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Regulation of E2F4 mitogenic activity during terminal differentiation by its heterodimerization partners for nuclear
1Laboratory of Gene Expression, Fondazione Andrea Cesalpino, Policlinico Umberto I, University of Rome La Sapienza, Italy.
Abstract:
E2F/DP heterodimers play a pivotal role in the regulation of cell growth and differentiation. A decrease in E2F/DP activity occurs during cell cycle arrest and differentiation. However, very little is known about the specific role of the various E2F/DP members along the transition from proliferation to terminal differentiation. We have previously shown that E2F4 accounts for the vast majority of the endogenous E2F in differentiating muscle cells. Here, we show that E2F4, which lacks a nuclear localization signal (nls), is distributed in both the nucleus and the cytoplasm, in either asynchronously growing myoblasts or differentiated myotubes. E2F4 nuclear accumulation is induced by the binding in the cytoplasm with specific partners p107, pRb2/p130, and DP3delta, an nls-containing spliced form of DP3, which provide the nls. Although overexpression of E2F4/DP3delta reactivates the cell cycle in quiescent cells, the E2F4 nuclear accumulation induced by pRb2/p130 and p107 correlates with cell growth arrest Moreover, E2F4/DP3delta-induced cell cycle reactivation is efficiently counteracted by either p107 or pRb2/p130 overexpression. Reinduction in quiescent cells of DNA synthesis by E2F1/DP1 overexpression is abrogated by coexpression of pRb and is hampered by MyoD overexpression. Both pRb2/p130 and pRb, as well as MyoD, are up-regulated in myotubes. Accordingly, multinucleated myotubes, which are induced to reenter the S-phase by oncoviral proteins, are refractory to cell cycle reactivation by forced expression of E2F4/DP3delta or E2F1/DP1. Thus, E2F/DP repression represents only one of multiple redundant circuits that control the postmitotic state in terminally differentiated cells and that are targeted by adenovirus E1A and SV40 large T antigen.
Insights
E2F4 protein localization and nuclear accumulation are regulated by binding partners, influencing cell cycle progression during muscle differentiation. These interactions contribute to cell cycle arrest and the postmitotic state in terminally differentiated cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Developmental Biology
Background:
- E2F/DP heterodimers are crucial for cell growth and differentiation.
- E2F/DP activity decreases during cell cycle arrest and differentiation.
- The specific roles of E2F/DP members in the proliferation-to-differentiation transition are not fully understood.
Purpose of the Study:
- To investigate the role of E2F4 in muscle cell differentiation.
- To elucidate the mechanisms regulating E2F4 localization and activity.
- To understand how E2F/DP proteins contribute to terminal differentiation and cell cycle exit.
Main Methods:
- Western blotting to assess protein levels.
- Immunofluorescence microscopy to determine protein localization.
- Overexpression studies to analyze cell cycle effects.
- Co-immunoprecipitation to identify binding partners.
Main Results:
- E2F4 localizes to both nucleus and cytoplasm in myoblasts and myotubes.
- Nuclear accumulation of E2F4 is mediated by binding to p107, pRb2/p130, and DP3delta.
- E2F4/DP3delta overexpression can reactivate the cell cycle in quiescent cells.
- E2F4 nuclear accumulation induced by p107 or pRb2/p130 correlates with cell growth arrest.
- Terminally differentiated myotubes are refractory to cell cycle re-entry induced by E2F/DP overexpression.
Conclusions:
- E2F4's localization and activity are tightly regulated by its binding partners during muscle differentiation.
- E2F/DP repression is one of several redundant mechanisms maintaining the postmitotic state in terminally differentiated cells.
- Viral oncoproteins targeting E2F/DP pathways can override these differentiation-induced cell cycle blocks.