Oncogene (1997) 14, 1171 ± 1184 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 MyoD prevents cyclinA/cdk2 containing E2F complexes formation in terminally dierentiated myocytes Pier Lorenzo Puri1,3, Clara Balsano2, Vito Lelio Burgio3, Paolo Chirillo1,3, Gioacchino Natoli1,3, Letizia Ricci4, Elisabetta Mattei4, Adolf Graessmann5 and Massimo Levrero1 1Fondazione A Cesalpino and 3I Clinica Medica, University of Rome `La Sapienza', Viale del Policlinico 155, 00161 Roma, Italy; 2Dipartimento di Medicina Interna, University of L'Aquila; 4Istituto di Tecnologie Biomediche CNR, Roma; 5Institute fur Molekularbiologie und Biochemie der Freien Universitat Berlin, 14195 Berlin, Germany Withdrawal from the cell cycle of dierentiating for myogenic dierentiation is the irreversible with- myocytes is regulated by the myogenic basic helix ± drawal from the cell cycle (Nadal-Ginard, 1978; loop ± helix (bHLH) protein MyoD and the pocket Stockdale and Holtzer, 1961). Master regulators of proteins pRb, p107 and pRb2/p130. Downstream muscle cell commitment and dierentiation are the eectors of `pocket' proteins are the components of the members of the MyoD family of myogenic basic E2F family of transcription factors, which regulate the Helix ± Loop ± Helix (bHLH) proteins (Lassar and G1/S-phase transition. We analysed by EMSA the Munstemberg, 1994; Olson and Klein, 1994), the composition of E2F complexes in cycling, quiescent retinoblastoma protein (pRb) (Gu et al., 1993) and undierentiated and dierentiated C2C12 skeletal muscle its related proteins p107 and pRb2/p130 (Mayol et al., cells. An E2F complex containing mainly E2F4 and 1993; Schneider et al., 1994; Shin et al., 1995). pRb2/p130 (E2F-G0/G1 complex) appears when DNA In conditions permissive for dierentiation, pRb synthesis arrests, replacing the cyclinA/cdk2 containing binds MyoD via a region overlapping the `pocket' E2F complex of proliferating myoblasts (E2F-G1/S region (Gu et al., 1993), a conserved domain, also complex). Serum stimulation reinduces DNA synthesis present in the pRb-related proteins p107 and pRb2/ and the re-appearance of E2F-G1/S complexes in p130 and required for binding with viral transforming quiescent myoblasts but not in dierentiated C2C12 proteins and the members of the E2F/DP family myotubes. In dierentiating C2C12 cells, E2F complexes (DeCaprio et al., 1988; Whyte et al., 1989; Dyson et switch and DNA synthesis in response to serum are al., 1990; LaThangue, 1994; Wolf et al., 1995). pRb also prevented when MyoD DNA binding activity and the binds and inactivates the ubiquitously expressed HLH cdks inhibitor MyoD downstream eector p21 are protein Id2 (Iavarone et al., 1994), which upon high induced. Thus, during myogenic dierentiation, forma- mitogens concentration sequestrates the E proteins and tion of E2F4 and pRb2/p130 containing complexes is an inhibits MyoD binding to the E-box (Benezra et al., early event, but not enough on its own to prevent the re- 1990). Like pRb, also p107 interacts with MyoD in a activation of DNA synthesis. Using a subclone of similar fashion, suggesting that redundance might exist C3H10T1/2 mouse ®broblasts stably expressing Estro- to ensure the dierentiation program under appropriate gen Receptor-MyoD (ER-MyoD) chimerae, we found conditions. However, pRb de®cient cells ± where p107 that estrogen directed MyoD activation prevents the re- myogenic functions are recruited, undergo phenotypic association of cyclinA/cdk2 to the E2F4 containing myogenic dierentiation, although retaining their ability complex following serum stimulation and this correlates to re-enter into the cell cycle in response to serum with suppression of E2F activity and the inability of cells stimulation (Schneider et al., 1994). This suggests that to re-enter the cell cycle. Our data indicate that, in pRb is critical to prevent DNA synthesis in differ- dierentiating myocytes, one mechanism through which entiated myotubes in response to mitogens. MyoD induces permanent cell cycle arrest involves p21 pRb's and the other `pocket' proteins role in the upregulation and suppression of the proliferation-asso- permanent exit from the cell cycle and terminal ciated cdks-containing E2F complexes formation. dierentiation is also suggested by the evidence that the products of the viral oncogenes, i.e. polyomavirus Keywords: MyoD; E2F; pRb2/p130; p21; cell cycle large T antigen, adenovirus E1A and SV40 large T antigens are all able to prevent dierentiation of both primary myoblasts and myogenic cell lines (Graess- mann et al., 1973; Webster et al., 1988; Braun et al., Introduction 1992; Maione et al., 1992, 1994; Caruso et al., 1993) and to reinduce DNA synthesis in C2C12 myotubes In vitro skeletal muscle dierentiation consists in (Endo et al., 1986; Cardoso et al., 1993; Crescenzi et mononucleated myoblasts fusion into multinucleated al., 1995). myotubes (phenotypical dierentiation) which express pRb and pRb-related proteins bind and negatively a coordinate set of muscle-speci®c genes (biochemical regulate the activity of E2F (Chellapan et al., 1991; dierentiation) (Olson and Klein, 1994). Prerequisite Hiebert et al., 1992; Weintraub et al., 1992; Cobrinik et al., 1993; Helin et al., 1993a; Zamanian and LaThangue, 1993; Tommasi and Pfeier, 1995). E2F Correspondence: PL Puri is a family of heterodimeric transcription factors made- Received 31 July 1996; revised 8 November 1996; accepted 8 up of E2F-like and DP-like subunits (LaThangue, November 1996 1994). Many genes involved in DNA replication and MyoD-mediated repression of E2Finmyocytes PL Puri et al 1172 cell proliferation that are sequentially activated during Results the cell cycle ± i.e. c-myc,c-myb, cdc2, thymidine kinase (tk), dihydrofolate reductase (DHFR) ± contain E2F complexes in cycling, G /G arrested and E2F binding sites in their regulatory region (LaThan- 0 1 dierentiated C2C12 cells gue, 1994; Nevins, 1992). At least seven genes encoding for components of the E2F family have been cloned C2C12 skeletal muscle cells were used to study the E2F (E2F from 1 to 5, DP1 and DP2) (Helin et al., 1992, complexes composition in dierent culture conditions. 1993b; Kaelin et al., 1992; Shan et al., 1992; Ivey- In high serum (20% FCS) C2C12 myoblasts actively Hoyle et al., 1993; Beijersbergen et al., 1994; Ginsberg proliferate, as demonstrated by high levels of 5-Bromo- et al., 1994; Li et al., 1994; Hijmans et al., 1995; Zhang 2 deoxy-Uridine (BrdU) incorporation (Figure 1A). and Chellapan, 1995). Multiple evidences indicate that Full dierentiation into mature multinucleated myo- E2F1 plays a critical role in regulating G1/S phase tubes is obtained by culturing con¯uent C2C12 cells in transition (Johnson et al., 1993; Sala et al., 1994; low serum (2% FCS; DM, dierentiation medium). Kowalik et al., 1995) and deregulated expression of C2C12 myotubes do not incorporate BrdU (Figure 1F) E2F1 leads to apoptosis in certain cell types (Shan and and display several muscle-speci®c markers, such as Lee, 1994; Qin et al., 1994). In muscle cells, E2F1 myosin heavy chain (MHC) (Figure 1C). When C2C12 overexpression inhibits myogenic dierentiation (Wang cells are kept in the absence of serum (0.1% FCS) et al., 1995). In association with DP1, E2F1 binds before con¯uence is reached, quiescent undierentiated underphosphorylated pRb and forms a complex which mononucleated myoblasts can be observed between 24 retains its DNA-binding property but is not able to and 48 h after starvation. These quiescent myoblasts activate transcription (Weintraub et al., 1992; Helin et do not incorporate BrdU (Figure 1E) and do not al., 1993a). In many cell types the pRb-E2F complex is express muscle speci®c markers (Figure 1B), although a minor component of the G0 E2F-DNA binding retaining the ability to proliferate in response to activity (Chittenden et al., 1993; Dou et al., 1994; mitogens (Puri et al., 1995). Schwarz et al., 1993). In unstimulated human T cells For gel retardation assays, cell extracts were and in growth arrested murine ®broblasts an E2F prepared from noncon¯uent C2C12 cells cultured in complex, formed by E2F4 and pRb2/p130, has been 20% serum enriched medium (cycling myoblasts), from detected (Cobrinik et al., 1993; Vairo et al., 1995). This noncon¯uent C2C12 cells cultured in 0.1% serum complex can play a role in inducing and/or maintaining (quiescent myoblasts) and from C2C12 dierentiated cellular quiescence (Claudio et al., 1994; Vairo et al., myotubes. We detected four E2F complexes whose 1995). Functional studies have demonstrated that formation is dierentially regulated along the cell cycle pRb2/p130 suppress E2F4 but not E2F1 mediated and during dierentiation (Figure 2a). The binding trans-activation and that E2F4 coexpression is able to speci®city of each E2F complex was con®rmed by overcome pRb2/p130 mediated G1 arrest more competition experiments with excess unlabelled wild eciently than Rb-induced G1 blockade (Vairo et al., type and mutant E2F speci®c oligonucleotides (Figure 1995). E2F4 also forms heterodimers with members of 2b) and unrelated unlabelled DNA (data not shown). the DP family and p107 (Beijersbergen et al., 1994; The slower migrating band (E2F-G1/S complex), Ginsberg et al., 1994), an association occurring in late detected in cycling myoblasts, disappears when cells G1 or at the G1/S boundary (Schwarz et al., 1993). In enter G0 (C2C12 quiescent) being replaced by a faster addition, both p107 and pRb2/p130 have been migrating band (the E2F-G0/G1 complex) also found in described to associate with cyclin E and A and their dierentiated C2C12 myotubes. Two additional faster dependent kinase cdk2 to form E2F-containing migrating bands were observed in all cell cycle phases, complexes in late G1 and S-phase, respectively but were more evident in cycling myoblasts then in (Devoto et al., 1992; Lees et al., 1992; Pagano et al., quiescent cells. Fully dierentiated myotubes display 1992; Hannon et al., 1993; Li et al., 1993). However, faster migrating bands similar in abundance to those the functional consequences of these multimeric detected in quiescent cells.