activation of transcription of genes essential for cell proliferation. While the other two activating E2Fs (E2F1 and E2F3) appear to be broadly expressed, E2F2 expression is largely restricted to cells of the hematopoietic lineage. Indeed, previous work has pointed to a role for E2F2 in the function and development of various hematopoietic lineages. S-phase progression is impaired in B, erythroid, and myeloid lineages in the absence of E2F1 and E2F2, consistent with a role for these E2F products as positive regulators of cell proliferation [50]. Nevertheless, the possibility of a more complex role for E2F2 in hematopoietic cells emerged from studies demonstrating that the loss of E2F2 appears to enhance the proliferation of T cells following antigenic stimulation, suggesting a negative role for E2F2 in defining a threshold for Ag-stimulated proliferation [51,52]. Although this latter observation is not consistent with the results we present here regarding proliferative capacity in E2f2-null B lineage cells, it certainly is true that with Myc overexpression, the absence of E2F2 function may accelerate tumor onset by increasing the proportion of progenitor B lineage populations. Opavsky and colleagues have recently used a bitransgenic model of Myc-induced T cell lymphomagenesis to probe the importance of E2F1, E2F2 and E2F3 for Myc activities [25]. Analogous to our findings for B lineage lymphomagenesis, they found that E2f1 or E2f3 deficiencies have no effect on T cell lymphoma progression. Most importantly, E2f2 deficiency accel- erates tumor onset in the T cell model as it does for the Em-myc model. In addition, other aspects of their findings are reminiscent of our results, namely that E2f2 heterozygotes have an intermediate phenotype, suggesting some haploinsufficiency, and that with E2f2 deficiency there is an increased incidence of multiple tumor clones. The salient difference is that in their study, E2f2 deficiency is associated with reduced apoptosis. Specifically, Opavsky and colleagues detected a reduced percentage of annexin-positive T cells in moribund mice that were E2f22/2 versus E2f2+/+ whereas in our experiments, E2f2 deficiency was not associated with reduced apoptosis, whether assessing the bulk of B lineage cells from pretumorous mice, or when looking at B220+ cells isolated from tumorous lymph nodes (Figure 3A and Figure S9C). In marked contrast to the study of Baudino et al. [23], the work described here did not demonstrate that E2f1 deficiency influenced lymphoma development or p27Kip1 regulation. This discrepancy could be the consequence of the different knockout alleles utilized and possible differences in strain background and breeding strategies. But we also note that the wild type E2f1 Em-myc mice in the Baudino et al. study [23] exhibit a more precipitous and overall earlier tumor-onset curve than reported in most other studies with C57BL/6 congenic, wild type Em-myc mice. Developmental effects that influence the outcome of oncogenesis Our results indicate that E2f2 deficiency enhances the emergence of the ‘‘early-standard’’ form of lymphoma likely because the absence of E2F2 activity expands the population of cells that is the usual target for the oncogenic process in the Em-myc model. As a result, the population of E2f22/2 tumors is also more homogeneous with respect to their phenotype, as reflected by the gene expression profiles. In contrast, we propose that the loss of E2f4 results in the decrease of this population of cells and thus the frequency of appearance of standard morphology early onset tumors. There is not a complete absence of these cells since a few tumors do arise in the absence of E2F4 that cluster with the ‘‘early- standard’’ wild type and E2f22/2 tumors. But the consequence of this depletion is enrichment for tumors with a late chronological E2F and Myc-Induced Lymphoma onset, whether to the extreme of the ‘‘early’’ cluster or in the ‘‘late’’ cluster, likely due to an opportunity for these tumors to develop because of the reduced frequency of the ‘‘early-standard’’ variety. Taken together, these results point to a role for E2F activities in determining the population of B lineage cells that contribute to the development of tumors and highlights the interplay between two cell regulatory activities, E2F and Myc, in determining the outcome of the oncogenic process. Materials and Methods Mouse strains and tumor monitoring Mice were housed in a Duke University Medical Center Division of Laboratory Animal Resources facility and experiments approved by the Duke University Institutional Animal Care and Use Committee. The generation of the specific lines of E2F- deficient mice has been previously described [19,28,53]. The original 129 substrain background was 129/SvJae for the E2F1, E2F2 and E2F3 cohorts and 129/OlaHsd for the E2F4 cohort. Based on breeding history, the E2F1 cohort mice used in this study were predominantly C57BL/6 (backcrossed five generations into C57BL/6) while the E2F2, E2F3 and E2F4 cohorts were mixed C57BL/66129. The four E2F cohorts were maintained separately and breeding involved crossing heterozygous mice to yield wild type, heterozygous and mice in each generation. The Em-myc transgenic mouse line 292-1 [5] extensively backcrossed into C57BL/6 and originally from Dr. Alan Harris (Walter and Eliza Hall Institute, Melbourne, Australia), was kindly provided by Dr. Scott Lowe (Cold Spring Harbor Laboratory). For each E2F cohort, stock Em-myc positive C57BL/6 congenic males were bred to E2Fn+/2 females and of the progeny only the Em-myc positive E2fn+/2 males, designated the F1 males, were kept. These F1 males, E2fn+/2 myc+, were then bred to E2fn+/2 myc2 females. The Em-myc positive progeny of this cross, E2fn+/+, E2fn+/2, and E2fn2/2 were then compared. Because maternal transmission is associated with reduced latency [34], transmission of the Em-myc transgene was exclusively paternal in this breeding scheme. Em-myc negative siblings were also kept as a source of related mice that lacked myc transgene effects. Em-myc positive mice were monitored weekly to identify any mice with malignant disease. Mice were evaluated for any visible or palpable lumps, a hunched posture, tachypnea, a swollen belly, or ruffled fur and sacrificed promptly upon the appearance of any such symptoms. Lymphomas that emerged were dissected from sacrificed mice, washed in PBS, and frozen in liquid nitrogen or processed for flow cytometric analysis. The frozen tissue provided material for Southern and western analysis. Statistical analysis Tumor onset data refer to the time in days between birth and the first indication of illness. Using GraphPad’s Prism program, the time values were plotted to generate Kaplan-Meier survival curves and the curves compared by a logrank test. For comparisons of means and standard deviations, the paired student t-test was performed and statistical significance was determined if the p,0.05. Western analysis To assess alterations in p19ARF, Mdm2 and p53 protein expression, lymphoma samples were dissected from morbid mice and immediately frozen. Samples were then weighed, ground to a powder in liquid nitrogen and resuspended in 60 mM Tris (pH 6.8)/1% SDS at 1 ml per 0.2 g tumor weight, boiled, sonicated, and any remaining debris removed by centrifugation. PLoS Genetics | www.plosgenetics.org 12 September 2009 | Volume 5 | Issue 9 | e1000640