E2F and Myc-Induced Lymphoma intermediate phenotype of the E2f2 heterozygotes suggests a degree of haploinsufficiency. In addition, the E2f2+/2 lymphomas showed no loss of heterozygosity demonstrating that E2F2 does not behave like a classic tumor suppressor in the Em-myc context (data not shown). Finally, a deficiency in E2F4 also had a dramatic effect on Myc- induced lymphoma development (Figure 1D). A comparison of E2f4+/+, E2f4+/2 and E2f42/2 mice revealed that E2f42/2 mice remained tumor-free for significantly longer than siblings (p,0.0001) with a median tumor-free span of 375 days past birth. Lymphoma onset may be modestly delayed in E2f4+/2 mice (p = 0.0465). Taken together, these data would suggest roles for two E2F proteins, both positive and negative acting, in affecting the onset of Myc-mediated lymphomas. A role for these two E2F family members also coincides with the prominent expression of these proteins in hematopoetic tissues (Figure S1). Loss of E2F2 or E2F4 function does not alter Myc- mediated proliferation or apoptosis Previous work has shown that in the B cell lineage Myc induces proliferation and apoptosis and retards differentiation [24,29]. As such, the effects of E2F loss of function on Myc oncogenesis could result from alterations in one or more of these processes. To address the potential for differential effects on proliferation, weanling mice were injected with BrdU, three hours later bone marrow was isolated, and the cell cycle distribution of B lineage cells (B220+ CD19+) determined. As shown in Figure 2A, proliferation of B lineage cells was similar for non-transgenic E2f2 wild type and cells. Importantly, the effect of Myc on cell cycle entry of B lineage cells, with increased S-phase cells and reduced G0/G1 cells, was independent of E2F2 status. As shown in Figure 2B, E2f4 wild type and B lineage cells were similarly proliferative in non-transgenic mice. As well there was still the expected increase in proliferation associated with bearing the Em- Figure 2. The cell cycle distribution of pretumorous B lineage cells is not altered by E2F2 or E2F4 status. (A) The cell cycle distribution of bone marrow B lineage cells (B220+ CD19+) from pretumorous E2F2 cohort mice. The error bars indicate standard deviation. (B) The cell cycle distribution of bone marrow B lineage cells (B220+ CD19+) from pretumorous E2F4 cohort mice. The error bars indicate standard deviation. doi:10.1371/journal.pgen.1000640.g002 PLoS Genetics | www.plosgenetics.org 4 September 2009 | Volume 5 | Issue 9 | e1000640