RESEARCH ARTICLE 513 α-Skeletal actin induces a subset of muscle genes independently of muscle differentiation and withdrawal from the cell cycle Peter W. Gunning1,3,4, Vicki Ferguson1,3, Karen J. Brennan2,* and Edna C. Hardeman2,‡ 1Cell Biology Unit and 2Muscle Development Unit, Children’s Medical Research Institute, Locked Bag 23, Wentworthville, NSW, 2145, Australia 3Oncology Research Unit, The New Children’s Hospital, PO Box 3515, Parramatta, NSW, 2124, Australia 4Department of Paediatrics and Child Health, University of Sydney, Sydney, NSW 2006, Australia *Present address: EMBL Heidelberg, Meyerhofstraße 1, Postfach 102209, D-69012 Heidelberg, Germany ‡Author for correspondence (e-mail: [email protected]) Accepted 14 November 2000 Journal of Cell Science 114, 513-524 © The Company of Biologists Ltd SUMMARY Muscle differentiation is characterized by the induction of characteristic of muscle differentiation were induced. genes encoding contractile structural proteins and the Stable transfectants displayed a substantial reduction repression of nonmuscle isoforms from these gene families. in cell surface area and in the levels of nonmuscle We have examined the importance of this regulated order tropomyosins and β-actin, consistent with a relationship of gene expression by expressing the two sarcomeric muscle between the composition of the actin cytoskeleton and cell actins characteristic of the differentiated state, i.e. α- surface area. The transfectants displayed normal cell cycle skeletal and α-cardiac actin, in C2 mouse myoblasts. progression. We propose that α-skeletal actin can activate Precocious accumulation of transcripts and proteins for a regulatory pathway linking a subset of muscle genes that a group of differentiation-specific genes was elicited by operates independently of normal differentiation and α-skeletal actin only: four muscle tropomyosins, two withdrawal from the cell cycle. muscle actins, desmin and MyoD. The nonmuscle isoforms of tropomyosin and actin characteristic of the undifferentiated state continued to be expressed, and no Key words: Muscle, Actin, Differentiation, Contractile protein, myosin heavy or light chain or troponin transcripts Isoform INTRODUCTION Munsterberg, 1994; Olson and Klein, 1994). The four members of this family, MyoD, Myf5, myogenin and MRF4, have At the onset of myogenesis, mononucleated myoblasts been shown to be sufficient to induce skeletal muscle gene withdraw from the cell cycle and fuse to form multinucleated transcription when ectopically expressed in a variety of cell myotubes. This profound change in cell shape is accompanied types. MyoD specifically has been shown to be involved in by a downregulation of the nonmuscle isoforms of the actin, myogenic differentiation in at least a two-step process. tropomyosin (Tm) and myosin genes and an induction of the Initially, MyoD causes cycling myoblasts to withdraw from the corresponding skeletal muscle isoforms (Devlin and Emerson, cell cycle without initiating differentiation (Crescenzi et al., 1978; Buckingham and Minty, 1983; Wade and Kedes, 1989). 1990; Sorrentino et al., 1990). Its subsequent activation of the The induction of these contractile proteins drives the assembly cyclin-dependent kinase inhibitor p21 coordinates terminal of the sarcomeric structures that allow contraction of the withdrawal from the cell cycle, resulting in differentiation muscle cell. The contractile proteins accumulate with a fixed (Halevy et al., 1995; Parker et al., 1995; Guo et al., 1995). The stoichiometry that reflects their physical relationships in the current model for myogenesis (Zhang et al., 1995), based on sarcomere (Devlin and Emerson, 1978). Despite the extensive gene targeting of the bHLH factors in mice (Rudnicki et al., isoform switching in each contractile protein gene family that 1992; Braun et al., 1992; Rudnicki et al., 1993; Hasty et al., accompanies muscle maturation (Sutherland et al., 1991; Esser 1993; Nabeshima et al., 1993; Venuti et al., 1995), indicates et al., 1993), the relative total output between gene families that MyoD and Myf5 establish myoblasts and activate remains similar at different stages of maturation (Wade et al., myogenin. Myogenin in turn is involved in myotube formation 1990). This suggests that regulation of output between gene and activation of muscle structural genes. Therefore, data from families may contribute to the maintenance of stoichiometry. both cell culture studies and transgenic mice suggests that the Such a mechanism, however, may require communication bHLH factors function prior to the expression of muscle pathways to directly link expression between the different gene structural protein genes. families. These studies have provided a hierarchical view of Our understanding of muscle differentiation has been myogenesis in which the master regulators activate muscle dominated by the discovery of the myogenic family of genes, but do not respond to, or monitor, the function of these bHLH transcription factors (Weintraub, 1993; Lassar and muscle products. Studies of structural protein gene expression 514 JOURNAL OF CELL SCIENCE 114 (3) in a variety of systems including muscle provide convincing undifferentiated myoblasts. Recent evidence for the presence evidence that feedback regulatory pathways operate in these of actin in the nucleus and the potential roles it plays in nuclear gene families. Gene transfection studies have shown that functions suggests that specific actin isoforms can influence fibroblasts and myoblasts usually regulate nonmuscle actin gene expression (Rando et al., 2000). Alternatively, synthesis (Leavitt et al., 1987a; Ng et al., 1988) and mRNA transcriptional regulation by actin isoforms could occur via the levels (Lloyd et al., 1992) in order to maintain a constant actin G-actin pool; modulations in G-actin regulate a subset of SRF pool size. The nonmuscle actin monomer pool size has been target genes (Sotiropoulos et al., 1999). directly implicated as a regulator of actin gene expression (Bershadsky et al., 1995). Recently, the G-actin level was shown to control serum response factor (SRF), a MATERIALS AND METHODS transcriptional regulator of nonmuscle actins, serum-inducible and muscle-specific genes (Sotiropoloulos et al., 1999). A DNA constructs α naturally occurring mutation in the α-cardiac actin (αca-actin) The human sk-actin gene was subcloned as a 9.5 kb HindIII fragment gene in some mouse strains reduces output from this locus in into pBR322 (Taylor et al., 1988). This DNA fragment contains 2 kb ′ ′ ′ the heart, which results in a compensating upregulation of the of 5 flanking and 4 kb of 3 flanking sequences. The 2 kb of 5 α-skeletal actin (α -actin) gene (Garner et al., 1986). In flanking region to the termination codon was isolated as a HindIII- sk XbaI fragment and ligated to an XbaI-BamHI fragment of the human transgenic studies, ectopic expression of myosin light chain γ-actin gene containing the 3′ half of the 3′UTR plus 5 kb of 3′ flank (MLC) 2 fast-elicited a reduction in transcript accumulation (Erba et al., 1988). The ligation product was inserted between the from the endogenous alleles (Shani et al., 1988). Forced HindIII and BamHI sites of pcDVI (Okayama and Berg, 1983). The expression of βTm in the heart resulted in the downregulation resulting construct, αsk-γ3′, is shown in Fig. 1. The HindIII-XbaI of αTm protein (Muthuchamy et al., 1995). Finally, the fragment of the αsk-actin gene was also cloned between the HindIII- finding that the relative mRNA output between contractile XbaI sites of pGEM-3, re-isolated as a HindIII-BamHI fragment and protein gene families is similar at different stages of cloned between the HindIII-BamHI sites of pcDVI. This resulted in maturation, independently of isoform switching, suggests the the placement of an SV40 3′UTR plus transcription termination signal α α ′ existence of some form of communication between these adjacent to the sk-actin termination codon as shown for sk-SV3 in genes (Wade et al., 1990). Therefore, it is conceivable that Fig. 1. An in-frame deletion between amino acids (aa) 22-367 was created by digestion of α -SV3′ with NaeI followed by religation to feedback regulatory pathways are a feature of structural sk produce αsk-SV3′ (Fig. 1). The αsk-actin promoter driving the CAT protein gene family expression. reporter gene was as described by Muscat and Kedes (Muscat and Actin gene transfections have also suggested the existence Kedes, 1987) and is shown in Fig. 1 (αsk-CAT). The human αca-actin of regulatory pathways between actin genes and genes gene was isolated as an EcoRI-BglII fragment containing 5 kb of 5′ associated with actin function. A mutant β-actin gene has been flanking and 1.5 kb of 3′ flanking sequence (Engel et al., 1982) and found to cause downregulation of synthesis of Tm isoforms cloned into the EcoRI-BamHI region of pSV2-neo (Southern and Tm1, 2 and 3 in fibroblasts (Leavitt et al., 1987b) and reduced Berg, 1982) (αca in Fig. 1). protein and mRNA levels of Tm2 and 3 in myoblasts Cell culture and transfection (Schevzov et al., 1993). In both studies, the Tm5 isoform was unaffected, demonstrating a clear isoform specificity of this C2 cells originally isolated by Dr D. Yaffe (Yaffe and Saxel, 1977) and subcloned in the laboratory of Dr H. Blau (Blau et al., 1983) were response. The normal γ-actin gene produced a similar result in β grown in DMEM medium (Gibco Laboratories, Grand Island, NY, myoblasts (Schevzov et al., 1993). Both the mutant -actin and USA) supplemented with 20% FCS (Commonwealth Serum Labs, normal γ-actin genes also impact on the protein and mRNA Melbourne, Australia) and 0.5% chicken embryo extract (Flow levels of vinculin and talin, two proteins involved in anchorage Laboratories Australia Pty Ltd., North Ryde, Australia). of actin filaments to focal contacts (Schevzov et al., 1995). DNA transfection and isolation of transfected clones were These studies are compatible with the previous suggestion that performed as described by Graham and van der Eb (Graham and van some muscle structural gene products may influence der Eb, 1973).