbit polyclonal IgG anti-MyoD was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Fluorescence In Situ Hybridization

Methods used here have been published in detail elsewhere (Carter et al., 1991; Johnson et al., 1991; Tam et al., 2002). In brief, DNA probes were nick translated with either biotin-11-dUTP or digoxigenin-16-dUTP (Roche Diagnostics, Indianapolis, IN). DNA-specific hybridization was accomplished by simultaneously hydrolyzing nuclear RNAs and denaturing target DNAs in 0.07 N NaOH for 5 min, followed by application of probe. RNA-specific hybridization involved applying probe to nondenatured cells such that cellular DNA is not available for hybridization. Cells subjected to immunofluorescence analysis after hybridization were fixed for 10 min before application of antibody. Total nuclear DNA was stained using 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI) for 30 s, which in mouse preferentially stains the A-T–rich DNA present in all mouse centromeric heterochromatin. The chromosome 14 interphase territory was painted, as described previously (Clemson et al., 1996). Cells were permeabilized with 0.5% Triton X-100 for 7 min before a heat denaturation in 70% formamide, 2 × SSC at 85°C for 5 min. Ten microliters of whole chromosome paint, denatured for 5 min at 75°C and prehybridized at 37°C for 2 h, was applied to dehydrated cells, and hybridized overnight. The painting probe was detected using rhodamine antidigoxigenin antibody (Roche Diagnostics).

Microscopy and Analysis

An Axioplan microscope (Carl Zeiss, Thornwood, NY) designed for imaging multicolor fluorescence was used. Variable excitation using a filter wheel (Ludl Electronic Products, Hawthorne, NY) in conjunction with multiband-pass emission filters (Chroma Technology, Brattleboro, VT) was used for two or three color in-register imaging. High-resolution digital image capture was performed using a Photometrics 200 Series camera (Photometrics, Tucson, AZ), a high-resolution, low-depth of field objective (100×, numerical aperture 1.4; Carl Zeiss), and image acquisition and analysis software (GW Hannaway and Associates, Boulder, CO; and MetaMorph, Universal Imaging, Dowingtown, PA).

Signal was scored as associated with an SC-35 domain when there was no visible separation between the signal and the SC35 domain boundary. Signal was occasionally associated with small concentrations of SC-35 (<0.5 μm), but this was not scored as associated with a domain unless a large (typically 0.5–3 μm) prominent domain was present. All experiments were viewed and, when possible, analyzed directly through the microscope. Data represents multiple experiments analyzed independently by at least two investigators. For analysis of association with the peripheral heterochromatin, gene signals were scored as associated if they were in contact with or very close (<0.6 μm) to the edge of the nucleus.

Localization of Muscle-specific and Nonmuscle-specific Sequences in Myotube Nuclei

Before comparing the distribution of genes in different cell types, we first examined the distribution of seven different sequences, including muscle and nonmuscle genes, in fully differentiated human skeletal muscle, by using fluorescence in situ hybridization to detect specific nuclear RNAs and/or the corresponding gene (see MATERIALS AND METHODS). In >98% of the postmitotic nuclei within multinucleated skeletal myotubes, the RNA accumulations from the β-cardiac myosin heavy chain genes (cMyHC) associate with SC-35 domains. As described in MATERIALS AND METHODS, any signal that seemed to contact the domain with no visible separation is scored as “associated,” whereas even a slight visible separation is considered nonassociated. Typically, both of these slightly elongated accumulations overlap much of the domain (Figure 1, A and C), as described previously and termed a “type I” RNA pattern (Smith et al., 1999). The cMyHC gene, in contrast, consistently localizes immediately adjacent to or partially overlapping the edge of the SC-35 domain, similar to COL1A1 gene and its nuclear RNA (Xing et al., 1995).

The positions of two other muscle-specific genes, myogenin (myf-4) and MyoD, were also examined in differentiated myofibers. Human myogenin and MyoD are muscle regulatory proteins that are expressed at similar levels from genes of similar size and complexity, both of which are less complex and less highly expressed than cMyHC (Braun et al., 1990; Goldhamer et al., 1992). In contrast to the more globular or track-like morphology of cMyHC RNA, myogenin and MyoD nuclear RNAs produced two small bright foci (Figure 1, B, D, and G), similar to the round hybridization spots from a single copy gene. For >200 myogenin RNA foci examined, 88% were associated with a prominent (0.5–3.0 μm in diameter) SC-35 domain (Figure 1, B and D). The domain was much larger than the round spot of RNA signal, which contacted or slightly overlapped the domain periphery but did not occupy its central regions. This RNA distribution pattern has been termed “type II” (Smith et al., 1999). Because the RNA foci are smaller and at the edge of the domain, the domain clearly represents a distinct entity, a large accumulation of factors with which the gene and its RNA associates. Hybridization to myogenin DNA yielded results comparable with the RNA, with the gene also localizing to the domain periphery (Figure 1E). Note that in some nuclei both alleles associated with the same domain (Figure 1, E and F), supporting the idea that multiple genes cluster at these structures, as further demonstrated in Shopland et al. (2003).

In the same experiments, the MyoD RNA foci, virtually indistinguishable in size and intensity to myogenin RNA foci, did not associate with prominent splicing factor domains in the majority of myotube nuclei (Figure 1G, inset). In some experiments, MyoD RNA was associated with very small concentrations of SC-35, but only infrequently (12%) seemed to contact one of the 20–40 prominent (0.5–3.0 μm) SC-35 domains. These low rates of apparent association are in keeping with that expected for a “random” distribution within the limits of our analytical approach (Xing et al., 1995). Similar to its nuclear RNA, only 12% of MyoD DNA signals associated with SC-35 domains (Figure 1H). Hence, despite close similarities in the size of their genes and nuclear RNA accumulations, the myogenin gene is expressed at the periphery of a large domain of RNA metabolic factors, whereas the MyoD gene is not. These results support our earlier suggestion that genes are expressed in different nuclear environments, and are consistent with previous evidence that there are sufficient splicing factors in the interdomain space to process even highly complex pre-mRNAs (Smith et al., 1999).

For comparison to a broader view of sequence localization relative to SC-35 in muscle nuclei, we examined other nonmyogenic sequences in differentiated myotube nuclei, including sequences that are not expressed or do not encode pre-mRNA. As summarized in Figure 2, hybridization to the RNA polymerase III transcribed gene for human 5S rRNA (Little and Braaten, 1989) indicated only infrequent association with SC-35 domains. Similarly, the transcriptionally inactive human albumin gene (HSA-10; ATCC) (Figure 2), and a chromosome 17 alphoid repeat sequence, p3.6 (our unpublished data; Waye and Willard, 1986) revealed very low levels of apparent association, consistent with random expectation. These signals also frequently localized to a different focal plane of the nucleus than do the SC-35 domains and their associated genes (Figure 1, A–E) (Xing et al., 1993, 1995). An interesting case is that of collagen 1A1 RNA (COL1A1), previously shown to consistently associate with one of the largest SC-35 domains in fibroblast nuclei, a cell-type in which it is highly expressed (Xing et al., 1995). Because this gene is also expressed in myoblasts but down-regulated to low or negligible levels after cells fuse to form postmitotic skeletal muscle (Beach et al., 1985), it was interesting to determine whether the gene would remain associated with an SC-35 domains. The myotube nuclei exhibited RNA foci much smaller in developing muscle fibers than in fibroblasts. However, these still associated with a prominent SC-35 domain, consistent with the domain representing a separate entity. More than 85% of collagen gene signals remained positioned at the periphery of a prominent domain in myotube nuclei, down just slightly from the ∼99–100% association seen in fibroblasts (Xing et al., 1995) and below.

The above-mentioned results demonstrate a specific association of genes with SC-35 domains in muscle fibers, including genes that encode both a regulatory factor and more abundant structural proteins. These results also support that this spatial localization is locus specific and not a property of all genes expressing intron-containing RNAs, as discussed further below.

Are Genes That Localize with SC-35 Domains in Human Skeletal Muscle Organized in the Same Way within Human Diploid Fibroblasts?

For those muscle genes that are associated with SC-35 domains, is the association related to cell type and/or expression of that gene? Because cMyHC and myogenin genes associated at high frequency with domains in expressing muscle cells, we addressed whether the same spatial location of these genes was observed in other nonexpressing cell-types, e.g., possibly as a coincidence of chromosomal localization. To address this, we examined human diploid fibroblasts that neither express these genes nor are committed to do so.

In striking contrast to the gene localization observed in differentiated muscle, only 13% of cMyHC gene signals and 8% of myogenin gene signals seemed to contact an SC-35 domain in fibroblast cultures. The MyoD gene signal showed 11% association, similar to that seen in myotube nuclei (Figure 2). Hence, these results support that the cMyHC and myogenin genes do not associate with domains in this nonmyogenic cell-type, but become specifically positioned at the domain periphery in nuclei of differentiated muscle. To rule out that this was due to some global rearrangement of the nucleus upon muscle differentiation, four other nonmyogenic sequences examined in muscle cells were reexamined here. As shown in Figure 2, none of these showed a change in their relationship to the SC-35 domains; only the expressed cMyHC and myogenin genes showed this change in position. Hence, these results demonstrate that association of specific genes with domains is cell type dependent, occurring in differentiated cells that express the genes but not in nonmyogenic cells that do not.

Does Localization with SC-35 Domains Correlate with Muscle Differentiation or Myoblast Commitment to the Myoblast Lineage?

At what point in development of specific cell types does the difference in nuclear gene position between muscle and fibroblasts arise? Is terminal differentiation necessary or might the genes become positioned adjacent to an SC-35 domain during commitment to a myoblast lineage, e.g., in cells determined to form muscle but that have not yet differentiated and expressed these genes. To address this, sequences were assayed in committed but undifferentiated satellite myoblasts, still in proliferative phase. Both nonexpressed cMyHC and myogenin genes showed very little (<12%) association with SC-35 domains, not significantly different from random expectation (Figures 1, I and J, and 2). MyoD, even though expressed early in satellite myoblasts grown in culture, (Smith et al., 1994; de La Serna et al., 2001a), did not preferentially associate, in keeping with its localization to nondomain space in myotubes (Figure 1K). The frequency of association for the nonmyogenic sequences was very similar to that in fibroblasts. A summary table of all results is presented in Figure 2. These results show that these genes are not prepositioned in myoblasts that are still proliferative and nonexpressing. Hence, developmental commitment to a muscle lineage does not itself result in repositioning of muscle genes relative to factor rich SC-35 domains.

To further establish a link between splicing factor domain association and changes in gene expression during muscle differentiation, we examined the myogenin gene in a different model system, using mouse NIH3T3-derived B22 cells induced to differentiate to muscle by introduction of a retrovirus expressing MyoD. It has been previously documented that expression of this master muscle regulatory factor can “convert” nonmuscle cells to a muscle pathway and induce muscle-specific gene expression, including myogenin (Davis et al., 1987; Novitch et al., 1996). As shown previously for both cMyHC and myogenin (de La Serna et al., 2001a), introduction of MyoD into the B22 cells converts them to myoblasts which, when cultured under differentiation-promoting low serum conditions (see MATERIALS AND METHODS), withdraw from the cell cycle and express muscle-specific genes (in cells that remain mononucleated). The expression of muscle markers was confirmed using both Western blots (our unpublished data) and immunofluorescence (Figure 3E). In several experiments the frequency of cells expressing MyoD or myogenin was typically ∼70%. In the mock-infected, nonmuscle cells on average 19% of mouse myogenin gene loci seemed to contact an SC-35 domain (Figure 3, A and B). This is in keeping with a “random” apparent association, but may be made slightly higher because the B22 cells tend to have smaller but more numerous SC-35 domains. Interestingly, in B22 cells induced to undergo myogenesis by MyoD, the SC-35 domains became fewer but larger, as seen in several experiments (supporting earlier observations in differentiating rat muscle; Lawrence et al., 1993). Because infection did not result in MyoD expression in 100% of cells, it was important to identify myogenin-expressing cells by hybridizing to myogenin nuclear RNA, which showed that the level of association in these cells was 89% (Figure 3, C and D). This is in keeping with our scoring of the association level of the myogenin gene in cultures with a ∼70% infection rate. This fourfold or more increase in the localization of the myogenin gene with SC-35 domains was seen in numerous experiments, demonstrating that MyoD-induced differentiation of B22 cells largely recapitulates the repositioning of this muscle-specific gene adjacent to SC-35 domains.

Does Association with SC-35 Domains Require Transcriptional Activation?

We cannot rule out that these genes become “poised” adjacent to an SC-35 domain in a small fraction of postmitotic myoblasts just before their expression. However, results clearly demonstrate that repositioning of both alleles of myogenin and cMyHC relative to SC-35 domains typically occurs concomitant with or very close to initiation of mRNA expression.

The above-mentioned findings clearly demonstrate that there is a repositioning of two muscle specific genes relative to SC-35 domains concomitant with cell type differentiation and gene expression. The temporal resolution is sufficient to conclude that gene expression and domain association occur close in time during muscle differentiation. However, location of genes adjacent to an SC-35 domain may occur just before, at the same time, or just after gene expression begins. A priori, the most likely possibility may seem to be that active RNA synthesis and a sufficient level of RNA on the gene may be a prerequisite for this association to occur. However, the results mentioned above do not rule out the possibility that the gene becomes positioned with the domain as terminally differentiating cells withdraw from the cell cycle, but just before gene expression. Hence, the gene might become “poised” for expression during a narrow window of time.

Although this is a difficult question to address conclusively, we present initial results that suggest that active gene expression might not be required and that prepositioning of the gene before expression remains a viable possibility. Again, we used the same system as described above in which NIH3T3 cells are induced by MyoD, but in this case, we examined the consequences of inducible dominant negative expression of BRG1, which forms dominant-negative SWI/SNF, a chromatin remodeling complex that has been shown necessary for muscle-specific gene expression in these cells. Expression of the mutant SWI/SNF has been shown to inhibit expression of myogenin mRNA and protein to undetectable or negligible levels, while at the same time allowing withdrawal from the cell cycle (de La Serna et al., 2001b). We had anticipated that the interference with transcription would prevent any change in location of the myogenin gene relative to the SC35 domains after MyoD induction. Surprisingly, in duplicate experiments there was an increased association of the myogenin gene with an SC35 domain, significantly above the typical “background” random association of ∼15–20% (average 19%) in mock infected cells (no MyoD). Myogenin gene association with an SC-35 domain in MyoD-infected cultures expressing the mutant SWI/SNF ranged from 34 to 51%. Because muscle-specific gene expression is inhibited in these cells, we could not use the presence of myogenin nuclear RNA foci to distinguish expressing cells from those that were not infected with MyoD or not effectively induced to express muscle genes, as in the above-mentioned experiments with normal SWI/SNF. Immunofluorescence analysis of parallel cell samples indicated that in these experiments the average MyoD infection rate was 55–60%, thus the frequency of SC35 association of 34–51% in the total population remains approximate but likely underrepresents the association rate in cells induced by MyoD.

Further analyses of this and other genes would need to be examined to conclude that gene expression is not required for gene relocalization relative to SC35 domains. However, these initial findings do indicate that domain association increases under conditions in which myogenin gene expression is repressed, although the extent of association may not be as great as it would otherwise be when RNA synthesis was more active. These results are sufficient to indicate that it should not be presumed that significant RNA synthesis is required. Rather, these results raise the possibility that MyoD induction of terminal differentiation, in the absence of significant RNA synthesis, can cause reorganization of certain muscle-specific genes proximal to factor-rich domains. In either case, what is most clear is that collectively our findings show a definitive reorganization of two muscle-specific genes adjacent to SC35 domains during a short window of time during which myoblasts differentiate into muscle, whereas no such nuclear repositioning for several other sequences occurred.

Does Gene Position Change within the Chromosome Territory or Relative to Centromeric Heterochromatin?

The above-mentioned results demonstrate that the cMyHC and myogenin genes become repositioned with cell type relative to SC-35 domains. We briefly examined positioning of these genes relative to other features of nuclear topography that have been implicated in gene expression, to assess whether these change concomitantly with SC-35 domain association. Previous evidence indicates that SC-35 domains most frequently reside between chromosome territories (Zirbel et al., 1993; Clemson and Lawrence, 1996) and that gene loci may localize specifically at the outer periphery of this territory. Hence, changes in position relative to the chromosome territory and interchromosome space might coincidentally impact position relative to SC-35 domains, or vice versa. Some evidence indicates that certain active genes position at the periphery of the territory (Dietzel et al., 1999; Volpi et al., 2000), whereas analysis of other genes indicates that they maintain a peripheral position irrespective of activity or cell type (Kurz et al., 1996; Dietzel et al., 1999). We examined the localization of the cMyHC gene relative to its chromosome 14 territory in both the active state in differentiated myotubes and the inactive state in myoblasts. As shown in Figure 3, F and G, the gene was clearly and consistently positioned at the outer boundary of the chromosome 14 territory in both cell types regardless of activity status. The percentage of cMyHC genes localized at or just internal to the territory periphery was 81 and 84%, respectively, for myotubes and myoblasts. There was no significant difference in the small percentage of signals, which seemed slightly separate from the chromosome territory. This confirms prior evidence that muscle genes are peripherally localized within the chromosome territory in different cell types (Kurz et al., 1996). Hence, the positioning of the MyHC gene adjacent to an SC-35 domain is not a consequence of a repositioning relative to the chromosome territory.

In recent years, there has been increasing interest in the idea that inactive genes may be specifically positioned with respect to centromeric or peripheral heterochromatin and that close association of certain genes with this heterochromatic compartment may causally inactivate their expression (Brown et al., 1997; Francastel et al., 1999). To examine the proximity of the myogenin gene to centric heterochromatin, we took advantage of the fact that the A-T–rich centromeres of mouse chromosomes are easily visualized with DAPI staining. That DAPI staining demarcated regions of inactive heterochromatin was confirmed by hybridization to heterogenous nuclear RNA (hnRNA) by using a probe to Cot-1 DNA, which was previously shown to delimit inactive heterochromatin (Hall et al., 2002). Figure 3H demonstrates that DAPI-bright A-T–rich centromeres occupy regions of low hnRNA concentration and further illustrates that these cells have a relatively thin rim of heterochromatin at the nuclear periphery. The association of the myogenin gene to centromeric heterochromatin was examined in expressing and nonexpressing B22 cultures, by using the model system described above. In non-MyoD–induced cell cultures in which no cells express the myogenin gene, 16% of the myogenin gene signals seemed associated with the centromeric heterochromatin (in contact at the periphery) (Figure 3, A and B). In MyoD-induced cultures, in which most cells express this gene, the frequency of association with centromeric DNA decreased slightly to 14% (Figure 3, C and D). Frequently, one allele in a nucleus would seem to contact heterochromatin and the other would not, consistent with a potentially random relationship (Figure 3D). Infrequently a gene seemed to contact both an SC-35 domain and centric heterochromatin simultaneously (Figure 3D). Furthermore, <1% of signals were found to be at the nuclear periphery. We conclude that the myogenin gene, in 3T3 cells that normally will never express muscle genes, is not preferentially positioned in a domain of centric heterochromatin, nor does its position relative to centric heterochromatin change upon induction, in contrast to the sharp increase in SC-35 domain association concomitant with MyoD-induced expression.

These results support that in differentiated cells cMyHC and myogenin genes reposition specifically with respect to SC-35 domains and that this localization is not a coincidental result of global DNA reorganization or changes in gene position relative to either the chromosome territory or, in this case, centric heterochromatin. Rather, these results indicate that all regions of the nuclear periphery or the broader euchromatic nucleoplasm are not equivalent and that a key parameter that distinguishes different nuclear regions is proximity to SC-35 domains.

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