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 II␤ 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.The E2F transcription factors E2F1 to E2F6 bind to E2F sites in promoters and regulate the expression of a large array of genes that encode proteins important for DNA replication and cell cycle progression. In response to growth signals, activated G 1 cyclin-dependent kinases phosphorylate retinoblastoma protein (pRb) and release E2F from pRb binding. This event is critical in controlling G 1 /S transition (9, 23). Among the E2F family members, E2F1, E2F2, and E2F3 are transcriptional activators and are induced in response to growth stimulation, with peak accumulation at G 1 /S. Together, they are essential for cellular proliferation since a combined mutation of E2F1, -2, and -3 completely blocks cellular proliferation (31), whereas E2F4 and E2F5 function mainly as transcriptional repressors (9, 23).Among the same subclass of E2F family, each individual E2F member has its unique biological properties. The unique feature for E2F1 is its activity in apoptosis induction (8,14,21) and its function as a tumor suppressor. E2F1 Ϫ/Ϫ mice are viable but develop a broad spectrum of tumors (10, 37). This unique tumor suppressor function could be partly attributed to the apoptotic activity of E2F1. In comparison, overexpression of E2F2 or E2F3 was shown to induce apoptosis as well, but to a lesser extent than with E2F1 (21, 28).Recently, we showed that DNA damage specifically induces E2F1 but not the other E2F family proteins (17). In response to DNA damage, two key regulators of DNA damage checkpoints, ATM and ATR (ATM-Rad3-related protein) kinases, phosphorylate E2F1 but not E2F2, E2F3, or E2F4. The specific phosphorylation of E2F1 at serine 31 by ATM/ATR leads to stabili...

Diversity within the pRb pathway: is there a code of conduct?

“…E2F-1 is phosphorylated on S31 by ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related (ATM/ATR) kinases (Lin et al, 2001). A yeast two hybrid screen in which a small N-terminal region of E2F-1 (containing S31) was used as the 'bait' isolated TopBP1 (DNA topoisomerase IIb binding protein 1) as a protein that could interact with E2F-1 (Liu et al, 2003). TopBP1 contains eight BRCT motifs and it is through the sixth motif that it forms an interaction with E2F-1, an association which is induced by DNA damage and dependant upon phosphorylation of S31 (Liu et al, 2003).…”

“…TopBP1 contains eight BRCT motifs and it is through the sixth motif that it forms an interaction with E2F-1, an association which is induced by DNA damage and dependant upon phosphorylation of S31 (Liu et al, 2003). Many E2F-1 activities are downregulated upon its association with TopBP1, including transcription, induction of S phase and apoptosis (Liu et al, 2003). Mechanistically, it is believed that the recruitment of Brg1/Brm (a subunit of the SWI/SNF chromatin-remodelling complex) to the TopBP1/E2F-1 complex leads to the repression of E2F-1 regulated promoters .…”

Regulation of epidermal apoptosis and DNA repair by E2F1 in response to ultraviolet B radiation

“…In addition to transcriptionally regulating DNA repair genes, E2F1 also physically associates with a number of DNA repair proteins including DDB2, NBS1 and TopBP1 (Hayes et al, 1998;Maser et al, 2001;Liu et al, 2003). E2F1 has also been shown to colocalize with BRCA1 in foci that are presumptive sites of DNA repair .…”

“…These include transcriptional upregulation of DNA repair factors such as DDB2 and XPC (Adimoolam and Ford, 2002;Amundson et al, 2002;Tan and Chu, 2002;Fitch et al, 2003), recruitment of repair factors to sites of DNA damage through physical interaction with p53 (Wang et al, 2003) and the ability of p53 to function as a chromatin accessibility factor to allow access of repair factors to sites of damage (Rubbi and Milner, 2003). Given that E2F1 can upregulate the expression of a number of DNA repair genes (Wang et al, 1999;Polager et al, 2002;Ren et al, 2002) and that E2F1 physically associates with several DNA repair factors (Hayes et al, 1998;Maser et al, 2001;Liu et al, 2003), it is quite possible that like p53, the antiapoptotic effect of E2F1 is related to a stimulation of DNA repair.…”

Life and death decisions by the E2F transcription factors

“…The N-terminus of E2F1, when phosphorylated by ATM, provides a binding site for the BRCT domains of TopBP1 [59]. TopBP1-mediated inhibition of E2F1 transcriptional activity, including its ability to induce cell death, occurs both by recruitment of a repressive complex containing E2F1, TopBP1 and Brg/Brm to E2F-responsive promoters, as well as sequestration of E2F1 in BRCA1-containing foci in the nucleus [59,60]. DNA damage also induces E2F1-mediated activation of the NAD-dependent deacetylase SirT1, which in turn binds to and inhibits the activity of E2F1, in an apparent negative-feedback mechanism [61•].…”