Author Summary The diversity of human cancers reflects the variety of genetic changes that cause tumors to emerge and progress. Even for mice engineered with a specific cancer-causing mutation, the resulting tumors are often divergent, reflecting different additional mutations. We wanted to investigate how activities that work together can collaborate in tumorigenesis. Specifically, we are interested in Myc and the E2F family of proteins, intersecting activities that influence a cell’s decision to replicate, rest, or die. We made use of an engineered mouse that develops pre-B and B cell lymphoma initiated by Myc and tested whether the loss of particular E2F family members influences these lymphomas. We found that tumor emergence was accelerated by E2F2 loss and delayed by E2F4 loss. We attributed these results to the finding that the mice lacking E2F2 have a greater proportion than usual of the most susceptible, early-stage B lineage cells and the mice lacking E2F4 have fewer of these cells. Distinct tumor types emerged with their relative proportions influenced by E2F2 and E2F4 status. We conclude that the variety of tumors probably reflect different stages of B lymphoid development that respond to Myc and that E2F proteins can influence the proportions of these different stages. studies have demonstrated a central role for the Rb-E2F pathway in the regulation of cellular proliferation. The majority of genes encoding DNA replication and mitotic activities are under the control of E2F proteins. Indeed, recent experiments provide evidence for a role for E2Fs in coordinating transcriptional regulatory events at G1/S and G2/M [9–11]. Other work has shown that E2Fs also link this critical proliferative pathway with the p53 response through a capacity to induce the p19ARF/ Mdm2 pathway leading to the accumulation of p53 protein [12,13]. As such, E2Fs provide a mechanism to directly link the control of cell proliferation with the determination of cell fate. In addition to the connection between Myc and E2F in the control of cellular proliferation, Myc expression couples cellular proliferation with the induction of apoptosis under specific growth conditions where survival growth factors are limiting. Myc-induced apoptosis is largely dependent upon p53 signaling and, similar to E2F1, involves the induction of p19ARF, inhibition of Mdm2, and elevated p53 [14]. The shared functional properties of the Myc and E2F transcription factors, coupled with the finding that Myc can induce E2F gene expression [15,16], raise the possibility that Myc function might be mediated, at least in part, through the action of the E2F transcription factors. Indeed, work by the Bernards laboratory revealed that in addition to targeting p27Kip1, the mitogenic activity of Myc likely involves regulation of E2Fs [17]. This possibility has been more directly assessed using mouse embryo fibroblasts (MEFs) from embryos deleted for specific E2F genes to evaluate the functional relationship between Myc and various E2F proteins [18]. Experiments using these E2F-deficient MEFs showed that the ability of Myc to induce S phase in the absence of other mitogens is severely impaired in MEFs deleted for E2f2 or E2f3, but not E2f1 or E2f4. In contrast, Myc induced apoptosis in primary serum-deprived MEFs was delayed in cells deleted for E2f1, but not affected by E2f2 or E2f3 deletion. Thus, at least in cell culture, the induction of specific E2F activities is an essential downstream event in the Myc pathway that controls cell proliferation versus apoptosis, and some of the functions of Myc, E2F and Myc-Induced Lymphoma such as the induction of p19ARF and p53 could be explained, at least in part, with one pathway leading through E2F activation. To address the significance of the Myc-E2F connection in a relevant, in vivo setting, we have made use of a series of E2F- deficient mouse strains, in combination with the Em-myc transgenic model of lymphomagenesis (MGI:2448238), to investigate whether deficiencies in E2F1, E2F2, E2F3 or E2F4 (MGI:1857424, 2179111, 2177428 and 1888951) can influence Myc’s oncogenic potential. We find that there is a critical role for two E2F activities in affecting the potential for Myc-induced oncogenesis. Results As a prelude to the investigation of a role for E2F activity in Myc-mediated oncogenesis, we analyzed the pattern of tissue- specific expression of the various E2F genes. RNA was prepared from selected tissues of wild type mice and then analyzed by quantitative RT-PCR. E2f1 and E2f3 genes were widely expressed whereas expression of the E2f2 gene was largely restricted to the hematopoietic tissues assayed including bone marrow, spleen, and thymus (Figure S1A). Please note that Text S1 (Supporting Materials and Methods) describes the procedures specific to Figures S1, S2, S3, S4, S5, S6, S7, S8, S9, and S10. This restricted E2f2 expression pattern at the RNA level was recapitulated at the protein level (Figure S1B). E2f4, while expressed widely at the RNA level, was also particularly strongly expressed in hemato- poietic tissues at the protein level (Figure S1C) [19]. While we did not measure E2F5 in these assays, it has previously been shown that the expression of this E2F family member is restricted to the differentiating epithelial layers of the skin, intestine and brain [20,21]. As such, we chose to focus our studies on the E2f1-4 genes and gene products. A role for E2F2 and E2F4 in Myc-induced lymphomagenesis Male Em-myc transgenic mice (backcrossed and maintained in the C57BL/6 strain) were bred into the four different E2F- deficient mouse lines. E2F2, E2F3 and E2F4 cohorts were maintained as C57BL/66129 while the E2F1 cohort was predominantly C57BL/6 in background. In particular, the E2F3 and E2F4 cohorts required maintenance on a mixed, rather than inbred, background because the yield of the E2f3-null and E2f4- mice was severely compromised upon inbreeding (data not shown). Sibling mice, wild type, heterozygous or for a particular E2F gene, and bearing the Em-myc transgene, were examined weekly for any sign of lymphoma emergence. Each mouse was checked for enlarged lymph nodes, a swollen abdomen, a hunched posture, ruffled fur and/or tachypnea [22]. Upon the appearance of any of these symptoms, the mouse was sacrificed, dissected to identify any lymph node enlargement, and tumor tissue harvested for analysis. For studies assessing the pre-tumor phenotype, mice were sacrificed within three to five weeks after birth and bone marrow and spleen recovered. Such samples were characterized as pre-neoplastic only if lymph node and spleen enlargement was nil or modest at the time of dissection and/or Southern analysis of B lineage cell DNA revealed no specific heavy chain rearrangements indicative of the emergence of tumor clones. Tumor emergence was evaluated in the four E2F cohorts (Figure 1), as well as for our Em-myc C57BL/6 congenic stock mice (Figure S2A). When the wild type mice in each cohort were compared to the stock transgenic mice, median onsets did diverge (Figure S2B), with earlier onsets associated with greater 129 strain contribution based on breeding history. In spite of this, the overall PLoS Genetics | www.plosgenetics.org 2 September 2009 | Volume 5 | Issue 9 | e1000640