MyoD prevents cyclinA/cdk2 containing E2F complexes formation in terminally differentiated myocytes

Abstract

Withdrawal from the cell cycle of dierentiating myocytes is regulated by the myogenic basic helix ± loop ± helix (bHLH) protein MyoD and the pocket proteins pRb, p107 and pRb2/p130. Downstream eectors of`pocket' proteins are the components of the E2F family of transcription factors, which regulate the G 1 /S-phase transition. We analysed by EMSA the composition of E2F complexes in cycling, quiescent undierentiated and dierentiated C2C12 skeletal muscle cells. An E2F complex containing mainly E2F4 and pRb2/p130 (E2F-G 0 /G 1 complex) appears when DNA synthesis arrests, replacing the cyclinA/cdk2 containing E2F complex of proliferating myoblasts (E2F-G 1 /S complex). Serum stimulation reinduces DNA synthesis and the re-appearance of E2F-G 1 /S complexes in quiescent myoblasts but not in dierentiated C2C12 myotubes. In dierentiating C2C12 cells, E2F complexes switch and DNA synthesis in response to serum are prevented when MyoD DNA binding activity and the cdks inhibitor MyoD downstream eector p21 are induced. Thus, during myogenic dierentiation, formation of E2F4 and pRb2/p130 containing complexes is an early event, but not enough on its own to prevent the reactivation of DNA synthesis. Using a subclone of C3H10T1/2 mouse ®broblasts stably expressing Estrogen Receptor-MyoD (ER-MyoD) chimerae, we found that estrogen directed MyoD activation prevents the reassociation of cyclinA/cdk2 to the E2F4 containing complex following serum stimulation and this correlates with suppression of E2F activity and the inability of cells to re-enter the cell cycle. Our data indicate that, in dierentiating myocytes, one mechanism through which MyoD induces permanent cell cycle arrest involves p21 upregulation and suppression of the proliferation-associated cdks-containing E2F complexes formation.

E2F activates late-G1 events but cannot replace E1A in inducing S phase in terminally differentiated skeletal muscle cells

“…None of these infections elicited DNA synthesis, while the E1A-carrying dl520 adenovirus reactivated the myotubes in a dosedependent fashion (multiplicities of infection (MOIs) from 100 ± 1000 induced DNA synthesis in 20 ± 595% of the myotubes). Since the prevailing E2F form in myotubes is E2F-4 (Puri et al, 1997), it was conceivable that E1A reactivates myotubes by releasing this speci®c protein which might exert activities possibly not shared by its relatives. To test this possibility, we transfected into satellite-cell derived myotubes a plasmid encoding an E2F-4 chimeric protein carrying a nuclear localization signal (E2F-4-NLS) (Muller et al, 1997).…”

E2F4 (E2F transcription factor 4, p107/p130-binding)

“…Likewise, overexpression of E2F4 in mice epidermis under the K5 promoter leads to E2F4 expression in the nucleus of cycling keratinocytes in the basal cell layer and the hair follicle resulting in hyperplasia and increased tumor formation in a mouse skin model of multistage carcinogenesis (Wang et al, 2000). Moreover, endogenous E2F4 is observed in the nucleus of many differentiated cells including ciliated epithelial cells (Danielian et al, 2007), myotubes (Puri et al, 1997;Puri et al, 1998) and neurons (Persengiev et al, 1999). Overall, these results suggest that E2F4 can act as either an activator or an inhibitor of transcription, proliferation and differentiation.…”

“…Of note, Apostolova et al suggested that E2F4 may also travel to the nucleus on its own (Apostolova et al, 2002). A number of studies have highlighted the importance of regulating the subcellular localization of E2F4 (Magae et al, 1996;Lindeman et al, 1997;Muller et al, 1997;Verona et al, 1997;Puri et al, 1997;Puri et al, 1998;Gill and Hamel, 2000;Deschenes et al, 2004). In immortalized fibroblasts and certain cancer cells, E2F4 is expressed in the nucleus of quiescent cells and as cells progress through G1 and enter the S phase, E2F4 translocates to the cytoplasm (Lindeman et al, 1997;Muller et al, 1997;Verona et al, 1997).…”

“…However, other publications reported an ubiquitous expression of E2F4 (Gill and Hamel, 2000;D'Souza et al, 2001). Furthermore, it was suggested that E2F4 accounts for the vast majority of E2F cellular activity (Moberg et al, 1996;Puri et al, 1997). Of note, E2F4 expression changes according to cellular status.…”

Differences in DNA binding properties between E2F1 and E2F4 specify repression of the Mcl-1 promoter

“…In contrast, the growth-restraining E2Fs do not possess this extended N-terminal region and are constitutively expressed throughout the cell cycle (He et al, 2000;Moberg et al, 1996;Sardet et al, 1995). Furthermore, these growth-restraining E2Fs (in particular, E2F4) have been demonstrated to promote di erentiation in a variety of systems, whereas the growth-promoting E2Fs cannot promote di erentiation (Paramio et al, 2000;Persengiev et al, 1999;Puri et al, 1997).…”