Regulation of E2F1 by BRCT Domain-Containing Protein TopBP1

Abstract

The E2F transcription factor integrates cellular signals and coordinates cell cycle progression. Our prior studies demonstrated selective induction and stabilization of E2F1 through ATM-dependent phosphorylation in response to DNA damage. Here we report that DNA topoisomerase IIβ binding protein 1 (TopBP1) regulates E2F1 during DNA damage. TopBP1 contains eight BRCT (BRCA1 carboxyl-terminal) motifs and upon DNA damage is recruited to stalled replication forks, where it participates in a DNA damage checkpoint. Here we demonstrated an interaction between TopBP1 and E2F1. The interaction depended on the amino terminus of E2F1 and the sixth BRCT domain of TopBP1. It was specific to E2F1 and was not observed in E2F2, E2F3, or E2F4. This interaction was induced by DNA damage and phosphorylation of E2F1 by ATM. Through this interaction, TopBP1 repressed multiple activities of E2F1, including transcriptional activity, induction of S-phase entry, and apoptosis. Furthermore, TopBP1 relocalized E2F1 from diffuse nuclear distribution to discrete punctate nuclear foci, where E2F1 colocalized with TopBP1 and BRCA1. Thus, the specific interaction between TopBP1 and E2F1 during DNA damage inhibits the known E2F1 activities but recruits E2F1 to a BRCA1-containing repair complex, suggesting a direct role of E2F1 in DNA damage checkpoint/repair at stalled replication forks.

ACKNOWLEDGMENTS

We thank Graeme Bolger for the HeLa cDNA library, Kazusa DNA Research Institute for the KIAA0259 construct, Joe Nevins for the Δ1-88 E2F1 and Δ283-358 E2F1 constructs, Karen Vousden for the p14^ARF promoter-Luc (E1β-Luc) construct, and Fumio Hanaoka for pKL12 and pKL12 E2FAB. We also appreciate critical reading of the manuscript by Joe Nevins and Graeme Bolger. We thank Marion Spell at the UAB Flow Cytometry core facility for the flow cytometry analyses and Kun-Sang Chang for suggestions on immunostaining.

The work was supported by grants from a General Motors Cancer Research Scholar Award (W.-C.L.), American Cancer Society UAB Research Grant Program (IRG-6000141, P. I. Albert F. LoBuglio), NIH/NCI K12 CA 7693705 (W.-C.L.), and a UAB Avon/Breast Cancer SPORE Career Development Award (W.-C.L. and F.-T.L.). W.-C.L. and F.-T.L. are recipients of an HHMI/UAB Faculty Development Award.