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Pax3 plays critical roles during developmental and postnatal myogenesis. We have previously shown that levels of Pax3 protein are regulated by monoubiquitination and proteasomal degradation during postnatal myogenesis, but none of the key regulators of the monoubiquitination process were known. Here, we show that Pax3 monoubiquitination is mediated by the ubiquitin-activating/conjugating activity of Taf1, a component of the core transcriptional machinery that was recently reported to be down-regulated during myogenic differentiation. We show that Taf1 binds directly to Pax3 and overexpression of Taf1 increases the level of monoubiquitinated Pax3 and its degradation by the proteasome. A decrease of Taf1 results in a decrease in Pax3 monoubiquitination, an increase in the levels of Pax3 protein, and a concomitant increase in Pax3-mediated inhibition of myogenic differentiation and myoblast migration. These results suggest that Taf1 regulates Pax3 protein levels through its ability to mediate monoubiquitination, revealing a critical interaction between two proteins that are involved in distinct aspects of myogenic differentiation. Finally, these results suggest that the components of the core transcriptional are integrally involved in the process of myogenic differentiation, acting as nodal regulators of the differentiation program.
Pax3 is a key regulator of myogenesis during development (Buckingham et al., 2003). In splotch (Sp) mice, which carry spontaneous mutations in the Pax3 locus, limb muscles are absent (Goulding et al., 1994; Bober et al., 1994). The formation of these muscles requires Pax3 for the induction of expression of c-Met, a tyrosine kinase receptor essential for the delamination and migration of muscle progenitor cells (Bladt et al., 1995; Epstein et al., 1996; Yang et al., 1996). Similar to what occurs in the process of melanocyte stem cell differentiation (Lang et al., 2005), Pax3 protein expression seems to be associated with an intermediate precursor cell of the myogenic lineage. While Pax3 appears to maintain an uncommitted state, it also directly regulates Myf5 which plays a major role in the determination of the myogenic cell fate (Bajard et al., 2006). The embryonic progenitors that express Pax3, and its close homolog Pax7, give rise to a population of adult muscle stem cells (Relaix et al., 2005; Gros et al., 2005; Kassar-Duchossoy et al., 2005). Both before and after expression of the myogenic regulatory factors Myf5 and MyoD, muscle precursor cells undergo extensive proliferation in the limb. Pax3 is most likely involved in maintaining this proliferative phase, directly or indirectly, through the activation of c-Met (Delfini et al., 2000; Buckingham et al., 2003). The regulation of the transition from proliferative progenitor cell to differentiating myoblast is poorly understood but is associated with a marked downregulation of Pax3.
In postnatal myogenesis, Pax3 is transiently expressed during muscle stem cell (“satellite cell”) activation in a highly proliferative intermediate progenitor cell population (Conboy and Rando, 2002). We have shown that when the cells are transitioning from intermediate progenitors to myoblasts, Pax3 levels decline due to protein ubiquitination and proteasomal degradation and that Pax3 degradation is a necessary step for the terminal differentiation to occur (Boutet et al., 2007). Surprisingly, Pax3 is degraded through monoubiquitination, not polyubiquitination, and shuttled to the proteasome by Rad23B (Boutet et al., 2007). Therefore, to understand the transition from a Pax3+ immature progenitor to a Pax3− mature myoblast both during satellite cell activation, we sought to identify the protein(s) responsible of the monoubiquitination of Pax3.
Monoubiquitination of nuclear proteins is important in the regulation of replication and transcription through histone monoubiquitination (Hicke, 2001). Polyubiquitination requires the concerted action of member of the E1 ubiquitin-activating, E2 ubiquitin-converting, and E3 ubiquitin ligase families, whereas monoubiquitination requires only E1 or E2 enzymes (Ciechanover et al., 2000). Taf1 (previously TafII250) is a major subunit of the TFIID transcriptional initiation complex and is an unusual multifunctional protein that possesses, in addition to a protein kinase activity (Dikstein et al., 1996) and a histone acetyltransferase activity (Mizzen et al., 1996), both E1 ubiquitin-activating and E2 ubiquitin conjugating (UBAC) activities (Pham and Sauer, 2000). It is responsible for the monoubiquitination of Histone H1 (Pham and Sauer, 2000), a linker histone that binds DNA between two nucleosomes. In the Drosophila embryo, Taf1 mediated monoubiquitination of Histone H1 appears to be important for the proper regulation of transcriptional activity (Pham and Sauer, 2000). Inactivation of Taf1 in yeast (Walker et al., 1997) and in hamster cell lines (Nishimoto et al., 1982) results in cell cycle arrest at the G1 phase. Null mutations in Drosophila result in lethality in early larval development (Wassarman et al., 2000). These knock-out studies in different organisms suggest a very broad role of Taf1 in cell proliferation and/or cell survival (Wassarman and Sauer, 2001).
In addition, Taf1 has been recently demonstrated to be differentially expressed during myogenic differentiation (Deato and Tjian, 2007). Whereas Taf1 and core subunits of the TFIID complex are dramatically downregulated during myogenic terminal differentiation, Taf1 appears to be upregulated during the transition from quiescence (“reserve cell”) to proliferation (“myoblast”) of myogenic progenitors in vitro (Deato and Tjian, 2007). This result suggests that Taf1 could regulate the transition from myogenic progenitor to myoblasts during postnatal myogenesis. Overall, the nuclear localization of both Pax3 and Taf1, the ability of Taf1 to mediate protein monoubiquitination through its E1 and E2 activities, and the expression pattern of Taf1 protein during the myogenic differentiation all pointed to Taf1 as a candidate for the UBAC activity mediating the monoubiquitination of Pax3 during myogenesis.
In the present study, we investigated the mechanism by which Pax3 protein levels are controlled by monoubiquitination and found that Taf1 is indeed a primary mediator of Pax3 monoubiquitination. Using in vitro and cell-free systems, we show that Taf1 is both necessary and sufficient for Pax3 monoubiquitination. These studies reveal the mechanism of Pax3 monoubiquitination. In addition, these results suggest a regulation of the myogenic differentiation program that is integrated with the core transcriptional machinery.
Based on the ability of Taf1 to catalyze nuclear protein monoubiquitination, we hypothesized that Taf1 might be responsible for Pax3 monoubiquitination. To test this, we transfected satellite-cell-derived (“primary”) myoblasts with either Taf1 siRNA or control siRNA. The siRNA oligonucleotides were transfected with extremely high efficiency (>99%) and were highly effective in reducing Taf1 transcript and protein levels without affecting Pax3 levels (Figure 1A and Figure S1A,B). Since the monoubiquitination of Pax3 leads to its rapid degradation by the proteasome, we first analyzed the steady-state levels of Pax3 which increase when ubiquitination is inhibited (Boutet et al., 2007). We previously demonstrated that Pax3 protein is undetectable in myoblasts but becomes detectable when the cells are treated with the proteasome inhibitor, MG132, to block proteasome-mediated degradation (Boutet et al., 2007). Likewise, in myoblasts in which Taf1 protein was knocked down, Pax3 protein was clearly detectable (Figure 1A).
As members of the core transcriptional complex have recently been shown to vary depending on the state of cellular differentiation (Deato and Tjian, 2007), we sought to determine if Taf1 is expressed throughout the stages of satellite cell activation and lineage progression when Pax3 protein levels initially increase, due to transcriptional upregulation (Lagha et al., 2008), and then subsequently decline due to monoubiquitination and proteasomal degradation (Boutet et al., 2007). Taf1 is expressed at the transcript and protein level in quiescent cells and increases as the cells begin to proliferate and progress along the myogenic lineage, prior to the onset of differentiation (Figure 1B,C). During this time, Pax3 protein increases transiently, peaking during the transit amplifying stage of muscle stem cell activation (Figure 1C and (Boutet et al., 2007)). Thus, during the critical transition when Pax3 protein levels are regulated by monoubiquitination and proteasomal degradation, Taf1 is clearly expressed in myogenic progenitors. In fact, Taf1 protein increase significantly as Pax3 protein levels decrease to almost undetectable levels even though Pax3 transcript levels remain persistently elevated (Figure 1B,C; Figure S1C). Using a reserve cell model of satellite cell quiescence and activation (Kitzmann et al., 1998), we also found that Taf1 is expressed in the quiescent state and the expression is increased at both the transcript and protein levels following activation (Figure S1 D,E).
Based on the hypothesis that Taf1 regulates Pax3 protein levels by monoubiquitination, the finding that the reduction of Taf1 leads to an increase in Pax3, and the fact that Taf1 and Pax3 are co-expressed in myogenic progenitors, we next determined whether the two proteins interact in cells. We transfected C2C12 myoblasts with plasmids expressing epitope-tagged Pax3 alone or with epitope-tagged Taf1 and performed co-immunoprecipitation studies. Using an antibody against either tag, we found that endogenous Taf1 could be pulled down with Pax3 (Figure 1D), and that, conversely, Pax3 could be pulled down with Taf1 (Figure 1E). Interestingly, Taf4, another member of the TFIID complex, could be pulled down with an antibody to the Pax3 tag (Figure 1D). Therefore, Pax3 binds to Taf1 in the context of the TFIID complex, although it is possible that Pax3 also binds to free Taf1.
To test whether endogenous Taf1 and Pax3 proteins interact, we treated primary myoblasts for 24 hours with MG132 to prevent the degradation of Pax3 and performed co-immunoprecipitation studies. Using an antibody against endogenous Taf1, we could pull down endogenous Pax3 (Figure 1F). We previously demonstrated that only the monoubiquitinated form of Pax3 accumulates in myoblasts when degradation is inhibited (Boutet et al., 2007). Indeed, the form of Pax3 that was pulled down in these studies was the monoubiquitinated form (Figure 1F). These results suggest that there is a physical association between Taf1 and Pax3 in myoblasts. To test whether this interaction was direct, we performed pull-down experiments using purified recombinant Taf1 and Pax3 proteins. We found that Pax3 could be pulled down with Taf1 in a cell free system (Figure 1G), suggesting that the interaction between the two proteins is direct.
To assess whether Taf1 could regulate the level of Pax3 protein by ubiquitination and proteasomal degradation, we compared the stability of Pax3 protein when Taf1 was overexpressed in C2C12 myoblasts in pulse chase experiments. Taf1 overexpression dramatically increased the rate of degradation of Pax3 protein (Figure 2A). As we had demonstrated that it is the monoubiquitinated form of Pax3 that is subject to protein degradation (Boutet et al., 2007), we tested directly for the ability of Taf1 to mediate Pax3 monoubiquitination using a cell-free assay of protein ubiquitination (Boutet et al., 2007). To test the reliability of the reconstituted ubiquitination assay, we used Histone H2B as a negative control and Histone H1 as a positive control for Taf1 UBAC activity (Pham and Sauer, 2000; Belz et al., 2002). In this assay, Histone H2B was not ubiquitinated by Taf1 in the presence or absence of ATP, whereas, as previously described (Pham and Sauer, 2000), Histone H1 was monoubiquitinated by Taf1 in the presence of ATP but not when ATP was not present in the reaction (Figure S2). When tested with purified Pax3 in the reaction mixture, Taf1 could monoubiquitinate Pax3 protein in presence of ATP but not in its absence (Figure 2B). Purified Taf1 proteins also demonstrated a strong ubiquitin signal suggesting that it activates ubiquitin efficiently in the presence of ATP (Figure 2B). This assay of protein ubiquitination in a cell-free system clearly demonstrates that Taf1 alone is sufficient to monoubiquitinate Pax3 protein.
Conversely, we assessed whether Taf1 is necessary for the monoubiquitination of Pax3. We first examined whether the level of endogenous Pax3 protein could be altered by inhibiting the expression of Taf1 using siRNA. Treatment with increasing amounts of siRNA against Taf1 revealed a dose-dependent increase of endogenous Pax3 protein (Figure 2C). Control siRNA treatment had no effect on Pax3 levels. Taf1 siRNA treatment had no effect on Pax3 mRNA levels (Figure S1B). These data strongly implicate Taf1 as an essential regulator of Pax3 protein stability.
As an independent test of the necessity of Taf1 for Pax3 ubiquitination, we inhibited Taf1 expression and analyzed the extent of Pax3 protein monoubiquitination. Specifically, in primary myoblasts treated with MG132 to allow the accumulation of ubiquitinated Pax3, we tested whether the inhibition of Taf1 by siRNA could promote the accumulation of non-ubiquitinated Pax3 protein. Compared to control myoblasts which accumulate predominantly monoubiquitinated Pax3, Taf1 siRNA-treated myoblasts accumulate both monoubiquitinated and non-ubiquitinated Pax3 (Figure 2D,E). The data suggest that Taf1 is the primary UBAC for Pax3 since the reduction of Pax3 monoubiquitination parallels the reduction of Taf1 levels (Figure 2D,E), and that the residual Taf1 could certainly account for the residual UBAC activity. However, the presence of another UBAC that might contribute to Pax3 monoubiquitination if Taf1 could be completely eliminated (which it cannot because that is lethal to the cell) cannot be excluded. Nevertheless, these results suggest that Taf1 is necessary for normal and full Pax3 monoubiquitination.
In Drosophila, Taf1 protein carrying V1072D or R1096P mutations display a reduced UBAC activity in vitro and in vivo (Pham and Sauer, 2000). We took advantage of the homology between the mammalian and Drosophila Taf1 proteins and generated the homologous mutations, Taf1V1049D and Taf1R1070P, in the mammalian Taf1. Consistent with a reduced UBAC activity, neither mutant, when overexpressed in C2C12 myoblasts, was as effective as wild-type Taf1 in increasing the degradation rate of Pax3 (Figure 3A).
Furthermore, we analyzed the level of monoubiquitinated Pax3 in control myoblasts or in myoblasts overexpressing Taf1, Taf1V1049D or Taf1R1070P. After treating Taf1-expressing cells with MG132 for 3 hours, monoubiquitinated Pax3 was clearly detectable (Figure 3B). There was less monoubiquitinated Pax3 protein when the Taf1 mutants were expressed (Figure 3B). MG132 treatment of control transfected cells for the same amount of time resulted in an even lower level of monoubiquitinated Pax3. Longer treatments (6 hours) with MG132 resulted in even higher levels of Pax3 in these cells, as shown previously (Boutet et al., 2007). Therefore, mutation in the Taf1 UBAC activity domain reduced markedly but not completely the UBAC activity of Taf1. The reduced monoubiquitination of Pax3 paralleled the increased stability of Pax3 proteins.
To assess quantitatively the level of monoubiquitination of Pax3, lysates were subjected to Nickel-agarose pull-down to purify monoubiquitinated proteins with histidine-tagged ubiquitin. Quantitation of the level of monoubiquitination of Pax3 with the expression of wild-type and mutant Taf1 proteins showed that there was less Pax3 monoubiquitination in the presence of Taf1 mutants than in the presence of wild-type Taf1 (Figure 3C). These data further implicate the role of Taf1 as a key regulator Pax3 monoubiquitination through its UBAC activity.
The preceding studies clearly demonstrate that Taf1 is capable of monoubiquitinating Pax3 and that inhibition of Taf1 leads to increases in Pax3 protein levels. In order to test directly for the functional role of Taf1 in Pax3-mediated processes, we examined two well-established roles of Pax3 in myogenesis – the inhibition of differentiation and the promotion of myogenic progenitor migration (Epstein et al., 1995; Epstein et al., 1996; Boutet et al., 2007). We therefore hypothesized that the inhibition of Taf1 expression with siRNA would inhibit myogenic differentiation because of the resulting increase in Pax3. To test this, we transfected primary myoblasts with either Taf1 siRNA or control siRNA and then induced those cells to undergo differentiation. In Taf1 siRNA treated cultures, markers of terminal differentiation were repressed compared to control siRNA treated cultures (Figure 4A). To address whether this effect was specifically due to an increased level of Pax3, we transfected Taf1-siRNA-treated cells with either control or Pax3 siRNA. Pax3 siRNA efficiently downregulated Pax3 transcript and protein levels (Figure S3A,B). Compared to control cultures (cells treated with a control siRNA and Taf1 siRNA), cultures transfected with both Taf1 and Pax3 siRNA resulted in a significant increase in the expression of markers of terminal differentiation (Figure 4B), demonstrating a function role of Taf1 in regulating myogenic differentiation by the regulation of Pax3 levels. It should be noted that the effects of Taf1 siRNA treatment on differentiation are similar to those seen following the expression of mutant forms of Pax3 that are resistant to monoubiquitination (Boutet et al., 2007), supporting the hypothesis that Taf1 regulates myogenic differentiation by its role as a UBAC for Pax3. These data further support the hypothesis that Taf1 regulates myogenic differentiation by regulating Pax3 monoubiquitination.
Pax3 has been shown to be essential during limb development for the migration of the muscle progenitor cells (Epstein et al., 1996; Yang et al., 1996). We tested whether the inhibition of Taf1 expression and the subsequent increase of Pax3 expression would have an effect on the migration of primary myoblasts in addition to the effects observed on differentiation. We used time-lapse microscopy to assess myoblast mobility and found that myoblasts treated with Taf1 siRNA migrated twice as fast as myoblasts treated with control siRNA (Figure 5A,B). To test whether this enhanced mobility was due to the maintenance of higher Pax3 protein levels, we transfected primary myoblast cultures with Taf1 siRNA together with either Pax3 or control siRNA. Compared to cultures treated with Taf1 siRNA alone, cultures treated with both Taf1 and Pax3 siRNA demonstrated a reduced motility, similar to the motility of cells treated with no siRNA (Figure 5C,D). These results demonstrate that Taf1 plays a functional role in regulating this Pax3-mediated process, analogous to its regulation of myogenic differentiation.
In order to complement the functional studies of the regulation of Pax3 by Taf1 in satellite cell-derived myogenic progenitors, we tested the functional effects of down regulation of Taf1 in embryonic progenitors. Using lineage tracing (see Methods), we were able to monitor myogenic progenitors in limb explants (Figure S4A,B). FACS-purified cells from embryonic limbs were highly enriched for myogenic cells (Figure S4C,D).
Around E10.5, myogenic progenitors, which are Pax3 positive, delaminate from the somitic dermomyotome and migrate into the limb buds (Bober et al., 1994). In the following two days, as the cells reach their destination in the limb, most of the myogenic progenitors down-regulate Pax3, express myogenic regulatory factors (MRFs) to become myoblasts, and differentiate (Buckingham et al., 2006). The delamination, migration and proliferation of skeletal muscle progenitors are all dependent on Pax3 function (Relaix et al., 2004). We chose to examine embryonic progenitors at E11.5 when a majority are still migrating and express high level of Pax3 and at E12.5 when most embryonic myoblasts have ceased migrating and express low level of Pax3 and high levels of MRFs (Buckingham et al., 2006).
Indeed, in the transition from E11.5 to E12.5, Taf1 levels increased while Pax3 levels declined in purified embryonic myoblasts (Figure 6A). Pax3 protein levels declined much more dramatically than did Pax3 transcript levels (Figure 6A–C). The absence of Pax3 protein in cells with intermediate levels of the transcript is similar to the pattern seen in postnatal progenitors as they progress along the myogenic lineage when Pax3 protein levels are regulated post-transcriptionally by protein degradation (Boutet et al., 2007). Indeed, treatment of embryonic myoblasts with MG132 resulted in much higher steady-state levels of Pax3 protein (Figure 6D). Corresponding to the decline in Pax3 protein levels from E11.5 to E12.5, E12.5 myoblasts expressed higher levels of Myogenin and had lower motility rates than E11.5 myoblasts (Figure 7A,B and Figure S5A,B).
To test whether Taf1 regulates these key Pax3-mediated functions of embryonic myoblasts, we knocked down Taf1 in E12.5 myoblasts when Taf1 is high and when Pax3 is low. Indeed, as in postnatal myogenic progenitors, the reduction of Taf1 in embryonic myoblasts resulted in an increase in the percentage of Pax3+ cells and a decrease in the percentage of Myogenin+ cells (Figure S6A). As with postnatal progenitors, reduction of Taf1 levels and the associated increase in Pax3 levels resulted in enhanced motility of embryonic myoblasts (Figure 7C–E). These data further support the importance of Taf1 in the regulation of Pax3 as a key regulator of myogenesis.
The results of the present study demonstrate that the UBAC involved in the monoubiquitination of Pax3 is Taf1, a major subunit of the initiation complex TFIID. We show that Taf1 directly interacts with Pax3, is sufficient for the monoubiquitination of Pax3, and regulates Pax3 protein levels. Reduction of Taf1 levels in myogenic progenitors results in increased Pax3 protein. Maintenance of Pax3 protein levels in myogenic progenitors inhibits differentiation, as previously shown (Boutet et al., 2007).
It is remarkable that the control of myogenic differentiation is deeply rooted in the regulation of the composition of the core promoter recognition complex with Taf1 as one of its major subunits (Ruppert et al., 1993). Discovery of cell-type specific TATA-binding protein associated factors (TAFs) suggest that modified TFIID complexes may be involved in mechanisms that regulate tissue specific program of gene expression (Guermah et al., 2003; Hochheimer and Tjian, 2003; Hiller et al., 2004; Indra et al., 2005; Chen et al., 2005). While tissue specific TAFs play an active role by trapping repression complexes (Chen et al., 2005) or by regulating a subset of specific differentiation genes (Indra et al., 2005; Fadloun et al., 2007), all these TAFs coexist with the canonical TFIID complex and seem to add specificity to the TFIID complex. Taf1 is especially important as alteration of its activities such as its kinase activity (Siegert and Robbins, 1999) or its HAT activity (Weissman et al., 1998) can alter the formation of the initiation complex. Thus, regulation of Taf1 activities by interactions with activators and transcription factors is integral to the process of transcriptional activation. Upon terminal myogenic differentiation, the TFIID complex is replaced by a novel core promoter recognition apparatus in which Taf1 and other core TFIID subunits disappear and are replaced with other subunits such as Taf3 and TRF3 (Deato and Tjian, 2007). In order for myogenic differentiation to proceed, Taf1, through its UBAC activity, must directly regulate the degradation of the transcription activator Pax3. As such, Taf1 appears to regulate the transition of progenitors to myoblasts and their subsequent differentiation.
The regulation of the stability of transcription factors, particularly with regard to transcription factors with acidic rich domains, appears to be important for transcriptional activity. Regions of the proteins where ubiquitination occur often overlap with transcription activation domains (Salghetti et al., 2000). Although polyubiquitination may be the canonical signal for proteasomal degradation, monoubiquitination appears to enhance transcriptional activity in some cases (Salghetti et al., 2001). In the case of Pax3, it is possible that the process of monoubiquitination enhances the transactivation potential of Pax3 by recruiting proteasome associated proteins such as the 19S proteasome, which, itself, appears to be capable of potentiating transcription (Gonzalez et al., 2002). Alternatively, monoubiquitination of Pax3 and subsequent proteasomal degradation could initiate transcription, following the model of “activation by destruction” proposed when the destruction of the transcription factor is a requirement for the initiation of transcription to occur (Tansey, 2001; Lipford et al., 2005).
It remains to be determined where the interaction of Taf1 and Pax3 occurs. If the monoubiquitination of Pax3 is a requirement for its activity, it is possible that Pax3 monoubiquitination occurs on the DNA during initiation of transcription of target genes. Taf1 is also known to be found TBP-free on the chromatin but not part of the pre-initiation complex in the nucleus (Bertolotti et al., 1996; Saurin et al., 2001; Lin et al., 2002), suggesting that Taf1 might be a direct negative regulator of Pax3 independent of any transcriptional context.
Taf1 is responsible for the monoubiquitination of Histone H1 (Pham and Sauer, 2000), a linker histone that binds DNA between two nucleosomes. In this study, we have identified a second substrate, Pax3, for Taf1-mediated monoubiquitination. In the case of Pax3, unlike that of Histone H1, Taf1 mediates monoubiquitination for the degradation of its substrate. As our reconstituted ubiquitination assay shows (Figure 2B), Taf1 alone is sufficient to monoubiquitinate Pax3. Previously we have shown that monoubiquitinated Pax3 is recognized by Rad23B and shuttled to the proteasome by binding of monoubiquitinated Pax3 to S5a via Rad23B (Boutet et al., 2007). To this model we now add Taf1 as the UBAC that mediates the monoubiquitination of Pax3 (Figure S6B).
Due to the pleiotropic activities of Taf1, it is difficult to selectively knockout the UBAC activity to establish whether Taf1 is the only enzyme responsible for the monoubiquitination of Pax3. To attest of this difficulty, the ts13 or tsBN462 cell lines, which lack the acetyltransferase activity of Taf1, display cell cycle arrest and apoptosis (Sekiguchi et al., 1995). Based on a mutation in Drosophila with reduced UBAC activity (Pham and Sauer, 2000), we generated homologous mutations in the mammalian Taf1 protein. Like the Drosophila mutant, UBAC activity of the mammalian Taf1 mutants was reduced but not eliminated (Figure 3A–C). Therefore, it is possible that Pax3 monoubiquitination is mediated by proteins with UBAC activity other than Taf1.
In summary, our results demonstrate a novel role of Taf1 as a key regulator of Pax3 monoubiquitination during myogenic differentiation. The molecular mechanisms coordinating myogenic lineage progression and differentiation appear to be deeply rooted in the regulation of the composition of the core promoter recognition complex with Taf1. These results provide a better understanding of the regulation of Pax3 and how myogenic lineage progression and differentiation are controlled post-transcriptionally.
The HA-hTaf1 mammalian expression construct was provided by Dr. R. Tjian (U. C. Berkeley) and the pEGFP-N3-Luciferase construct was provided by Dr. C. Bertoni (UCLA). The pEGFP-N3-Pax3 construct was previously described (Boutet et al., 2007). To mutate HA-hTaf1 at valine 1049 to generate Taf1V1049D, we used the forward primer 5′-CGCTGGGAAGTGATTGATGATGTGCGCACAATGTCAAC-3′ and its reverse complement. To mutate HA-hTaf1 at arginine 1070 to generate Taf1R1070P, we used the forward primer 5′-GCCCGTGGATCACCGTTTTCTGTGGCTGAGCATC-3′ and its reverse complement. Both mutations were generated using the Quickchange PCR directed mutagenesis Kit (Stratagene) according to manufacturer’s instructions.
Satellite cells were purified from bulk fibers and activated in vitro as described previously (Conboy and Rando, 2002; Brack et al., 2007). Primary myoblast cultures were maintained as proliferating mononucleated cells or were induced to differentiate as previously described (Quach and Rando, 2006). To prepare reserve cells, primary or C2C12 myoblasts were placed into differentiation medium for 5 days and reserve cells were prepared, according to the method of Kitzmann et al. (Kitzmann et al., 1998), except that cells were fully trypsinized and plated on a new dish. We took advantage of the differential adhesion between fibers and reserve cells. After the reserve cells adhered, the cultures were washed twice in PBS and the differentiation medium was added.
Cells were harvested and washed twice in phosphate buffered saline (PBS). Cells were lysed and total RNA was extracted using Triazol (Invitrogen) according to the manufacturer’s instructions. Two μg (for siRNA treated myoblasts) of total RNA were reverse transcribed using Superscript II kit (Invitrogen) and quantitative RT-PCR was carried out on a MyiQ real time PCR (BioRad) using Pax3, Taf1, and GAPDH TaqMan probes (Applied Biosystems). Relative quantification of gene expression normalized to GAPDH was carried using the comparative CT method (Pfaffl, 2001). Each measurement was performed in triplicate in three independent experiments.
Primary myoblasts were transfected with Taf1 or control siRNA (Invitrogen) at 15, 30, 45 or 60 nM. For analysis of differentiation, Taf1 or control siRNA oligonucleotides were used at the highest concentration of 60 nM. In the case of double transfections for differentiation, Taf1 and Pax3 siRNA (Invitrogen) or Taf1 and control siRNA were used at 60 nM each. 24 hours after transfection, cells were harvested or placed in differentiation medium for 24 hours and then harvested. Lysates were analyzed by Western blots. In specific experiments as noted, cells were transfected with Taf1 or control siRNA at a concentration of 60 mM and, 24 hours post-transfection, and were then treated with MG132 (10 μM) for 4 hours.
C2C12 myoblasts were transfected with plasmids and treated as indicated. Cycloheximide (5 μM) was added 24 hours after transfection, cells were harvested at different time points, and lysates were analyzed by Western blots. The films were scanned and quantified with ImageJ (http://rsb.info.nih.gov/ij/).
C2C12 myoblasts were transfected with pEGFP-N3-Pax3 and with either Taf1 constructs (wild-type (HA-hTaf1), Taf1V1049D, or Taf1R1070P) or with empty vector constructs. Transfected cells were pre-treated with 10 μM MG132 for 3 hours to block Pax3 degradation and extracts were prepared in lysis buffer containing 100 μM MG132, 20 μM ubiquitin aldehyde and 100 μM N-ethylmaleimide (NEM, Sigma). Lysates were denatured by boiling for 5 min in the presence of 0.1% SDS. Immunoprecipitation was carried out by adding 10 μg of anti-GFP monoclonal antibody (Santa Cruz Biotechnology). For co-transfection of the Pax3, His6-tagged ubiquitin, and HA-hTaf1 wild-type and mutant (Taf1V1049D or Taf1R1070P) expression vectors, cells were treated with 10 μM MG132 for 6 hours and extracts were prepared in CelLytic M reagent (Sigma) lysis buffer containing 100 μM MG132 and 20 μM ubiquitin aldehyde. His-tagged proteins were purified on Nickel affinity gel using His-Select M affinity Capture Kit (Sigma) according to the manufacturer’s instructions. Immunoprecipitated and purified proteins were analyzed by Western blotting using an anti-GFP monoclonal antibody and anti-ubiquitin antibodies (FK-2; Biomol International)
Recombinant Pax3 was produced as GST-fusion proteins in E. coli BL21, extracted in MT-PBS (10% glycerol, 1 mM DTT, 0.5 mM PMSF) containing 1% Triton X-100, and purified on glutathione-Sepharose resin (GE). GST tags were excised with Thrombin (GE) (5 U/ml for 6 hours at room temperature). Recombinant human Taf1 protein was produced in Sf21 cells with a baculovirus expressing hemagglutinin (HA)-tagged human Taf1 (Orbigen) and extracted using anti-HA antibody resin (Roche). Beads were washed and bound proteins were eluted with 20 μl of SDS loading buffer. Ubiquitination assays were performed after purification of HA-hTaf1 and carried out directly on the agarose bound Taf1 proteins in 50 mM Tris-Cl pH 7.6, 5 mM MgCl2 at 25°C for 2 hours in the presence of ubiquitin (5 μg) (Biomol International) and Histone H1 (Roche), Histone H2B (Roche) or Pax3 proteins with or without 2 mM ATP. Pulldown assays were performed after purification of HA tag and HA-hTaf1 and carried out in the same buffer as the ubiquitination assay at 4°C for 4 hours in the presence of Pax3 proteins. Reactions were stopped by the addition of sample buffer and subjected to SDS-PAGE followed by transfer and Western blot analysis.
C2C12 myoblasts were transfected either with expression vectors for GFP (pEGFP-N3-Pax3), Taf1 (HA-hTaf1), or respective control vectors. Extracts were prepared in lysis buffer containing 100 μM MG132 (Sigma). Co-immunoprecipitation was carried out by either adding 10 μg of anti-GFP monoclonal antibody (Santa Cruz Biotechnology) or anti-HA antibody resin (Roche). To study endogenous proteins, primary myoblasts were treated with 10 μM MG132 for 4 hours. Co-immunoprecipitation was carried out using 10 μg of anti-Taf1 antibody (Santa Cruz Biotechnology). After incubation at 4°C for at least 4 hours, beads were washed three times in lysis buffer and proteins were eluted by the addition of sample buffer and subjected to SDS-PAGE followed by transfer and Western blot analysis.
Immunoprecipitated and purified proteins were analyzed by Western blotting using the following antibodies: anti-GFP monoclonal antibody (1:1000, Clontech), anti-DsRed2 polyclonal antibody (1:1000, Clontech), anti-ubiquitin monoclonal antibody (FK-2; 1:1000), anti-ubiquitin polyclonal antibody (1:1000, Sigma), anti-Pax3 monoclonal antibody (1:100, DSHB), anti-Taf1 polyclonal antibody (1:100, Santa Cruz Biotechnologies), anti-HA monoclonal antibody (1:1000, Roche), anti-Sarcomeric α-Actinin monoclonal antibody (1:500, Sigma), anti-Myosin Heavy Chain monoclonal antibody (1:500, Sigma), anti-Myogenin monoclonal antibody (1:500, BD-PharMingen) and anti-GAPDH monoclonal antibody (1:5000, Ambion).
Primary myoblasts and sorted embryonic myogenic cells were transfected overnight in growth medium with either siRNA oligonucleotides (60 nM each) as indicated. Cells were plated on laminin/collagen (Sigma) coated dishes at the concentration of approximately 3,000 cells/cm2. Cultures were analyzed using a Zeiss Axiovert 200M inverted microscope (Carl Zeiss) fitted with an incubation chamber to provide a controlled environment (CTI Controller, Tempcontrol; Carl Zeiss; humidified 5% CO2). Phase contrast images were acquired every 5 minutes for 3 hours with a Zeiss camera MRm (Carl Zeiss) integrated in the Axiovision system (Carl Zeiss). Tracking of cells and measurements of distances were done with ImageJ (http://rsb.info.nih.gov/ij/) with a manual cell tracker plug-in. A minimum of 46 different cells from at least three independent transfections or at least three different sorts were collected for final analysis.
To purify myogenic populations that represent both migrating progenitors and differentiating myoblasts, we generated strains of mice in which the reporter gene dsred was expressed specifically in those cells. This was accomplished by crossing Z/Red mice, in which DsRed expression is induced in cells (and all their progeny) expressing Cre recombinase (Vintersten et al., 2004), with transgenic lines in which Cre is expressed in the myogenic lineage. Two different Cre-expressing strains were used. The M-Cre line expresses Cre in Pax3-expressing progenitors of somitic hypaxial origin (Brown et al., 2005). At E11.5, an enriched population of such progenitors migrate to the limbs (Brown et al., 2005). The Myf5-Cre-NN line expresses Cre in Myf5-expressing progeny of the Pax3-expressing cells, as Pax3 directly activates Myf5 in muscle progenitors of the limb (Haldar et al., 2007). Therefore, at E12.5, the population of Cre-expressing cells in the M-Cre and the Myf5-Cre-NN mice overlap since previously Pax3-expressing cells are then expressing Myf5. At this stage, these myogenic cell populations are enriched in myoblasts that are no longer migratory and are Pax3 negative and MRF positive (Buckingham et al., 2006). Crossing each Cre-driver with the Z/Red line (resulting in two strains that we will refer to as Pax3DsRed and Myf5DsRed) thus yields DsRed-expressing myogenic cells that can be purified at different embryonic stages by FACS and studied in vitro.
M-Cre and Myf5-NN-Cre drivers were crossed with heterozygous Z/RED reporter mice. Myogenic cells were isolated from E11.5 or E12.5 embryos. Forelimb and hindlimb buds were dissociated as previously described (Biressi et al., 2007) before sorting using a Vantage Sorter SE (Becton-Dickinson). Forward scatter and side scatter parameters were used to gate out cell clumps and debris. Cells dissociated from DsRed negative littermates were used to set the gating to exclude autofluorescence. Sorted myogenic cells were resuspended in Opti-MEM (Invitrogen) supplemented with 20% FBS, 20 mM Hepes and 5 ng/ml FGF (Peprotech) and plated on laminin/collagen (Sigma) coated dishes. The purity of the sorted cells was evaluated by immunofluorescence after 2 days of culture in Dulbecco’s Modified Eagle Medium, 2% Horse Serum (Invitrogen), 20 mM HEPES. For protein content analysis, sorted cells from 8 E11.5, 9 E12.5 Pax3DsRed and 15 E12.5 Myf5DsRed embryos were pooled.
For comparisons of two groups, Student’s t-tests (unpaired, non parametric and two tailed p values) were used. In all figures, error bars represent ± SD.
Figure S1. Regulation and modulation of Taf1 and Pax3 levels (related to Figure 1). (A) Immunofluorescence of primary myoblasts transfected with 60 nM of fluorescein-labeled siRNA oligonucleotides. Cells were fixed 24 hours post-transfection, stained for nuclei with DAPI, and examined by epifluorescence microscopy. More than 99% of the cells were positive for the presence of the fluorescein-labeled oligonucleotides. (B) Quantitative analysis by qRT-PCR of Taf1 mRNA levels in primary myoblasts treated with Taf1 siRNA at the indicated concentrations. When no Taf1 siRNA was present, control siRNA (60 nM) was transfected instead. Each is normalized to GAPDH and to the level of transcript in cells transfected with control siRNA, which was arbitrarily set at 1. Pax3 mRNA levels were also quantified to test for any effect of the Taf1 siRNA treatment; no effect was observed. (C) Quantitative analysis of Pax3 and Taf1 expression as determined by Western blot analysis (representative example shown in Figure 1C). Satellite cells were harvested and cultured from 0–5 days and assessed for Pax3 and Taf1 protein levels each day. Each protein is normalized to GAPDH; the level at the peak of expression was arbitrarily set at 1. (D) Quantitative analysis by qRT-PCR of Taf1 and Pax3 mRNA levels in quiescent and activated reserve cells from C2C12 myoblast cultures. Each is normalized to GAPDH mRNA level and the level in quiescent cells was arbitrarily set to 1. Error bars represent ± SD. (E) Western blot analysis of Taf1 and Pax3 protein levels in quiescent and activated reserve cells. Quantitative analysis of replicate experiments is shown below. Each is normalized to GAPDH protein level and the level in quiescent cells was arbitrarily set to 1.
Figure S2. Analysis of Histone H1 and H2B ubiquitination by Taf1 in a cell-free ubiquitination assay (related to Figure 3). Reactions containing purified ubiquitin, Histone H1 (as a positive control) or Histone H2B (as a negative control) in the presence or absence of ATP were analyzed by immunoblots with anti-Histone H1, anti-Histone H2B, and anti-ubiquitin antibodies. Arrows show the positions of Histone H2B, Histone H1 and monoubiquitinated H1 (H1-Ub1).
Figure S3. Modulation of Pax3 levels by Pax3 siRNA (related to Figure 4). (A) Quantitative analysis by qRT-PCR of Pax3 mRNA levels in primary myoblasts treated with Pax3 or control siRNA at indicated concentrations. Each is normalized to GAPDH and the level for control siRNA treated myoblasts for each concentration was arbitrarily set at 1. (B) Western blot analysis of the expression levels of Pax3 protein after Pax3 siRNA treatment. Primary myoblasts were transfected with Taf1 siRNA (in order to enhance the levels of Pax3 protein (see Figure 1)) and either Pax3 or control siRNA (60 nM each). Cells were harvested 24 hours after transfection and analyzed by immunoblots with anti-Taf1, anti-Pax3 and anti-GAPDH antibodies.
Figure S4. Pax3DsRed and Myf5DsRed myogenic cells at E11.5 and E12.5 (related to Figure 5). (A) Myogenic progenitors and myoblasts from limbs at E11.5 (Pax3DsRed) and at E12.5 (Pax3DsRed and Myf5DsRed). Representation of the position of the migrating progenitors (purple arrow heads) and myoblasts (red dots) in the limb buds. (B) Limbs were dissected and observed by fluorescence microscopy at E11.5 (Pax3DsRed) and at E12.5 (Pax3DsRed and Myf5DsRed). Migrating progenitors were located at the proximal (p) part of both the forelimb and the hindlimb at E11.5, whereas myoblasts reaching their destination were located at the distal(d) part of both forelimb and hindlimb at E12.5 (C) After enzymatic digestion, cells were sorted for DsRed. (D) Immunofluorescence of FACS-sorted DsRed positive cells plated in growth medium for 24 hours and placed into differentiation medium for 2 days. Cells were fixed and stained with a cocktail of antibodies against MyHC, MyoG, MyoD and Pax3 (Alexa 488) and counterstained for nuclei with DAPI. The percentage of myogenic cells was established by dividing the number cells positive for Pax7, MyoD, MyoG or MyHC by the total number of DAPI+ cells. The myogenicity of the cells isolated at E11.5 (Pax3DsRed) and at E12.5 (Pax3DsRed and Myf5DsRed) was comparable, with values of 85%, 81% and 86%, respectively. Scale represents 20 μm
Figure S5. Myogenin expression in E11.5 and E12.5 embryonic myogenic progenitors (related to Figure 6). (A) Expression of Myogenin (MyoG) protein in Pax3DsRed and Myf5DsRed sorted myogenic cells at E11.5 and E12.5. Myogenic progenitor cells isolated at E11.5 (Pax3DsRed) and at E12.5 (Pax3DsRed and Myf5DsRed) were analyzed by Western blots with anti-MyoG and anti-GAPDH antibodies. (B) Expression of Myogenin transcript levels in Pax3DsRed and Myf5DsRed sorted myogenic cells at E11.5 and E12.5. Transcript levels were analyzed by qRT-PCR and normalized to GAPDH and then to the values at E11.5.
Figure S6. Pax3 expression and distribution of cell motility of embryonic myogenic progenitors treated with Taf1 siRNA (related to Figure 7). (A) Immunofluorescence of FACS-sorted DsRed positive cells (E12.5) plated in growth medium and transfected with Taf1 or control siRNA (60 nM). Cells were fixed and stained with antibodies against Pax3 (red) and Myogenin (MyoG; green) and with DAPI (blue) to stain all nuclei. There was in marked increase in the number of cells expressing detectable Pax3 and a marked decrease in the percentage expressing Myogenin following treatment with Taf1 siRNA compared to control siRNA. Scale bar represents 20 μm. (B) Model of Taf1-mediated Pax3 monoubiquitination and subsequent degradation.
We thank all the members of the Rando laboratory for help, comments and discussion. We thank Dr. R. Tjian for the HA-hTaf1 mammalian expression construct. This work was supported by a Development Grant from the Muscular Dystrophy Association to SCB and by grants from the NIH (AG23806, AR056849, and an NIH Director’s Pioneer Award) and the Department of Veterans Affairs (Merit Review) to TAR.
Competing Interests statement The authors declare that they have no competing financial interests.
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