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 Because differentiated muscle cells (myotubes) are permanently withdrawn from the cell cycle, cellular mechanisms for the initiation and maintenance of the differentiated state must also exist (5).Studies with viral oncogenes have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...
Because of its expression in numerous cells, the herpes simplex virus thymidine kinase promoter (HSV-TK) is one of the best characterized promoters. Using the HSV-TK promoter as a model system, we have defined a new mode of E2F-1 transcriptional activation which utilizes the N-terminal region of E2F-1. We demonstrate that E2F-1 strongly activated HSV-TK, but in the absence of consensus E2F DNA elements. Nonetheless, E2F-1 could bind to GC-rich elements, which were conclusively identified in classic studies of HSV-TK as SP-1 sites. Second, the transcriptional activation of HSV-TK required the entire E2F-1 protein, including the Nterminal 89 amino acids. In contrast, the N-terminal 89 amino acids of E2F-1 were dispensable for transcriptional activation through consensus E2F sites. Third, we demonstrated that S phase entry is not sufficient for activation of HSV-TK by E2F-1, while the activation through consensus E2F sites is strictly linked to the cell cycle. Taken together, the activation of HSV-TK by E2F-1 proceeds by a different mechanism directed in part through the N-terminal region of E2F-1 and may be uncoupled from the known cell cycle regulatory role.The E2F transcription factors have a major role in cellular pathways, including the regulation of the G 1 /S transition and the activation of the S phase gene expression program (for reviews, see Refs. 1-4). Numerous growth control genes are regulated by E2F (e.g. N-myc, cdc-2, c-myc, dihydrofolate reductase). E2F function is regulated by protein-protein interaction with the retinoblastoma (RB) 1 family of tumor suppressor proteins (RB, p107, p130) and with the cell cycle machinery (cyclin E, cyclin A, CDK2). The regulation of E2F by RB has served as a paradigm for growth suppressor function. Expression of RB leads to arrest in G 1 , and one mechanism works through the inhibition of E2F and the majority of the S phase gene expression program. Conversely, disruption of the RB-E2F interaction leads to activation of S phase genes and progression through the cell cycle. Either displacement of RB by viral oncogenes (e.g. E1A) or the phosphorylation of RB by cell cycle-dependent kinases (CDKs) can dissociate the RB⅐E2F complex. Both events trigger S phase entry. Because E2F is a key regulatory target, we and others have demonstrated combinatorial complexity in E2F interactions with RB family members. Different E2F⅐RB family complexes dictate the G 1 /S and G 0 /G 1 cell cycle transitions in concert with the cell cycle machinery (5-9). E2F is a critical cellular regulatory protein since perturbation in expression levels lead to apoptosis or oncogenicity (10 -13).We and others have demonstrated that the N-terminal region of E2F-1 is an important regulatory region. We have demonstrated that cyclin A and CDK2 can interact directly with the extreme N-terminal region of E2F-1. The association results in direct phosphorylation of E2F-1 and the heterodimeric partner DP-1. The phosphorylation leads to inhibition of DNA binding and subsequent inactivation of E2F-1-dependent ...
Recent experiments in understanding the mechanism of the retinoblastoma protein (RB) function have revealed the existence of several cellular proteins that are complexed with RB. One of these cellular proteins is the E2F transcription factor, which was originally identified due to its inducibility by E1A during an adenovirus infection. The E2F recognition sequence is found in the promoters of several cellular genes involved in growth control, including several oncogenes. In this report, we provide evidence that the interaction of E2F and RB is mediated through a region on RB where viral oncogenes such as SV40 T antigen and adenovirus E1A bind and where tumorigenic mutations also cluster. Additional carboxy-terminal sequences are also required for the interaction with E2F. These observations provide evidence for a direct connection between tumor suppressor function and the gene expression program leading to cellular growth regulation.
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