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Compensation and Specificity of Function Within the E2F Family Oncogene (2007) 26, 321–327 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Compensation and specificity of function within the E2F family L-J Kong, JT Chang, AH Bild and JR Nevins Department of Molecular Genetics and Microbiology, Institute for Genome Sciences and Policy, Duke University Medical Center, Durham, NC, USA Functions encoded by single genes in lower organisms are maintained within the family of genes, including often represented by multiple related genes in the functional overlap.In many instances, this complexity mammalian genome. An example is the retinoblastoma of structure and function confounds genetic analyses and E2F families of proteins that regulate transcription that aim to associate discrete functions within individual duringthe cell cycle. Analysis of genefunction using genes.Further complicating the analysis is the use of germline mutations is often confounded by overlapping germline alterations that inactivate a gene of interest.If function resultingin compensation. Indeed, in cells deleted the particular gene is necessary for an essential function, of the E2F1 or E2F3 genes, there is an increase in the the imposed loss of function will either result in an expression of the other family member. To avoid inviable animal or will force a selection whereby another complications of compensatory effects, we have used function compensates for the loss of activity.The latter small-interferingRNAs that target individual E2F pro- is particularly relevant when dealing with a multigene teins to generate a temporary loss of E2F function. We family encoding related proteins with similar functions. find that both E2F1 and E2F3 are required for cells to An example of such complexity can be found in the enter the S phase from a quiescent state, whereas only retinoblastoma (Rb) and E2F families of proteins that E2F3 is necessary for the S phase in growing cells. We regulate transcription during the cell cycle.Whereas also find that the acute loss of E2F3 activity affects the C. elegans encodes a single Rb protein and Drosophila expression of genes encoding DNA replication and mitotic encodes two Rb proteins, three related genes can be activities, whereas loss of E2F1 affects a limited number identified in the mammalian genome (Dimova and of genes that are distinct from those regulated by E2F3. Dyson, 2005).Similarly, there are two E2F genes in We conclude that the long-term loss of E2F activity does the C. elegans and Drosophila genomes in contrast to the lead to compensation by other family members and that eight E2F genes in the mammalian genome (Attwooll the analysis of acute loss of function reveals specific and et al., 2004; Dimova and Dyson, 2005). Furthermore, a distinct roles for these proteins. clear example of the overlapping nature of function can Oncogene (2007) 26, 321–327.doi:10.1038/sj.onc.1209817; be seen in the analysis of Rb function using germline published online 14 August 2006 versus conditional loss-of-function mouse mutants. Mouse embryo fibroblasts (MEFs) isolated from ani- Keywords: E2F; cell cycle; expression signatures mals sustaining a permanent loss of Rb function retain a capacity to arrest in a quiescent state when deprived of growth factors, whereas fibroblasts in which an acute loss of Rb function was induced lose their capacity to arrest in G0 (Sage et al., 2003). Introduction Studies focused on delineating the function of individual members of the E2F family have identified The evolution of complex families of related activities is broad roles for several of the proteins.In particular, a common aspect of mammalian organisms.Functions E2F1–3 appear to function as transcriptional activators, that might be mediated by a single activity in Drosophila E2F4 and 5 appear to function as Rb-dependent or Caenorhabditis elegans are often seen to be encoded transcriptional repressors, and E2F6–8 may function by multiple related genes in the mammalian genome, as Rb-independent repressors of transcription (Dimova likely the result of duplications associated with unequal and Dyson, 2005).Nevertheless, attempts to clearly crossing over.These duplicated genes are then free to define specificity within the subfamilies, such as the evolve to acquire new functional roles, imparting greater E2F1–3 group, have been difficult.For instance, complexity of function within the mammalian organism. although the elimination of any single gene of this As a result, there is often considerable similarity group does not markedly affect cellular proliferation, double knockouts are impaired and the triple knockout Correspondence: Dr JR Nevins, Department of Molecular Genetics cannot grow (Wu et al., 2001). These results would thus and Microbiology, Institute for Genome Sciences and Policy, Duke suggest that these E2F proteins overlap in their University Medical Center, Durham, NC 27710, USA. function.Alternatively, they may possess quite distinct E-mail: [email protected] Received 26 January 2006; revised 8 May 2006; accepted 6 June 2006; functions but also have the capacity to compensate for published online 14 August 2006 the long-term loss of function as occurs in germline Compensation and specificity of function within the E2F family L-J Kong et al 322 knockouts.In an attempt to dissect the functional roles discrete phenotypes resulting from the long-term loss of of the individual E2F proteins, we have made use of E2F function.As an alternative approach to the study short hairpin RNA (shRNA) vectors that were gene- of E2F function, we have made use of shRNAs selected rated to target selectively either E2F1 or E2F3.We have to target either E2F1 or E2F3 to achieve an acute loss of then examined the consequence of an acute loss of E2F function.Each shRNA vector was inserted in a function of these two E2F proteins.The results suggest recombinant adenovirus to facilitate efficient expression that there are quite distinct functions of these proteins of the shRNA and loss of E2F function.Western that are revealed when cells only transiently lose their analysis demonstrates that the E2F1 shRNA specifically activity. reduces the level of the E2F1 protein without affect on the E2F3 level; likewise, the E2F3 shRNA specifically reduces E2F3, but not E2F1 protein levels (Figure 1b). Thus, the use of shRNAs allows for specific targeting of Results individual E2Fs and without evidence for compensatory effects from other family members. Various studies have highlighted a role for both E2F1 Given the previous results that no single E2F was and E2F3 in cell proliferation.Overexpression of either critical for cell cycle entry, but also the evidence for protein is sufficient to induce the S phase in otherwise compensation from long-term loss of function, we made quiescent cells (Cress et al., 1993; Johnson et al., 1993; use of the viruses expressing E2F1 or E2F3 shRNAs to Qin et al., 1994). Moreover, the combined loss of E2F1 evaluate the effects of acute loss of function of these and E2F3 does also impair the ability of cells to enter proteins on the process of cell cycle entry following the S phase, whereas knockout of either gene alone is serum stimulation.Cells were infected with control not sufficient to block the S phase (Humbert et al., 2000; virus, or the shRNA viruses, brought to a quiescent Wu et al., 2001). These results would suggest that either state by serum starvation for 48 h, and then stimulated E2F1 and E2F3 play overlapping roles in the transition to grow by the addition of serum.5-Bromo-2 0-deoxyur- into a cell cycle or that they are distinct, but the long- idine (BrdU) was added to the cultures 15 h after serum term loss of function of either protein leads to stimulation and the cells were harvested 3 h later.Each compensation by the other.In fact, an examination of virus expresses a green fluorescent protein (GFP) the levels of E2F proteins in mouse embryo fibroblasts protein to allow identification of virus-positive cells derived from knockout animals revealed that the level of and BrDU incorporation was detected by immunostain- E2F1 was increased in the E2F3 null MEFs, and ing.Many GFP-positive cells in the control virus likewise, the level of E2F3 was increased in the E2F1 infection were also positive for BrdU.In contrast, cells null MEFs (Figure 1a).These observations suggest the infected with either the shE2F1 virus or the shE2F3 possibility of a compensatory response to the loss of virus, as indicated by GFP, were negative for BrdU E2F function and might be a contribution to the lack of incorporation (Figure 2a). Quantitation of the results through examination of many fields revealed a greater than 90% reduction of BrdU incorporation in the E2F1 or E2F3 shRNA- expressing cells (Figure 2b).The same results were obtained with two independent shE2F1 and shE2F3 recombinant viruses, indicating that the result is not owing to nonspecific off-target effects of the shRNAs. The fact that each of the shRNAs blocks BrDU incorporation indicates that both E2F1 and E2F3 are important for driving quiescent cells into cell cycle, and thus must have distinct functions in this process.This is Figure 1 Effects of germline or acute loss of E2F function. in contrast to previously reported results using knockout (a) Compensation resulting from long-term loss of E2F1 or E2F3 cells, suggesting that the long-term loss of E2F1 or E2F3 function.Nuclear protein extracts were obtained from growing function can be compensated. / primary MEFs of wild type (WT), E2F1 null (E2F1À À) or E2F3 Previous
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