Multiple Changes in E2F Function and Regulation Occur upon Muscle Differentiation

Abstract

We have examined regulation of the E2F transcription factor during differentiation of muscle cells. E2F regulates many genes involved in growth control and is also the target of regulation by diverse cellular signals, including the RB family of growth suppressors (e.g., the retinoblastoma protein [RB], p107, and p130). The following aspects of E2F function and regulation during muscle differentiation were investigated: (i) proteinprotein interactions, (ii) protein levels, (iii) phosphorylation of the E2F protein, and (iv) transcriptional activity. A distinct E2F complex was present in differentiated cells but not in undifferentiated cells. The p130 protein was a prominent component of the E2F complex associated with differentiation. In contrast, in undifferentiated cells, the p107 protein was the prominent component in one of three E2F complexes. In addition, use of a differentiation-defective muscle line provided genetic and biochemical evidence that quiescence and differentiation are separable events. Exclusive formation of the E2F-p130 complex did not occur in this differentiation-defective line; however, E2F complexes diagnostic of quiescence were readily apparent. Thus, sole formation of the E2F-p130 complex is a necessary event in terminal differentiation. Other changes in E2F function and regulation upon differentiation include decreased phosphorylation and increased repression by E2F. These observations suggest that the regulation of E2F function during terminal differentiation may proceed through differential interaction within the RB family and/or phosphorylation.In many cells, the trigger event in differentiation is withdrawal from the cell cycle with subsequent distinct morphological transitions. Muscle cells are an excellent system for examining molecular mechanisms of differentiation because they exhibit both permanent cell cycle withdrawal and a distinctive phenotype. Differentiation involves a transition from myoblasts to myotubes. Myoblasts are undifferentiated cells and are characterized by rapidly growing, mononucleated cells. In contrast, differentiated cells, or myotubes, are multinucleated and tubular. Upon differentiation, myotubes also express several muscle-specific markers. Because differentiated muscle cells (myotubes) are permanently withdrawn from the cell cycle, cellular mechanisms for the initiation and maintenance of the differentiated state must also exist (5).Studies with viral oncogenes have indicated a critical role for the retinoblastoma family of growth suppressors (the retinoblastoma protein [RB], p107, and p130) in the differentiation pathway. The expression of E1A and polyomavirus large T antigen may block muscle differentiation (7,49,66). It is well established that these two divergent viral oncogenes can bind the RB family (69). Importantly, when the binding site for RB family members was mutated, both E1A and polyomavirus large T antigen mutants no longer blocked differentiation (7, 49). These studies suggest that RB family members function in the control of differenti...

pRb and p107 regulate E2F activity during lens fiber cell differentiation

“…While the binding partner of p107 in these complexes is not yet known, E2F-5 is a likely candidate, considering its structural similarity to E2F-4 (Hijmans et al, 1995;Itoh et al, 1995;Sardet et al, 1995). Although di erentiating lens ®ber cells contain a prominent p107/E2F complex, we found no evidence for a p130/E2F complex, using techniques that were su cient to detect such a complex in di erentiating C2C12 muscle cells (Shin et al, 1995). This contrasts with results from a variety of other cell types, which have established a strong correlation between p130/ E2F complex formation and cell cycle withdrawal.…”

“…Since formation of a p130/E2F protein complex is often associated with cell cycle arrest (Halevy et al, 1995;Corbeil et al, 1995;Vairo et al, 1995;Jiang et al, 1994;Shin et al, 1995), the failure of the p130 antibody to a ect any of the complexes formed by lens ®ber extracts was unexpected. We therefore tested the ability of this antibody to interfere with the formation of a previously identi®ed p130/E2F complex in di erentiated C2C12 myotubes.…”

Differential expression and functionally co-operative roles for the retinoblastoma family of proteins in epidermal differentiation

“…An increase in pRb during muscle and neuronal cell di erentiation has been reported (Shin et al, 1995;Corbeil et al, 1995). Contradictory results have been obtained concerning p107 and p130 expression, probably due in part to the Figure 4 Di erent members of the Retinoblastoma family of proteins co-operate to trigger speci®c stages of epidermal di erentiation.…”

“…p107 increases during neuronal (Corbeil et al, 1995) and erythroid di erentiation (Richon and Venta-Perez, 1996), and also during C2C12 myoblast cell di erentiation (Thorburn et al, 1993), but appears to decrease during L6 myoblast di erentiation (Kiess et al, 1995). Similarly, p130 has been reported to increase during C2 and L6 muscle di erentiation (Shin et al, 1995, Kiess et al, 1995, whilst its level remains constant during C2C12 muscle cell and neuronal di erentiation (Corbeil et al, 1995). However, to our knowledge, a sequential, coordinated expression of these three proteins as we have observed during HaCaT keratinocyte di erentiation has not previously been reported in other systems, suggesting that epidermal di erentiation may display speci®c features not present in muscle or neuron cell di erentiation.…”

“…Moreover, during mouse development pRb is expressed in the differentiating compartments of a number of tissues (Szekely et al, 1992). Finally, pRb, and its relatives p107 and p130, have also been associated with the processes of myogenesis and muscle di erentiation (Gu et al, 1993;Schneider et al, 1994;Shin et al, 1995;Throrburn et al, 1993;Kiess et al, 1995).…”