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MyoD prevents cyclinA/cdk2 containing E2F complexes formation in terminally differentiated myocytes
P L Puri1, C Balsano, V L Burgio
1Fondazione A Cesalpino and I Clinica Medica, University of Rome La Sapienza, Roma, Italy.
Abstract:
Withdrawal from the cell cycle of differentiating myocytes is regulated by the myogenic basic helix-loop-helix (bHLH) protein MyoD and the pocket proteins pRb, p107 and pRb2/p130. Downstream effectors of 'pocket' proteins are the components of the E2F family of transcription factors, which regulate the G1/S-phase transition. We analysed by EMSA the composition of E2F complexes in cycling, quiescent undifferentiated and differentiated C2C12 skeletal muscle cells. An E2F complex containing mainly E2F4 and pRb2/p130 (E2F-G0/G1 complex) appears when DNA synthesis arrests, replacing the cyclinA/cdk2 containing E2F complex of proliferating myoblasts (E2F-G1/S complex). Serum stimulation reinduces DNA synthesis and the re-appearance of E2F-G1/S complexes in quiescent myoblasts but not in differentiated C2C12 myotubes. In differentiating 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 effector p21 are induced. Thus, during myogenic differentiation, 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 fibroblasts 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 differentiating 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.
Insights
Myogenic differentiation involves MyoD and p21, which suppress cell cycle reentry by altering E2F complexes. This prevents proliferating myoblasts from re-entering the cell cycle after differentiation.
Area of Science:
- Cell Biology
- Molecular Biology
- Muscle Development
Background:
- Cell cycle withdrawal during myocyte differentiation is controlled by MyoD and pocket proteins.
- Pocket proteins regulate E2F transcription factors, which control the G1/S-phase transition.
Purpose of the Study:
- To analyze E2F complex composition during C2C12 skeletal muscle cell differentiation.
- To understand the role of MyoD and p21 in cell cycle arrest during myogenesis.
Main Methods:
- Electrophoretic mobility shift assay (EMSA) to study E2F complexes.
- Analysis of C2C12 skeletal muscle cells at different differentiation stages.
- Use of Estrogen Receptor-MyoD (ER-MyoD) chimerae in mouse fibroblasts.
Main Results:
- A shift in E2F complexes occurs during differentiation, with E2F4/pRb2/p130 replacing cyclinA/cdk2 complexes.
- MyoD activation and p21 induction prevent serum-stimulated DNA synthesis in differentiating cells.
- MyoD activation inhibits cyclinA/cdk2 reassociation with E2F4, suppressing E2F activity and cell cycle reentry.
Conclusions:
- MyoD induces permanent cell cycle arrest in myocytes through p21 upregulation.
- Suppression of proliferation-associated cyclin/cdk-containing E2F complexes is a key mechanism for cell cycle arrest.
- Formation of E2F4/pRb2/p130 complexes alone is insufficient to prevent cell cycle reactivation.