Cell cycle oscillators underlying orderly proteolysis of E2F8

Abstract

19E2F8 is a transcriptional repressor that antagonizes the canonical cell cycle 20 transcription factor E2F1. Despite the importance of this atypical E2F family member in cell 21 cycle, apoptosis and cancer, we lack a complete description of the mechanisms that control 22 its dynamics. To address this question, we developed a complementary set of static and 23 dynamic cell-free systems of human origin, which recapitulate inter-mitotic and G1 phases, 24 and a full transition from pro-metaphase to G1. This revealed an interlocking molecular 25 switch controlling E2F8 degradation at mitotic exit, involving dephosphorylation of Cdk1 26 sites in E2F8 and the activation of APC/C Cdh1 , but not APC/C Cdc20 . Further, we revealed a 27 differential stability of E2F8, accounting for its accumulation in late G1 while APC/C Cdh1 is 28 still active and suggesting a key role for APC/C in controlling G1-S transcription. Finally, we 29 identified SCF-Cyclin F as the ubiquitin ligase controlling E2F8 in G2-phase. Altogether, our 30 data provide new insights into the regulation of E2F8 throughout the cell cycle, illuminating 31 an extensive coordination between phosphorylation, ubiquitination and transcription in 32 promoting orderly cell cycle progression. 33 34 35 36 37 38 39 40 41 42 Ran et al., 2008, Ouseph, Li et al., 2012). Despite being part of the 'repressive' branch of E2F 57proteins, E2F7 and E2F8 belong to the pro-proliferative gene network underlying cell 58 proliferation (Cohen, Vecsler et al., 2013). 59 E2F1, as well as E2F7 and E2F8, are regulated post-translationally via temporal 60 proteolysis. The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit cell 61 cycle ubiquitin ligase and core component of the cell cycle machinery (King, Peters et al., 62 1995, Sudakin, Ganoth et al., 1995. The APC/C uses two related co-activators termed Cdc20 63 and Cdh1, which bind substrates and recruit them to the APC/C for ubiquitination and 64 subsequent degradation (Kernan, Bonacci et al., 2018). We previously identified both E2F7 65 and E2F8 as targets of the Cdh1-bound form of APC/C (APC/C Cdh1 ) (Cohen et al., 2013). 66These findings, together with other supporting studies (Boekhout, Yuan et al., 2016), shifted 67 the model by which the E2F1-E2F7-E2F8 circuitry communicates with the cell cycle clock to 68 regulate the transition from the G1-phase of the cell cycle into S-phase. Nevertheless, the 69 exact inter-dynamics of E2F1-E2F7-E2F8 circuitry throughout G1 and the mechanism by 70 which they are achieved are not entirely resolved. No less obscure is the interplay between 71 E2F1 and atypical E2Fs during G2-phase and mitosis. Dissecting these complex signaling 72 circuits is important for understanding the decision making mechanisms at two critical 73 points in the life of a proliferating cell -commitment to DNA replication and division. 74Cell-free systems are known for their capacity to reproduce complex cellular 75 processes in vitro while maintaining a physiologically relevant context, bridging the gap 76...

Cell cycle oscillators underlying orderly proteolysis of E2F8

“…We detected an interaction between the two proteins, irrespective of which protein was precipitated (Figure 8, I and J), suggesting that E2F8 binds Cyclin F. Taken together, these data suggest that E2F8 regulation by Cyclin F is direct and that SCF Cyclin F activity downregulates E2F8 prior to M-phase entry. These data (see also Wasserman et al, 2019) complement three parallel studies connecting SCF Cyclin F -mediated proteolysis to temporal dynamics of E2F members (Burdova et al, 2019;Clijsters et al, 2019;Yuan et al, 2019).…”

Cyclin F drives proliferation through SCF-dependent degradation of the retinoblastoma-like tumor suppressor p130/RBL2

“…We next examined endogenous proteins corresponding to the CDK-RB network genes that correlate most strongly with CCNF . We used two breast cancer cell lines, MCF-7 and T47D, engineered for doxycycline-inducible expression of cyclin F (Wasserman et al, 2020). Cells were treated with increasing amounts of doxycycline for 24 hours, then harvested for immunoblot to determine levels of CDK4, CDK6, cyclin D1, p107, and p130.…”

“…The ubiquitination of activator E2Fs allows for their degradation, and expression of mutant E2Fs that can no longer bind to cyclin F results in increased expression of E2F target genes. Further, during G2 phase, cyclin F was shown to regulate the atypical repressor E2Fs, E2F7 and E2F8, promoting their degradation(Wasserman et al, 2020; Yuan et al, 2019). Each of these substrates implicate cyclin F in indirectly controlling cell cycle gene expression, and our study uncovers a new mechanism by which cyclin F regulates transcriptional control at the start of the cell cycle, by ubiquitinating p130.…”

“…Further, cyclin F is involved in cell cycle gene transcription both through its non-catalytic inhibition of B-MYB after DNA damage (Klein et al, 2015), and its ubiquitination of activator and repressor E2Fs (Burdova et al, 2019; Clijsters et al, 2019; Wasserman et al, 2020; Yuan et al, 2019) and the stem-loop binding protein (SLBP) in G2-phase (Dankert et al, 2016). Together, its dynamic cell cycle regulation and numerous cell cycle substrates highlight the importance of cyclin F in cell cycle control.…”