reprogramming cell differentiation in the absence of dna...

Cell, Vol. 37, 879-887, July 1984, Copyright 0 1984 by MIT

Reprogramming Cell Differentiation in the Absence of DNA Synthesis

0092.8674/84/070879-09 $02.00/O

Choy-Pik Chiu and Helen M. Blau Department of Pharmacology Stanford University School of Medicine Stanford, California 94305

Summary

We examined whether the activation of muscle gene expression in nonmuscle cells required DNA synthesis. Human fibroblasts from amniotic fluid and fetal lung were fused with differentiated mouse muscle cells in the presence or absence of the DNA synthesis inhibitor, cytosine arabinoside. In the stable heterokaryons formed, the human contractile enzyme, MM-creatine kinase (CK), and the cell surface antigen, 5.1H11, were detected in comparable amounts regardless of whether DNA synthesis had occurred. A single cell analysis revealed that the efficiency of gene activation was high and that DNA synthetic activity was not affected by the ratio of muscle to nonmuscle nuclei in the heterokaryons. In addition, muscle gene expression was not restricted to the Gl phase of the cell cycle. We conclude that cell differentiation can be reprogrammed in heterokaryons regardless of cell cycle phase and in the absence of detectable DNA synthesis.

Introduction

The specialization of cells for the expression of differentiated functions involves two sequential steps: determination and commitment. First, a stem cell with multiple op- tions gives rise to a determined cell, which is restricted to a particular differentiation pathway. Second, under appropriate conditions, the determined cell becomes committed to express functions typical of that specific differentiated cell phenotype. Although it is evident that this sequence exists, the mechanisms controlling the expression of these cell states and the transitions between them are not well understood. This is largely due to a lack of model experimental systems in which to probe such biological questions (Ingram and Keane, 1980; Levenson and Housman, 1981).

To address questions regarding the mechanisms that regulate gene expression during cell specialization, we developed a heterokaryon system in which the stable fusion products of two differentiated cell types could be analyzed (Blau et al., 1983). A marked advantage of the heterokaryons over typical hybrids, or synkaryons, was that cell division did not occur following fusion so that the nuclei and cytoplasms of both cell types remained present. Furthermore, unlike hybrids, no selection was required to obtain the fused cells of interest and gene expression could be analyzed immediately following fusion. Using this system, we demonstrated that diploid human amniotic fibroblasts could be induced to express several different human muscle genes upon fusion with differentiated

mouse muscle cells and that this activation occurred via the cytoplasm (Blau et al., 1983).

This heterokaryon system is ideal for examining the extent to which the expressed potential of a determined or committed cell is heritable and stable as a result of changes in the genome itself and the extent to which it is reversible and subject to regulation by cytoplasmic factors. In myogenesis, cell proliferation and differentiation are distinct events which are mutually exclusive. As muscle cells differentiate and initiate muscle protein synthesis, they withdraw from the cell cycle and cease cell division (O’Neill and Stockdale, 1972). Consequently, a cell fused with a differentiated muscle cell is exposed to nuclei in the Gl phase of the cell cycle (Okazaki and Holtzer, 1966; Nadal-Ginard, 1978; Konigsberg et al., 1978) and a cytoplasm containing activators of muscle gene expression (Blau et al., 1983) and possibly inhibitors of mitosis (Ad- lakha et al., 1983).

In this study we have examined whether DNA replication is required for the reprogramming of a fibroblast, i. e., for the induction of muscle gene expression in a cell which normally never would express those genes. Toward this end, we monitored muscle gene expression and DNA synthesis in nonmuscle fibroblasts contained in heterokar- yens, both at the single cell level and in mass cultures. Our results demonstrated that DNA replication was not a prerequisite for the activation of two distinct muscle genes coding for a cell surface antigen and a contractile enzyme in two different strains of human fibroblasts isolated from amniotic fluid and fetal lung. In addition, muscle genes were activated in nonmuscle cells in cell cycle phases other than Gl These results indicate that significant DNA synthesis is not required for reprogramming cell differentiation in fibroblasts and that muscle gene expression is not restricted to specific phases of the cell cycle. They further suggest that alterations in chromatin configuration requiring DNA synthesis need not occur for the muscle genes in fibroblasts to be accessible to and respond to cytoplasmic regulatory factor(s) present in differentiated muscle cells. We conclude that there is considerable plasticity in the function of the nucleus of a specialized cell type and that the cytoplasm plays a very important role in the regulation of gene expression.

Results

DNA Synthesis and Creatine Kinase (CK) Activation in Heterokaryons The role of DNA synthesis in the activation of human CK gene expression was monitored in four separate experiments in which heterokaryons were cultured in the presence or absence of cytosine arabinoside (ara-c), an inhibitor of DNA synthesis (Cozzarelli, 1977; Fridland, 1978). As shown in the protocol in Figure 1, human amniotic fibroblasts or fetal lung fibroblasts (MRC-5) were plated onto cultures of mouse myotubes and fused with polyeth- ylene glycol. Cells were exposed to ara-c prior to fusion and continuously thereafter (Figure IB), and compared

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+ A or MRC-5 + OUA

I I I I I I I i

24 hr. 24 hr. 24 hr. 72 or 120 hr.

Figure 1. Experrmental Protocol for Studies of DNA Synthesis and Gene Activation in Heterokaryons

(A) Human nonmuscle cells, amniotic fibroblasts (A) or fetal lung fibroblasts (MRC-5) were plated onto sparse cultures of mouse myotubes for 24 hr and then fused wrth PEG. Twenty-four hours after fusion, the selective agents cytosine arabrnoside (ARA-C) and ouabain (OUA) were added to eliminate unfused parental cells. DNA synthesis was monitored by labeling heterokaryons with %thymrdine at time intervals immediately following fusion (see text). Human creatine kinase (CK) and/or 5.lHi 1 cell surface antigen were assayed 4 or 6 days after fusion. (6) The same protocol as in (A) except that the cultures were exposed to ara-c for 24 hr prior to fusion with PEG, and continuously thereafter.

with control cultures which were not exposed to the drug until 24 hr after fusion (Figure 1A). Heterokaryons were labeled with 3H-thymidine for the first 24 or 48 hr following fusion and cultured for a total of 4 or 6 days. This time interval was selected for analysis, since in the experiments in which gene activation was first detected, DNA synthesis had routinely been inhibited 1 day after fusion (Blau et al., 1983). Replicate cultures were then either processed for autoradiography or assayed for human creatine kinase (CK) activity. The mouse and human nuclei in individual heterokaryons were identified by their fluorescent staining patterns after reaction with Hoechst 33258 (Blau et al., 1983) and scored for their DNA synthetic activity by the presence of silver grains (Figure 2). Under our conditions of autoradiography, a nucleus that synthesized only 20% of its genomic DNA was detectable (see Experimental Procedures). Human and mouse CK isozymes were distin- guished by their electrophoretic mobiilty on nondenaturing polyacrylamide gels (Figure 3, lanes 2 and 3).

The four experiments entailed an analysis of a total of 2,306 human fibroblast and 4,419 mouse muscle nuclei contained in 761 individual heterokaryons (Table 1). The efficiency of fusion, or percent of total myotubes which were heterokaryons, was similarly high (77%+4%) and the average number of mouse nuclei inside heterokaryons exceeded the number of human fibroblast nuclei in each case. In contrast, the proportion of human fibroblast nuclei undergoing DNA synthesis during the labeling period differed, probably because of differences in the DNA synthetic activity of the fibroblasts prior to fusion, Generally, the percent of total human fibroblast nuclei that was labeled inside heterokaryons was low, regardless of the presence or absence of the inhibitor; with the exception of experiment 1, only 6%&l % of fibroblast nuclei were labeled. Closer inspection of the autoradiograms revealed a further decrease in the level of labeling or DNA synthetic

Figure 2. Detection of DNA Synthesis in Mouse-Human Heterokaryons

Heterokaryons were labeled with 3H-thymidine for 1 hr and immediately fixed, processed for autoradiography and stained with Hoechst 33258. A multinucleated heterokaryon containing two punctate mouse nuclei and one uniformly stained human nucleus is shown by fluorescence microscopy (top). The same heterokaryon (outlined by arrows) viewed with phase- contrast optics reveals the presence of silver grains over the human nucleus indicating that it synthesized DNA during the labeling period (bottom).

activity per human nucleus in ara-c-treated heterokaryons. In each pair of experiments (A and B) the proportion of human nuclei that had undergone an entire round of DNA replication and had more than 50 silver grains was much lower in heterokaryons exposed to the inhibitor. These results indicate that only a minority of all fibroblast nuclei inside heterokaryons was engaged in DNA synthesis during the first 1 or 2 days after fusion, and that the amount of DNA synthesis that did occur in these few nuclei was significantly reduced in the presence of ara-c. We therefore conclude that in the heterokaryons exposed to ara-c, a complete round of DNA replication occurred in at most 0.9%+0.5% of fibroblast nuclei.

Replicate cultures of those described above were analyzed for the production of CK isozymes either 4 or 6 days

Gene Activation Without DNA Synthesis 881

MM

MB

BB

Figure 3.

1234567 Acttvatron of Human Creatine Kinase in the Absence of DNA

Synthesis

Whole cell extracts were electrophoresed on nondenaturing polyacrylamide gels and the CK rsozymes detected wrth UV illumination using a coupled enzyme reactron producrng NADPH as Its end product. CK isozymes are shown for a coculture of mouse muscle and human amniotic fibroblasts after 6 days without PEG (lane l), cultured mouse muscle (lane 2) cultured human muscle (lane 3), heterokaryons of mouse muscle and human amniotic fibroblasts (lanes 4 and 5), and heterokaryons of mouse muscle and human fetal lung fibroblasts (MRC-5) (lanes 6 and 7) 6 days after PEG fusion (see Table 1, experiments 2 and 4). Cultures were either not exposed to ara-c until 24 hr after fusron as II- Frgure IA (lanes 4 and 6) or continuously exposed to ara-c as in Frgure 1 B (lanes 5 and 7). The efficiency of fusion of the heterokaryons analyzed were similar within each pair of fusrons and aliquots with equivalent enzyme actrvrtres were loaded on the gel. Arrows indicate CK isozymes containing human subunrts

after fusion. A typical example is shown in Figure 3. Creatine kinase is an enzyme composed of two subunits, M and B; therefore three isozymes are possible: BB, MB, and MM. The synthesis of the B subunit is characteristic of undifferentiated myoblasts and nonmuscle cells. M subunit synthesis is initiated upon muscle differentiation. Thus the MB isozyme is typical of early myotubes and the MM isozyme of mature myotubes. A difference in the mobility of the human and mouse MM-CK isozymes is clearly evident in standards prepared from cultured human and mouse muscle, respectively (lanes 2 and 3). When mouse muscle cells and fibroblasts are grown together for 6 days but not fused with PEG, the pattern of CK isozymes resembles that of mouse muscle alone (lane 1). Six days after fusion of mouse myotubes with either amniotic or MRCd fibroblasts, a band of intermediate mobility between the mouse and human MM-CK isozymes is apparent in both heterokaryon cultures exposed to ara-c (lanes 4 and 6) and in untreated controls (lanes 5 and 7). This is a

functional interspecific hybrid enzyme composed of one mouse and one human M subunit (Blau et al., 1983). In addition, bands in the regions of the human MB and MM isozymes are visible. It appears that within the limits of this assay, equal amounts of human isozymes are present in the heterokaryon cultures in which DNA synthesis was inhibited and in untreated controls. From these results we conclude that fetal fibroblasts from both amniotic fluid and lung, which normally never express muscle functions, can be induced to do so when fused with muscle cells. Fur- thermore, in both nonmuscle cell types, DNA synthesis is not required for M-CK gene activation to occur.

Lack of Influence of Nuclear Ratio on DNA Synthesis No significant effect of nuclear composition on DNA synthesis was observed. Muscle nuclei contained in multinucleated myotubes do not normally synthesize DNA (Stock- dale and Holtzer, 1961). In a total of 761 heterokaryons analyzed, these postmitotic nuclei were not induced to initiate DNA synthesis by the presence of fibroblast nuclei undergoing DNA replication. This was true even when a single muscle nucleus was present in a heterokaryon containing two or more nonmuscle nuclei which had undergone DNA replication and were labeled. Altogether, for experiments 1-4 in Table 1, of all the 4419 mouse muscle nuclei inside heterokaryons that were analyzed, only 0.5%+0.4% were labeled (data not shown). Similarly, the incidence of labeled human fibroblast nuclei was not affected by the number of nonreplicating muscle nuclei present in the heterokaryons. Both labeled and unlabeled fibroblast nuclei were observed in heterokaryons with a large excess of postmitotic nuclei, e.g., with mouse muscle to human fibroblast nuclear ratios as high as 21 :I.

Single Cell Assay for Activation of 5.1Hll Even when heterokaryons were treated with ara-c, a small proportion of the human nonmuscle nuclei still exhibited detectable DNA synthesis by autoradiography. This low level of DNA synthesis in ara-c-treated cultures was not evident in previous experiments using TCA precipitation (see Experimental Procedures). This is likely to be due to the increased sensitivity of the autoradiographic assay. To determine definitively that this amount of DNA synthesis was not associated with the human muscle gene activation observed, a single cell assay was necessary. For this purpose we used a monoclonal antibody which recognizes a muscle-specific cell surface antigen, 5.1 HI 1 (Walsh, 1980; Walsh and Ritter, 1981). As shown in Figure 4, this antibody reacts with an antigen present on cultured human myoblasts and myotubes, but not human fibroblasts. In addition, it is species-specific and does not recognize its mouse muscle counterpart; 5.1 HI1 was never detected in myotubes containing only mouse nuclei prior to or following PEG treatment. The specificity of this antibody for a human muscle membrane component makes it ideal for studies of gene activation in individual heterokaryons. As shown in Figure 5, when human fibroblasts were fused

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Table 1, DNA Synthesis and Human Creatine Kinase Activation

Nuclei Scoredd Nuclear % Labeled % Labeled Heterokaryons % Hetero- Ratio Human Nuclei Human Nuclei Human

Expt.” Ara-cb Scored (nr M H karyons” (M:H)’ (210 grains)g (250 grains)” CK’

1 (4 - 53 242 91 65 3:i 52 21 .o + (W + 61 253 91 58 3:1 38 1.0 +

2 (4 - 114 676 458 a7 2:l 5 4.0 + (B) + 94 806 291 79 4:i a 2.0 +

3 (4 - a4 427 223 76 3:l 5 3.0 + 63 + 114 643 333 at 3:l 7 0.3 +

4 6% - 125 828 471 a7 2:l 7 6.0 +

C3 + 116 544 348 a5 2:l 1 0.2 +

a Heterokaryons were produced by fusing mouse muscle cells with human fibroblasts from amniotic fluid (Expts. 1-3) or from fetal lung (Expt. 4). The cells ware labeled with 3H-thymidine either for the first 24 hr (Expts. 1-3) or 48 hr (Expt. 4) and then fixed and processed for autoradiography 6 days after fusion. (A) and (B) correspond to the protocols in Figures IA and 1 B, respectively. b (-) Heterokaryons were not exposed to ara-c until 24 hr after fusion. (+) Heterokaryons were exposed to ara-c prior to fusion for 1 hr (Expt. 1) or 24 hr (Expts. 2-4) and continuously thereafter. ‘Total number of heterokaryons scored (n). d Total number of mouse (M) and human (H) nuclei scored in all the heterokaryons. e Percent of total myotubes that were heterokaryons. f The ratro of mouse (M) to human (H) nuclei was determined for each heterokaryon and the mean ratio for each experiment is shown. g Percent of total human nuclei that were autoradiographically labeled with ten or more silver grains (I. e., 3-fold above background). h Percent of total human nuclei that were heavily labeled with more than 50 silver grains, and probably underwent a complete round of DNA replication (see Experimental Procedures). ’ Human CK gene activation assayed 4 and 6 days after fusion. (+) indicates that human MB, MM, and mouse-human hybrid MM bands were readily apparent (see Figure 3, lanes 4-7).

with mouse muscle cells to form heterokaryons, the expression of human 5.1 HI 1 was clearly induced.

DNA Synthesis and 5.1Hll Activation in Individual Heterokaryons We examined the activation of the gene coding for 5.1 HI 1 in individual mouse muscle-human fibroblast heterokaryons in which DNA synthesis had been continuously inhibited with ara-c (Figure 16). Control cultures were not treated with ara-c until 24 hr after fusion (Figure IA). Both sets of cultures were labeled with 3H-thymidine during the initial 48 hr after fusion and analyzed 4 days later for 5.1 Hl 1 expression and DNA synthetic activity.

An example of a heterokaryon analyzed in this manner is shown in Figure 5A. The heterokaryon contains one mouse muscle and three human fibroblast nuclei. None of these nuclei has a significant number of silver grains over it indicating that detectable DNA synthesis did not occur in the time period assayed. Yet the distinct pattern of immunofluorescence provides evidence that at least one gene coding for the 5.1 Hl 1 antigen was activated.

As shown in Table 2, 95%*1% of all 202 heterokaryons analyzed in four separate experiments expressed the 5.1 HI 1 antigen regardless of whether or not DNA synthesis was inhibited by ara-c. This frequency of 5.1Hll gene activation far exceeded the maximum proportion of heterokaryons (19%) that contained a labeled human nucleus. In fact, as many as 173 of the heterokaryons scored expressed 5.1H11 in the absence of any detectable DNA synthesis. These results clearly demonstrate that the induction of expression of the 5.1Hll muscle membrane

component, like the enzyme creatine kinase, can occur in the absence of DNA synthesis.

Activation of 5.1Hll in Phases of the Cell Cycle Other Than Gl Although DNA synthesis was not observed in the majority of heterokaryons in which 5.1 Hl 1 expression was detected, a small but significant proportion of cells did contain labeled fibroblast nuclei. Of particular interest is that group of heterokaryons that contained only one fibroblast nucleus which was labeled with 3H-thymidine. An example of a heterokaryon of this kind is shown in Figure 5B. The heterokaryon contains one mouse and one human nucleus The human fibroblast nucleus has more than 50 silver grains over it, indicating that it synthesized a significant amount of DNA after fusion with the mouse muscle cell. In addition, 5.1 Hl 1 expression is readily detectable by immunofluorescence. Since we have never observed mitotic figures in nuclei inside heterokaryons, the labeled human fibroblast nucleus was either in the S or G2 phase of the cell cycle. These results provide evidence that novel muscle gene expression can occur in phases of the cell cycle other than Gl

Discussion

Activation of Muscle Genes in the Absence of DNA Synthesis Studies of cell specialization for a differentiated pathway such as myogenesis have typically focused on the commitment step, the transition from determined cell (myo-


Figure 4. Specificity of 5.1 HI 1 for Human Muscle Cells

Cells were incubated with the monoclonal antibody, 5.1H11, and stained with a rhodamine-conjugated second antibody. Cultured human muscle cultured mouse muscle (middle), and MRC-5, human fetal lung fibroblasts (bottom) are shown in phase contrast (left) and fluorescence (right).

blast) to differentiated cell (myotube). A limitation of this approach is that myoblasts are already destined to differentiate as muscle and have undergone many biochemical and molecular changes required for myogenesis. The determination step, on the other hand, has not been analyzed because no means currently exists for identifying stem cells, the precursors of myoblasts (Pearson, 1980; Ingram and Keane, 1980). In order to study changes associated with cell specialization at an earlier stage of myogenesis, we induced the expression of muscle functions in cells that were not in the myogenic lineage. Normal diploid human fibroblasts from amniotic fluid or from fetal lung were fused with differentiated mouse muscle cells to form stable heterokaryons in which five previously silent human muscle genes were activated. The gene products included diverse functions: structural proteins of the contractile apparatus (myosin light chains) (Blau et al., 1983) a contractile enzyme (creatine kinase), and a component of the muscle cell surface (5.1Hll). A single cell analysis of the expression of 5.1 HI 1 revealed that the frequency of activation was very high; it was expressed in 95%&l % of the 202 heterokaryons analyzed. Since the nuclei of both cell types in a heterokaryon remained separate, gene activation was mediated by regulatory factors present in the differentiated muscle cell cytoplasm. Thus the nonmuscle nucleus expresses a number of different muscle functions in response to muscle cytoplasmic activators and it does so with high efficiency.

The heterokaryon system is uniquely suited to address- ing specific questions regarding cell specialization. In the experiments reported here we examined the possibility that determination and commitment are associated with heritable changes in the structure of the genome. For the nucleus of a fibroblast to respond to muscle cytoplasmic activators, modifications of the muscle genes such as changes in DNA methylation, DNAase sensitivity, DNA- associated proteins, and gene rearrangement might be necessary. Such modifications are known to be associated with changes in gene expression (Newman et al., 1976; Weintraub and Groudine, 1976; Seidman and Leder, 1978; Razin and Riggs, 1980; Jones and Taylor, 1980; Groudine and Weintraub, 1982; Weisbrod, 1982; Tonegawa, 1983). They are thought to affect the transcriptional accessibility of the genes, In certain cases, such changes have been shown to require DNA synthesis (Santi et al., 1983).

In this report, we have examined the requirement for DNA synthesis in the induction of muscle gene expression in fibroblast nuclei. The extent to which fibroblast nuclei exhibited DNA synthesis after fusion into myotubes differed in several experiments in which comparable amounts of the human muscle-specific product creatine kinase were detected. Furthermore, muscle gene activation was equally efficient when DNA synthesis was blocked by the inhibitor, cytosine arabinoside. By single cell analysis it was possible to identify 173 heterokaryons in which the human muscle cell surface antigen 5.1 Hl 1 was expressed in the absence

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Figure 5. Activation of 5.1Hll (A) in the Absence of DNA Synthesis and (B) in Cell Cycle Phases other than Gi

Heterokaryons of mouse muscle and human fetal lung fibroblasts were labeled for 48 hr with %thymidine and assayed 4 days later for 5.1Hll by immunofluorescence and for DNA synthesis by autoradiography. Heterokaryons are shown with phase-contrast optics (top), Hoechst fluorescence (middle), and rhodamine fluorescence (bottom). (A) Heterokaryon containing one mouse and three human nuclei (middle), none of which were autoradiographically labeled with silver grains (top), expressed 5.1 HI 1 (bottom) nevertheless. (B) Heterokaryon with one mouse and one human nucleus (middle), with >50 silver grains over the human nucleus (top), and expressron of 5.1Hll was evident (bottom).

of DNA synthesis detectable by autoradiography. Although our methods were sufficiently sensitive to detect one-fifth of a round of replication, it could be argued that a minor amount of DNA synthesis occurred that was highly specific for the muscle genes. However, no precedents for local- ized DNA synthesis in the activation of genes have been described and this possibility seems unlikely. Other studies have shown that the commitment of determined myoblasts to differentiated myotubes does not require DNA synthesis (Konigsberg, 1971; O’Neill and Stockdale, 1972; Doering and Fischman, 1974; Buckley and Konigsberg, 1974; Na- daCGinard, 1978). Our experiments demonstrate that DNA synthesis is also not a prerequisite for the induction of muscle gene expression in cells not destined to become muscle. Thus the chromatin conformation of the muscle genes in fibroblasts is accessible to muscle cytoplasmic

regulators in the absence of genetic modifications that require DNA synthesis.

Activation in Different Phases of the Cell Cycle Commitment has been thought to be limited to specific phases of the cell cycle. Bischoff and Holtzer (1970) suggest that the decision to initiate differentiation is made in the S-phase of a critical “quantal” cell cycle, which is a prerequisite for the irreversible expression of the muscle program in the following Gl. On the other hand, O’Neill and Stockdale (1974) Buckley and Konigsberg (1974) Konigsberg et al. (1978) Nadal-Ginard (1978) and Nguyen et al. (1983) maintain that the decision to initiate differentiation is made only when the cells are in Gi and is entirely dependent on environmental conditions. Each of these studies used determined myoblasts that were already


Table 2. DNA Synthesis and Human 5.1 HI 1 Activation

Nuclei Scoredd Nuclear % Heterokaryons % Heterokaryons Heterokaryons % Hetero- Ratio With Labeled Expressing

Expt.a Ara-cb Scored (ny M H karyons” (M:H)’ Human Nucleig 5.1Hll”

1 (4 - 43 188 134 96 2:i 19 93 09 f 64 348 195 a3 2:i 5 97

2 (4 - 43 203 123 a3 2:i 16 95 (B) + 52 196 153 a7 2:i 4 94

a Heterokaryons were produced by fusing mouse muscle ceils with human fetal lung fibroblasts (MRC-5). Cells were labeled with 3H-thymidine for the first 49 hr following fusion. Heterokaryons were processed for autoradiography and assayed for 5lH11 expression 4 days later. b-f Same as Table 1, g Percent of total heterokaryons that contained at least one labeled human nucleus. Nuclei with ten or more grains, or 3-fold above background, were scored as labeled. h Percent of total heterokaryons that were positive for 51H11 by immunofluorescence.

destined to differentiate into muscle. In contrast, in our experiments, fibroblasts which were not restricted to the myogenic pathway were examined. In most experiments, almost no DNA synthesis occurred and the majority of fibroblast nuclei in heterokaryons were in the Gi phase of the cell cycle and therefore diploid. In other experiments DNA synthesis occurred in as many as 52% of the fibroblast nuclei and these nuclei were tetraploid and in the G2 phase of the cell cycle, since mitotic figures were never observed inside a heterokaryon. Yet, in all cases, muscle gene activation was observed. In addition, the analysis of individual heterokaryons clearly revealed that gene activation could occur even when the only fibroblast nucleus present was heavily labeled and had undergone an almost entire round of replication (see Figure 5B). Finally, DNA synthesis in fibroblasts could be efficiently inhibited by ara- c without any effect on their ability to express the human CK or 5.1 HI 1 genes after fusion into heterokaryons. Since the fibroblasts comprised an asynchronous population of cells prior to fusion, they were probably arrested by the inhibitor at various times during the S-phase of the cell cycle (Fridland, 1978). In contrast with the previous studies cited above, we conclude from these experiments that the expression of differentiated muscle genes does not require precise nuclear ploidy or a specific phase of the cell cycle.

Activation in Other Heterokaryon Studies Our findings differ from those observed in previous studies of heterokaryons. First, no activation of differentiated genes has been reported in heterokaryons formed between ‘fibroblasts and differentiated hepatoma or muscle cells (Mevel-Ninio and Weiss, 1981; Wright and Aronoff, 1983; Lawrence and Coleman, 1984). In fact, the liver and muscle specific functions were extinguished altogether, and in the case of liver not re-expressed until fibroblast chromosomes were lost upon propagation of the heterokaryons as hybrids. These results suggest that fibroblasts contain repressor molecules which suppress the expression of other differentiated genes. In contrast, we have shown that two muscle-specific genes were frequently activated in fibroblast-muscle heterokaryons. Second, in

heterokaryons formed between proliferating cells and differentiated cells that were arrested in Gl , DNA synthesis was detected in the previously quiescent nuclei (Johnson and Harris, 1969; Gordon and Cohn, 1971; Johnson and Mullinger, 1975). A similar reinitiation of DNA synthesis in postmitotic muscle nuclei was also reported for myotube- fibroblast heterokaryons, and this reinitiation was dependent on the relative nuclear ratio of the two cell types (Schwab and Luger, 1980). In contrast, in our experiments no DNA synthesis was induced in the nuclei of postmitotic differentiated muscle cells which were fused with proliferating fibroblasts. This was true even when the fibroblast nuclei in the heterokaryons were present in 2- to 3-fold excess and actively replicating their DNA.

The difference in results observed by us and others is not readily apparent but may be due to the nature of the differentiated cells and the medium conditions used. It has been clearly shown that the expression of muscle gene products and the withdrawal from cell cycle in biochemi- cally differentiated muscle cells are reversible if the cells are given the appropriate mitogen-rich media (Devlin and Konigsberg, 1983; Nguyen et al., 1983). The studies of Wright (1983) and Lawrence and Coleman (1984) used mononucleated myocytes which had been prevented from fusion by EGTA and cytochalasin treatment. Although these myocytes were capable of synthesizing certain muscle proteins, they were not entirely postmitotic; a significant proportion (28%) of the cell population incorporated 3H- thymidine (Wright, 1983). Gordon and Cohn (1971) and Schwab and Luger (1980) cultured their heterokaryons in a nutrient-rich medium with 10% newborn calf serum or 10% horse serum, respectively. In our experiments, on the other hand, fibroblasts were fused with well-differentiated, spontaneously contracting muscle myotubes, and the heterokaryons formed were maintained in a mitogen-poor medium. In addition, ara-c was used to ensure that almost all muscle cells were postmitotic before they were fused with fibroblasts. Only 0.5% of the muscle nuclei in our heterokaryons synthesized DNA. Thus the degree to which the muscle parental cell type is terminally differentiated prior to fusion and the conditions used for culturing the heterokaryons may affect the function of the nuclei.

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Role of Cytoplasm in Gene Expression It is clear from the experiments reported here that the nucleus, rather than being impervious, is highly susceptible to regulation by the cytoplasm it creates. If the normal dialogue between the nucleus and the cytoplasm is altered or disrupted, e. g., by placing a specialized nucleus in a foreign cytoplasm, the function of that nucleus changes. Our experiments also demonstrate that DNA synthesis is not required for this reprogramming of nuclear function, i. e., the expression of a differentiated state characteristic of another specialized cell type. Consequently, modifications of the genome that require DNA synthesis need not occur in order for the muscle genes in a nonmuscle cell such as a fibroblast to respond to muscle cytoplasmic activator(s). From these findings we conclude that there is a remarkable plasticity in the function of the nucleus of a differentiated cell and that the cytoplasm plays a critical role in regulating the expression of the differentiated state.

Experimental Procedures

Cells and Tissue Culture Mouse muscle cells, C&, were a subclone isolated in our laboratory from the C2 cell line originally obtained by Yaffe (Yaffe and Saxel, 1977). Mouse myoblasts were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% fetal calf serum and 0.5% chick embryo extract. Multinucleated myotubes were obtained by changing the medium when the cells reached confluence to DMEM supplemented with 2% horse serum. The following day, cells were replated at low density and ara-c (10” M) was added to eliminate dividing myoblasts. Myotubes formed in this manner were used 3 days later for the production of heterokaryons.

Human amniotic fluid cells (amniocytes) were generously provided by Dr. W. Loughman. Amniocytes were of the fibroblast type (Hoehn et al., 1974; Crouch and Bornstein, 1978) previously identified by morphology and collagen synthetic pattern and shown to be capable of activation in heterokaryons (Blau et al., 1983). MRC-5 were diploid human fetal lung fibroblasts. Both fibroblast cell types were grown in the same growth medium described above for mouse muscle cells.

PEG Fusion Heterokaryons were produced by fusing CZC12 myotubes and human amniotic fibroblasts or MRC-5 with PEG as described by Blau et al. (1963). Briefly, the cultures were treated with PEG 1000 (50% w/v in DMEM [pH 7.41) for 60 set at 37°C and then thoroughly rinsed with DMEM three times in succession for 60 set each. To remove unfused cells, the selective agents, ara-c (10e6 M) and ouabain (10m5 M), were routinely added. Ara-c inhibits DNA synthesis and kills dividing cells (Cozzarelli et al., 1977); ouabain inhibits Na+-K+ ATPase and has a 199fold greater affinity for the human rather than the rodent enzyme (Thompson and Baker, 1973).

Inhibition of DNA Synthesis We determined from a dose-response curve that during a 24 hr exposure, a concentration of 10m5 M ara-c maximally inhibited DNA synthesis without impairing cell viability. A 1 hr exposure of fibroblast cultures to the drug at this concentration reduced the incorporation of ‘H-thymidine into TCA precipitable material to 1% of control levels,

Autoradiography and Nuclear Identification Heterokaryon cultures on collagen-coated 35 mm dishes were labeled with 3H-thymidine at 0.1 &i/ml (82.7 Ci/mmole, NEN) for the indicated period of time. The cells were fixed in 1% paraformaldehyde in phosphate buffered saline solution for 20 min at 37°C followed directly by 100% methanol for 20 min at -20°C. After rinsing extensively in dHZO and air drying, the dishes were coated with NTB-2 nuclear track emulsion (Kodak) diluted 1 :l with 2% glycerol at 40°C. Dishes were dried at room temperature, exposed at 4°C for 24 hr, and then developed in D-19 (Kodak).

A cell that replicated 20% of its genomic DNA could be detected by our methods. When proliferating fibroblasts alone were incubated with 0.1 pCi/ml of ‘H-thymidine for 24 hr. greater than 50 silver grains per nucleus were detectable. A nucleus was scored as labeled if it had IO or more grains over it, or 3.fold the level of background. A nucleus with more than 50 grains was scored as heavily labeled and considered likely to have undergone a complete round of replication.

Autoradiograms were stained with Hoechst 33258 (Riedel-de Haen, Hannover, Germany), 0.12 pg/ml 0.9% NaCl for 15 min at 37°C. Stained nuclei were visualized using a Leitz fluorescence microscope with illumination at 340-380 nm. The silver grains of the autoradiograms were examined using phase-contrast microscopy.

CK Activity Assay and Gel Electrophoresis Total CK activity was determined spectrophotometrically. CK isozymes were detected by electrophoresis of whole cell extracts on 5% nondenaturing gels and by visualizing the end-product of a coupled enzyme reaction (NADPH) under UV illumination as previously described (Blau et al., 1983).

lmmunofluorescence Assay The monoclonal antibody, 5.1 HI 1, generously provided by Dr. Frank Walsh, recognizes a muscle-specific cell surface antigen in human cells (Walsh, 1980; Walsh and Ritter, 1981). Live cells were preincubated at 37’C in DMEM containing 2% HS for 10 min and then exposed to 5.1Hll antibody in mouse ascites fluid at 1:400 dilution for 30 min. Cells were then washed twice for 5 min each with DMEM containing 2% HS. A second goat anti- mouse antibody conjugated to rhodamine (Zymed Co.) was then added at 1 :lOO dilution for 30 min. Cells were washed, rinsed with PBS and fixed with paraformaldehyde and methanol as described above. Antibody binding was visualized using a Leitz fluorescence microscope with illumination at 530-560 nm. Specificity of binding was determined by the lack of fluorescence when parallel dishes were exposed to second antibody only or to mouse gamma globulin (25 pg/ml) partially purified from nonimmune ascites fluid.

Acknowledgments

We wish to thank Dr. Frank Walsh for the use of his monoclonal antibody, 5.1 Hi 1, and Dr. David Yaffe for providing us with the mouse muscle cell line. We are grateful to our colleagues in the laboratory and to Dr. Frank Stockdale for critical evaluation of the manuscript, and to Karen Benight for expert secretarial assistance. This work was supported by grants to H. M. B. from the National Institutes of Health (GM2671 7 and HD18179), the Muscular Dystrophy Association of America, and the March of Dimes Birth Defects Foundation. H. M. B. is a Hume Faculty Scholar and the recipient of a Research Career Development Award from the National Institutes of Health. These studies were perfoned by C.-P. C. in partial fulfillment of the Ph.D. requirements of Stanford University.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

Received March 15, 1984; revised April 27, 1984

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reprogramming cell differentiation in the absence of dna...

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