Maintenance of cells in a quiescent state after terminal differentiation occurs through a number of mechanisms that regulate the activity of the E2F family of transcription factors. We report here that changes in the subcellular compartmentalization of the E2F family proteins are required to prevent nuclei in terminally differentiated skeletal muscle from reentering S phase. In terminally differentiated L6 myotubes, E2F-1, E2F-3, and E2F-5 were primarily cytoplasmic, E2F-2 was nuclear, whereas E2F-4 became partitioned between both compartments. In these same cells, pRB family members, pRB, p107, and p130 were also nuclear. This compartmentalization of the E2F-1 and E2F-4 in differentiated muscle cells grown in vitro reflected their observed subcellular location in situ. We determined further that exogenous E2F-1 or E2F-4 expressed in myotubes at levels fourfold greater than endogenous proteins compartmentalized identically to their endogenous counterparts. Only when overexpressed at higher levels was inappropriate subcellular location for these proteins observed. At these levels, induction of the E2F-regulated genes, cyclins A and E, and suppression of factors associated with myogenesis, myogenin, and p21(Cip1) was observed. Only at these levels of E2F expression did nuclei in these terminally differentiated cells enter S phase. These data demonstrate that regulation of the subcellular compartmentalization of E2F-family members is required to maintain nuclei in a quiescent state in terminally differentiated cells.
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