E2F1

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E2F1 promotes the recruitment of DNA repair factors to sites of DNA double-strand breaks

The E2F1 transcription factor plays a multifaceted role in cell proliferation and survival and can alternatively behave as tumor suppressor gene or an oncogene, depending on biological context.¹ E2F1’s role as a tumor suppressor can be explained by its ability to induce apoptosis; however, at least three emerging lines of evidence suggest that E2F1 may have a role in maintaining genomic integrity through direct participation in the DNA damage response. First, E2F1 is regulated by multiple DNA damage-induced signaling cascades. For one example, ATM can phosphorylate E2F1 on

Ser 31 in response to ionizing radiation (IR), leading to E2F1 protein stabilization.² Second, recent work has shown that E2F1 deficiency can impair various DNA damage response processes, such as nucleotide excision repair.³ Finally, E2F1 is found physically associated with DNA regions undergoing repair, which is consistent with a direct role in the assembly of DNA repair complexes.^3,4

Given these multiple lines of evidence implicating E2F1 in the maintenance of DNA integrity, Chen et al.⁵ recently asked if E2F1 deficiency would affect genome stability. They utilized the phosphorylation of histone variant H2AX, which occurs in chromatin that flanks a double-stranded break, as an indirect marker of DNA damage. They observed that primary adult fibroblasts derived from mice lacking E2F1 had an increase in the number of cells with spontaneous γH2AX foci as well as a dramatic increase the number cells having more than six foci. Likewise, when the E2F1-deficient cells were challenged with ionizing radiation (IR), there was evidence of significantly increased double-stranded breaks, as measured by single cell gel electrophoresis assay.

Next, Chen et al.⁵ investigated the mechanism underlying the increase of double-stranded breaks in E2F1-deficient cells. NBS1 is one of the first proteins to be recruited to double-stranded breaks as a component of the MRN (Mre11-Rad50-NBS1) complex. Using indirect immunofluorescence, they demonstrated a profound decrease in the number of NBS1 containing foci in E2F1-deficient cells compared with WT cells following IR. In contrast, γH2AX foci were increased in E2F1-deficient cells following irradiation. While the total levels of NBS1 protein were not affected by E2F1 deficiency, NBS1 phosphorylation in response to DNA damage was reduced in cells lacking E2F1.

Noting that E2F1 has been observed to be a physical component of foci representing sites of double-stranded breaks^3,4 and that E2F can physically interact with NBS1 near origins of replication,⁶ Chen et al. proposed that E2F1 and NBS1 might physically associate in response to DNA damage. This hypothesis was clearly supported by co-immunoprecipitation experiments following IR.

Double-stranded breaks are accurately repaired by homologous recombination. This process involves formation of single-stranded DNA at the breakage sites, stabilized initially by association with RPA (replication protein A). RPA is later displaced by the Rad51 recombinase in a BRCA2-dependent process. Chen et al. again used immunofluorescence to examine foci formation in WT and E2F1-deficient cells treated with IR. They discovered that E2F1 deficiency reduced the formation of both RPA- and Rad51-containing foci induced by IR. E2F1 deficiency did not affect total RPA protein expression levels, but reduced levels of the Rad51 protein by a post transcriptional mechanism. E2F1-defiency did not affect the formation of 53BP1-containing foci.

At this point there is insufficient evidence to predict how E2F1 influences the recruitment of NBS1, RPA and Rad51 to sites of DNA damage. Given recent work,⁷ it is interesting to speculate that E2F may recruit chromatin- modifying enzymes, such as GCN5, to double-stranded breaks, thereby facilitating repair. The observation that E2F1 deficiency does not reduce γH2AX foci suggests that E2F does not affect the initial direct binding of the MRN complex and ATM (which phosphorylates H2AX) to the sites of DNA damage. More likely, E2F1 affects subsequent waves of NBS1 recruitment and DNA damage signal amplification. Perhaps E2F1 facilitates the recruitment of NBS1 by MDC1 (mediator of DNA damage checkpoint 1). The observation that E2F1 deficiency did not affect the formation of 53BP1-containing foci suggests that E2F1 does not affect MDC1 recruitment of the ubiquitin ligases, which mark histone H2A as 53BP1-binding sites.⁸

Although much work remains to explain how E2F1 affects the assembly of DNA repair complexes, these findings clearly highlight the potential importance of E2F1 to the maintenance of genomic integrity. The work also leads to the very important question of whether E2F1 deficiency might contribute to human cancer. Although most clinical studies⁹ suggest increases in the activity of E2F1 and other E2Fs in human cancer, there are examples of E2F1 downregulation. It would be very interesting to examine clinical samples in which E2F1 is downregulated to determine whether these tumors would present evidence of increased genomic rearrangements compared with similar tumors expressing E2F1.