Portal de Periódicos da CAPES

The basic helix-loop-helix transcription factor Mist1 functions as a transcriptional repressor of MyoD

1998; Springer Nature; Volume: 17; Issue: 5 Linguagem: Inglês

10.1093/emboj/17.5.1412

1460-2075

Claudie Lemercier, Robert Q. To, Rosa A. Carrasco, Stephen F. Konieczny,

Signaling Pathways in Disease

Article2 March 1998free access The basic helix–loop–helix transcription factor Mist1 functions as a transcriptional repressor of MyoD Claudie Lemercier Claudie Lemercier Department of Biological Sciences, Purdue University, West Lafayette, IN, 47907-1392 USA Present address: Laboratoire de Greffe de Moelle, UMR 5540, Universite Bordeaux 2, 146 rue Leo Saignat, 33076 Bordeaux Cedex, France Search for more papers by this author Robert Q. To Robert Q. To Department of Biological Sciences, Purdue University, West Lafayette, IN, 47907-1392 USA Present address: The Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA, 94121 USA Search for more papers by this author Rosa A. Carrasco Rosa A. Carrasco Department of Biological Sciences, Purdue University, West Lafayette, IN, 47907-1392 USA Search for more papers by this author Stephen F. Konieczny Corresponding Author Stephen F. Konieczny Department of Biological Sciences, Purdue University, West Lafayette, IN, 47907-1392 USA Search for more papers by this author Claudie Lemercier Claudie Lemercier Department of Biological Sciences, Purdue University, West Lafayette, IN, 47907-1392 USA Present address: Laboratoire de Greffe de Moelle, UMR 5540, Universite Bordeaux 2, 146 rue Leo Saignat, 33076 Bordeaux Cedex, France Search for more papers by this author Robert Q. To Robert Q. To Department of Biological Sciences, Purdue University, West Lafayette, IN, 47907-1392 USA Present address: The Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA, 94121 USA Search for more papers by this author Rosa A. Carrasco Rosa A. Carrasco Department of Biological Sciences, Purdue University, West Lafayette, IN, 47907-1392 USA Search for more papers by this author Stephen F. Konieczny Corresponding Author Stephen F. Konieczny Department of Biological Sciences, Purdue University, West Lafayette, IN, 47907-1392 USA Search for more papers by this author Author Information Claudie Lemercier1,2, Robert Q. To1,3, Rosa A. Carrasco1 and Stephen F. Konieczny 1 1Department of Biological Sciences, Purdue University, West Lafayette, IN, 47907-1392 USA 2Present address: Laboratoire de Greffe de Moelle, UMR 5540, Universite Bordeaux 2, 146 rue Leo Saignat, 33076 Bordeaux Cedex, France 3Present address: The Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA, 94121 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:1412-1422https://doi.org/10.1093/emboj/17.5.1412 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info A good model system to examine aspects of positive and negative transcriptional regulation is the muscle-specific regulatory factor, MyoD, which is a basic helix–loop–helix (bHLH) transcription factor. Although MyoD has the ability to induce skeletal muscle terminal differentiation in a variety of non-muscle cell types, MyoD activity itself is highly regulated through protein–protein interactions involving several different co-factors. Here we describe the characterization of a novel bHLH protein, Mist1, and how it influences MyoD function. We show that Mist1 accumulates in myogenic stem cells (myoblasts) and then decreases as myoblasts differentiate into myotubes. Mist1 functions as a negative regulator of MyoD activity, preventing muscle differentiation and the concomitant expression of muscle-specific genes. Mist1-induced inhibition occurs through a combination of mechanisms, including the formation of inactive MyoD–Mist1 heterodimers and occupancy of specific E-box target sites by Mist1 homodimers. Mist1 lacks a classic transcription activation domain and instead possesses an N-terminal repressor region capable of inhibiting heterologous activators. Thus, Mist1 may represent a new class of repressor molecules that play a role in controlling the transcriptional activity of MyoD, ensuring that expanding myoblast populations remain undifferentiated during early embryonic muscle formation. Introduction During embryonic development, specific biochemical and morphological changes occur in response to extracellular signals that target individual cells within a particular organ or tissue. In many cases, the responses represent major changes in gene expression patterns. To ensure that gene activity is modulated correctly under these conditions, the transcription machinery must be regulated precisely by a number of positive- and negative-acting transcription factors. Among the different transcription factor families that have been identified, the basic helix–loop–helix (bHLH) family has been shown to be a key regulator of many different developmental pathways. In the case of skeletal muscle, four bHLH muscle regulatory factors (MRFs) (known as MyoD, myogenin, MRF4 and Myf-5) are involved in the differentiation and maintenance of the skeletal muscle phenotype (reviewed in Buckingham, 1994; Olson and Klein, 1994; Ludolph and Konieczny, 1995). Forced expression of any one MRF in non-muscle cells results in the conversion of the cells to a myogenic stem cell population that can be induced to express genes involved in terminal differentiation. The MRFs, like most bHLH proteins, form homodimers, but preferentially heterodimerize with the widely expressed E2A gene products, E12 and E47 (Blackwell and Weintraub, 1990; Lassar et al., 1991), or with the related bHLH protein HEB (Zhang et al., 1991; Hu et al., 1992). It is this high affinity, stable heterodimer complex that binds to a conserved DNA motif (E-box; -CANNTG-) which is present in the promoter or enhancer regions of many developmentally regulated genes, including genes involved in myogenic differentiation events. The MRFs are classified as myogenic 'activator' proteins because each contains at least one activation domain that is able to induce gene transcription. However, not all bHLH proteins exhibit activator properties. Many function instead as negative regulators of gene transcription. For example, Id (Benezra et al., 1990), which possesses a HLH motif but lacks a basic domain, is capable of forming heterodimers with bHLH proteins and thereby preventing DNA binding. Id inhibits MyoD activity in vivo by forming either transcriptionally inactive complexes of MyoD–Id or by forming heterodimers with E-proteins and effectively blocking the formation of active MyoD–E-protein complexes (Benezra et al., 1990; Jen et al., 1992). A second class of negative regulators are the HES proteins, which are bHLH factors that exhibit a repressor activity that manifests itself through binding to an E-box-related (N-box) DNA target sequence (Sasai et al., 1992). Similarly, the mouse bHLH protein Twist has been shown to alter the activity of the MyoD protein family by blocking DNA binding, by titrating E-proteins and by inhibiting trans-activation of the MyoD co-factor, MEF2 (Spicer et al., 1996; Hamamori et al., 1997). Thus, both bHLH and HLH proteins can modulate effectively the transcriptional activity of the MyoD family and regulate directly how MyoD controls myogenic events during development. The above studies have revealed the importance of bHLH proteins in regulating both gene transcription and cell fate determination. They have also demonstrated that active and negative strategies are involved in controlling the establishment and maintenance of a particular phenotype. In order to understand better how bHLH proteins interact and regulate each other's activities, we recently identified an additional member of the complex bHLH protein network, Mist1 (Mak et al., 1996). The Mist1 gene exhibits a complicated expression pattern, with transcripts being detected in several different tissues including skeletal muscle derivatives (Lemercier et al., 1997). Specifically, Mist1 transcripts are expressed in skeletal muscle-forming regions during embryonic days E12.5 and E16.5, but disappear rapidly as muscle cells differentiate. This partially overlapping expression pattern with the MyoD protein family prompted us to examine whether Mist1 interacts with the muscle regulatory factors and whether it modulates MRF activity. In this report, we show that Mist1 protein accumulates in undifferentiated myoblasts, but levels rapidly decrease as cells begin to differentiate. In addition, although Mist1 associates with MyoD, the heterodimer complex of Mist1–MyoD is unable to bind to muscle-specific E-box elements. Mist1 also inhibits MyoD from inducing muscle fiber formation in transfected C3H10T1/2 fibroblasts and represses MyoD from activating gene transcription in myogenic cells. This repression mechanism is complex, however, involving DNA binding competition as well as titration of E-proteins and the presence of a strong repressor domain in the N-terminus of the Mist1 protein. Our results suggest that Mist1 inhibits the activity of the muscle regulatory factors in skeletal muscle and also raise the possibility that Mist1 may serve as a regulator of other bHLH proteins in additional developmental systems. Results Mist1 protein is co-expressed with MyoD in proliferating myoblasts We previously reported the cloning and characterization of a cDNA encoding a novel bHLH protein, Mist1, which exhibits a unique expression pattern during embryonic development and in adult tissues (Lemercier et al., 1997). Although Mist1 is capable of binding to an E-box DNA target sequence as a homodimer, Mist1 protein complexes do not activate gene transcription when tested in a variety of experimental systems (Lemercier et al., 1997). Since the Mist1 gene is expressed in skeletal muscle-forming regions during embryonic development, we set out to investigate the possibility that Mist1 may regulate members of the MyoD MRF family. To address this possibility, a derivative of the C2 myogenic cell line (C2C7) was tested for Mist1 expression. C2 cells express MyoD in undifferentiated myoblasts as well as in differentiated myotubes (Vaidya et al., 1989). Examination of Mist1 protein accumulation in these cells reveals that Mist1 is present at relatively high levels in undifferentiated myoblasts, but as differentiation proceeds over a 3 day period Mist1 protein accumulation becomes reduced, with only very low levels, if any, detected in the myotube cultures (Figure 1). Conversely, as differentiation progresses, high levels of skeletal myosin accumulate. Thus, Mist1 is co-expressed with MyoD in myoblasts but not in differentiated myotubes, suggesting the possibility that Mist1 and MyoD may interact within a common regulatory network in proliferating myogenic stem cell populations. Figure 1.Mist1 protein accumulation decreases as myoblasts differentiate into myotubes. C2C7 myoblasts (Mb) were maintained in an undifferentiated state or induced to differentiate into myotubes (Mt) over a 3 day period (lanes 1–3) as described in Materials and methods. Protein extracts were harvested each day and then subjected to Western analysis using antibodies directed against Mist1 and skeletal myosin. Mist1 protein levels decrease as myoblasts differentiate into myotubes whereas skeletal myosin accumulates only in the myotube cultures. Download figure Download PowerPoint Mist1 forms homodimers and heterodimers with MyoD and E-proteins In order to examine potential interactions between MyoD and Mist1, electrophoretic mobility shift assays were performed using in vitro translated proteins and a 32P-labeled E-box oligonucleotide. As shown in Figure 2, Mist1 homodimers readily interact with the E-box sequence whereas MyoD homodimers do not bind to the DNA. When MyoD and Mist1 are co-incubated with the E-box probe, no new DNA-bound complexes are observed. Interestingly, addition of MyoD to these reactions produces a noticeable reduction in Mist1 homodimer binding, suggesting that MyoD interacts with Mist1, but that the MyoD–Mist1 heterodimer complex does not bind to the target DNA. Mist1 also forms a heterodimer with the E-protein E47, but in this instance the Mist1–E47 complex binds to the E-box site (Figure 2). Figure 2.Mist1 homodimers interact with E-box DNA targets. In vitro translated Mist1, MyoD and E47 proteins were incubated with a 32P-labeled TnI E-box oligonucleotide and subjected to electrophoretic mobility shift assays. Under these conditions, Mist1 forms homodimers which bind efficiently to the E-box site. In contrast, MyoD or E47 alone do not produce DNA-binding complexes. Incubation of Mist1 with MyoD leads to a decrease in Mist1 homodimer binding, suggesting that a Mist1–MyoD complex forms that does not bind to DNA. Similarly, Mist1–E47 heterodimers form but, in this instance, the heterodimer complex interacts with the E-box site. F, free probe. Download figure Download PowerPoint The reduction in Mist1–Mist1 DNA complexes observed in the presence of MyoD prompted us to investigate directly whether Mist1 interacts with this bHLH muscle regulatory factor. For these studies, in vitro protein–protein interaction assays were performed using a histidine-tagged Mist1 protein (His-Mist1) and a variety of 35S-labeled in vitro translated proteins. Nickel beads, with or without His-Mist1, were incubated with the 35S-labeled proteins, and bound proteins were subsequently resolved on an SDS–PAGE gel. When His-Mist1 is incubated with [35S]Mist1, a strong, specific 24 kDa band is detected by autoradiography (Figure 3), confirming that Mist1 homodimers readily form under these conditions. Similarly, Mist1 forms heterodimers with the [35S]MyoD and [35S]E47 proteins (as well as with [35S]MRF4, data not shown). However, no interactions are observed when His-Mist1 and [35S]c-Fos or [35S]MLP are tested. These in vitro studies demonstrate that Mist1 interacts with the bHLH proteins Mist1, MyoD, MRF4 and E47, and that formation of homodimers or heterodimers occurs independently of DNA target sites. In addition, Mist1 protein interactions are limited to bHLH factors since no interactions are observed with c-Fos (leucine zipper protein), MLP (LIM-domain protein) or with the MADS domain protein MEF-2 (data not shown). Figure 3.Mist1 forms both homodimers and heterodimers with MyoD and E47. A His-tagged Mist1 fusion protein (+) was conjugated to nickel agarose beads and mixed with [35S]Mist1, -MyoD, -E47, -c-Fos or -MLP. The bound complexes then were resolved on an SDS–polyacrylamide gel and visualized by autoradiography. As a negative control, the 35S-labeled proteins were incubated with nickel beads alone (−) in the binding reaction. Mist1 interacts efficiently with the bHLH proteins Mist1, MyoD and E47 but not with the control leucine zipper protein c-Fos or with the LIM domain protein MLP. The input [35S]proteins are shown in the last five lanes for reference. Download figure Download PowerPoint Mist1 functions as a potent inhibitor of MyoD activity Given that the Mist1 and MyoD genes exhibit partial overlapping expression patterns (Lemercier et al., 1997) and the proteins directly interact with each other, we next examined whether Mist1 influences MyoD activity when co-expressed in cells. C3H10T1/2 fibroblasts were transfected with MyoD, Mist1 or a combination of expression plasmids, and the number of MyoD-generated myocytes was measured. As expected, MyoD efficiently induces the formation of muscle fibers that express skeletal muscle myosin (Figure 4A). However, when Mist1 is co-transfected with MyoD, fiber formation and myosin expression are severely reduced (Figure 4A). Analysis of cell extracts prepared from these cultures confirmed that high levels of myosin protein accumulate in the MyoD-transfected cells but very little skeletal myosin is detected in cells co-transfected with the MyoD plus Mist1 cDNA constructs (Figure 4B). Importantly, the reduction in fiber formation and myosin accumulation is not due to a Mist1-dependent decrease in MyoD protein levels. Cells transfected with the MyoD expression plasmid, with or without the Mist1 expression plasmid, exhibit similar levels of MyoD (Figure 4B). Thus, Mist1 inhibits MyoD activity without altering the synthesis or stability of the MyoD protein. Figure 4.Mist1 inhibits MyoD-induced myogenesis. (A) C3H10T1/2 cells were transfected with expression plasmids containing MyoD or MyoD plus Mist1 and then induced to differentiate. Immunohistochemistry using a skeletal myosin-specific antibody reveals large differentiated myocytes in MyoD cultures whereas only rare, small myocytes (arrows) are detected in cultures co-expressing MyoD and Mist1. (B) Western analysis of protein extracts from cell cultures comparable with those in (A) reveal that skeletal myosin (MF-20) is detected in the MyoD-expressing cells but not in cells co-expressing MyoD and Mist1. Note that the level of MyoD protein (detected using an anti-HA antibody) is similar in both the MyoD and MyoD plus Mist1 groups. Download figure Download PowerPoint In an effort to identify the mechanism(s) by which Mist1 represses MyoD activity, we also examined the effect Mist1 has on MyoD-dependent transcription. For these studies, MyoD and/or Mist1 expression plasmids were co-transfected into C3H10T1/2 cells with troponin I luciferase (TnI-Luc) or (E-box)4-CAT (containing four copies of an E-box site) reporter genes. As expected, cells expressing MyoD generate high levels of TnI luciferase activity (Figure 5A). However, when MyoD and Mist1 expression plasmids are tested at a 1:1 ratio, TnI-Luc expression is repressed by 85%. A similar Mist1-dependent repression (75% reduction) is obtained when the (E-box)4-CAT reporter gene is tested (Figure 5A). At higher Mist1:MyoD ratios (5:1) (Figure 5B), TnI-Luc expression is completely absent, confirming that Mist1 functions as a potent inhibitor of MyoD activity. Once again, this inhibition is specific since Mist1 does not influence the transcriptional activity of non-bHLH factors nor does Mist1 affect transcription from control SV40-Luc, CMV-LacZ and MEF2-CAT reporter genes (data not shown). Figure 5.Mist1 inhibits MyoD from transcriptionally activating E-box-dependent reporter genes. (A) C3H10T1/2 cells were co-transfected with expression plasmids encoding MyoD or Mist1 and either the (E-box)4-CAT or TnI-Luc reporter genes as described in Materials and methods. No reporter gene expression is detected in the control transfections (without MyoD or Mist1) or in the Mist1 alone group, whereas high levels of reporter gene expression are detected with MyoD. However, when MyoD and Mist1 (1:1) are co-transfected into the cells, reporter gene expression is reduced ∼75–85%. Error bars indicate the standard deviation. (B) Mist1 and the HLH factor Id were co-transfected with MyoD (0.2 μg) into C3H10T1/2 cells as in (A) using increasing concentrations of expression plasmid DNA (0–2 μg). Although both Mist1 and Id inhibit MyoD-induced reporter gene expression, Mist1 is a more potent repressor in this assay system. Download figure Download PowerPoint The repressor function of Mist1 is somewhat analogous to the repressor activity associated with Id, a HLH protein that inhibits the activity of the MRFs by forming DNA-binding inactive complexes of Id–MyoD or Id–E-proteins (Jen et al., 1992). In order to compare the ability of Id and Mist1 to repress MyoD activity, we tested both proteins in MyoD transfection assays. When equal concentrations of Id and MyoD expression plasmids are tested in C3H10T1/2 cells, TnI-Luc activity is reduced ∼50%, and at a 5:1 molar ratio, only 10% of MyoD-dependent activity remains (Figure 5B). In contrast, at these concentrations, Mist1 represses TnI-Luc activity by 85 and 100%, respectively. Although it is difficult to compare directly the repressor activity associated with these two proteins, it is clear that Mist1 inhibits MyoD activity at least as well as Id represses MyoD activity. Additionally, since Mist1 is a bHLH factor with DNA-binding activity, and Id represents a HLH factor lacking DNA-binding activity, the mechanism of Mist1-induced inhibition may be substantially different from the mechanism by which Id represses the MRFs (see below). Mist1 repression partially occurs through a DNA-binding mechanism There are several mechanisms by which Mist1 may inhibit MyoD activity, including: (i) titration of E-proteins from MyoD, resulting in a decreased number of transcriptionally active MyoD–E-protein complexes, (ii) occupancy of essential E-box sites, thereby preventing MyoD–E-proteins from binding to DNA or (iii) dimerization with MyoD, forming a DNA-binding inactive complex. To understand further the contribution of protein dimerization versus DNA binding involved in Mist1 repression, C3H10T1/2 fibroblasts were transfected with expression plasmids encoding MyoD, E47 or a tethered MyoD∼E47 protein (Neuhold and Wold, 1993) in the presence or absence of Mist1. As shown previously, Mist1 efficiently represses MyoD-dependent TnI-Luc expression, whereas the related bHLH factor, Mash1 (Johnson et al., 1990), has little effect on MyoD activity (Figure 6A). Co-transfection of MyoD and E47 into the C3H10T1/2 cells produces the same high levels of TnI-Luc activity as observed with MyoD alone. Interestingly, inclusion of Mist1 in the MyoD + E47 group again generates an 85% reduction in reporter gene expression, suggesting that the additional E47 protein is not sufficient to reverse the Mist1-dependent inhibition of TnI-Luc gene expression. Thus, it seems likely that Mist1 repression is not dependent on a simple E-protein titration model. As a confirmation of this hypothesis, we also co-transfected Mist1 with a tethered MyoD∼E47 construct (Neuhold and Wold, 1993). MyoD∼E47 possesses the same activating properties that are associated with the individual MyoD and E47 proteins, but the tethered MyoD∼E47 protein is highly resistant to Id repression since stable intramolecular MyoD–E47 heterodimer complexes rapidly form (Neuhold and Wold, 1993). Surprisingly, when Mist1 is co-transfected with MyoD∼E47, a large decrease (90%) in reporter gene activity is observed, which also parallels the decrease in MyoD∼E47-induced muscle fiber formation (Figure 6B). Conversely, overexpression of Id has little effect on MyoD∼E47-induced myogenesis (Figure 6B), reinforcing the idea that Mist1 repression does not involve a strict titration of available bHLH factors but primarily involves a DNA-binding mechanism. Figure 6.Mist1 inhibits MyoD activity even in the presence of an excess of E-proteins. (A) C3H10T1/2 cells were co-transfected with the indicated MyoD, E47 and MyoD∼E47 expression plasmids, TnI-Luc and either Mist1 or Mash1. Analysis of TnI-Luc reporter gene activity reveals that Mist1 efficiently represses MyoD and the pre-formed tethered MyoD∼E47 complex from activating the reporter gene. Similarly, Mist1 repression of MyoD is not altered by an excess of E47, suggesting that Mist1 inhibits MyoD activity through a mechanism that is independent of dimerization with E-proteins. The bHLH factor Mash1 serves as a negative control in these studies. Error bars indicate the standard error of the mean. (B) Western analysis of skeletal myosin and MyoD∼E47 protein accumulation in C3H10T1/2 cells transfected with the MyoD∼E47 expression plasmid, with or without the Mist1 or Id expression plasmids. As predicted, Mist1 represses MyoD∼E47 function whereas Id has little affect on the myogenic-inducing activity associated with this pre-formed heterodimer complex. In all cases, the level of the MyoD∼E47 protein, as detected with an anti-MyoD antibody, remains roughly equivalent. Download figure Download PowerPoint To test this latter hypothesis, electrophoretic mobility shift assays were performed using a 32P-labeled E-box probe and in vitro translated proteins. Co-incubation of MyoD with E47 generates the predicted MyoD–E47 heterodimer complex that binds efficiently to the E-box site (Figure 7A). Incubation of Mist1 with E47 results in two distinct DNA-binding complexes, a Mist1–Mist1 homodimer and a Mist1–E47 heterodimer. When increasing amounts of Mist1 are added to the MyoD + E47 reactions, a progressive reduction in the formation of DNA-bound MyoD–E47 complexes occurs that coincides with an increase in the formation of DNA-bound Mist1–E47 heterodimers and Mist1–Mist1 homodimers. These results suggest that Mist1 is able to disrupt a transcriptionally active complex, such as MyoD–E47, to form two additional DNA-binding complexes (Mist1–Mist1, Mist1–E47) that, none the less, remain transcriptionally inactive on muscle genes. Interestingly, Mist1 has no effect on the DNA-binding activity associated with the MyoD∼E47 tethered protein (Figure 7B), even though MyoD∼E47 transcriptional activity is severely repressed by Mist1 (Figure 6A). Figure 7.Mist1 inhibits MyoD–E47 complexes from interacting with E-box targets. (A) In vitro translated Mist1, MyoD and E47 proteins were incubated with a 32P-labeled TnI E-box oligonucleotide and subjected to electrophoretic mobility shift assays as described in Figure 2. MyoD and E47 form heterodimers that interact with the E-box DNA site. Incubation with increasing concentrations of Mist1 inhibits the formation of a MyoD–E47–DNA complex. At the same time, a new E47–Mist1 complex is detected as well as an increase in Mist1–Mist1 homodimer binding. Each of the dimer–DNA complexes is indicated. DNA–protein complexes containing a truncated version of E47 (due to an internal translation initiation site) are indicated by (*). (B) Similar electrophoretic mobility shift assays as in (A) except that the pre-formed MyoD∼E47 complex was competed with increasing concentrations of Mist1. In this instance, Mist1 fails to block MyoD∼E47 DNA binding, even though Mist1 homodimers readily interact with the E-box site. F, free probe. Download figure Download PowerPoint The experiments described above strongly support the idea that Mist1 represses skeletal muscle differentiation primarily through occupancy of E-box sites and not through titration of specific bHLH factors in the cell. In order to test this hypothesis, we generated a mutant Mist1 cDNA (Figure 8A) that contains a substitution of the basic domain amino acids RER with GGG (Mist1mut basic) (see Materials and methods for details). We reasoned that an altered protein which retains dimerization but not DNA-binding activity would no longer function as a repressor if Mist1 repression occurs primarily through DNA interactions. As predicted, both the wild-type Mist1 and the Mist1mut basic proteins efficiently form homodimers when tested in an in vitro binding assay (Figure 8B). However, Mist1mut basic homodimers fail to bind to E-box sites whereas the wild-type Mist1 protein readily forms a Mist1–Mist1–DNA complex (Figure 8C). Figure 8.A basic domain mutant of Mist1 does not inhibit myogenesis. (A) The basic helix–loop–helix amino acid sequence of the wild-type (Mist1) and basic domain Mist1 (Mist1mut basic) proteins. The altered amino acids (RER→GGG) are indicated. (B) Protein–protein interaction assay as described in Figure 3. Both Mist1 and Mist1mut basic efficiently form homodimers in solution. (C) Electrophoretic mobility shift assay as described in Figure 2. In this instance, Mist1 homodimers interact with the targeted E-box oligonucleotide whereas Mist1mut basic homodimers are unable to bind to the DNA (B, bound complexes, F, free oligonucleotide probe). (D) C3H10T1/2 cells were co-transfected with expression plasmids encoding MyoD, Mist1 or Mist1mut basic and the TnI-Luc reporter gene as described in Figure 5. Mist1 efficiently inhibits MyoD from activating the TnI-Luc gene whereas Mist1mut basic has no effect. (E) Similar in vivo assays were performed in the myogenic cell line C2C7. In this instance, C2C7 myoblasts were transfected with 1.5 μg of TnI-Luc alone (control) or co-transfected with 0.5 μg of pcDNA3-Mist1 (Mist1) or 0.5 μg of pcDNA3–Mist1mut basic. The cells were induced to differentiate for 72 h and then assayed for luciferase activity. Whereas Mist1 efficiently represses the TnI–Luc reporter, the Mist1mut basic protein has no effect on C2C7 terminal differentiation. Error bars in (D) and (E) indicate the standard error of the mean. Download figure Download PowerPoint In order to test the new Mist1mut basic protein in vivo, we co-transfected Mist1 expression plasmids along with a MyoD expression plasmid and the TnI-Luc reporter gene into C3H10T1/2 fibroblasts. As shown in Figure 8D, wild-type Mist1 inhibits MyoD-induced activation of the TnI-Luc reporter whereas Mist1mut basic has no effect, despite the fact that the Mist1mut basic protein stably accumulates in the cell and translocates to the nucleus (data not shown). As a further test for in vivo activity, we examined the ability of Mist1 to block the activity of the endogenous MyoD protein in the myogenic cell line C2C7. As predicted, C2C7 cells expressing the wild-type Mist1 protein fail to activate expression of the TnI-Luc reporter gene (Figure 8E). In contrast, the Mist1mut basic protein has no effect on C2C7 terminal differentiation, even though Mist1mut basic is capable of dimerizing with MyoD and E47 (unpublished data). Taken together, these data suggest that Mist1 primarily inhibits MyoD in proliferating myoblasts through a DNA-binding mechanism and that the contribution of dimerization with endogenous bHLH proteins is not a significant factor given that the Mist1mut basic protein, which retains dimerization function, no longer represses MyoD in these myogenic cells. Mist1 possesses an N-terminus repressor domain The ability of Mist1 to