Akiko Kumagai

Eukaryotic transcription factors (TFs) form local, high‐concentration hubs at specific genomic loci through dynamic, multivalent interactions mediated by their low‐complexity domains, a behavior essential for transcriptional activation. Characterizing the dynamics and regulation of TF hubs requires high‐resolution imaging of TFs in their native cellular environment; however, such biophysical characterization remains limited. Here, we combine CRISPR/Cas9‐mediated genome editing and single‐molecule microscopy to investigate the behaviors of endogenous oncogenic fusion TF EWS::FLI1 in Ewing sarcoma cells. EWS::FLI1 forms sub‐diffraction‐limit hubs with dissolution mechanisms that prevent macroscopic liquid‐liquid phase separation. Hub formation is a neomorphic behavior of EWS::FLI1 not conferred by its parental proteins, EWSR1 and FLI1. During mitosis, EWS::FLI1 hubs dissolve, but EWS::FLI1 molecules continue to dynamically associate with mitotic chromosomes, consistent with a mitotic bookmarking role. Nascent RNA destabilizes EWS::FLI1 hubs without affecting their dimensions. Finally, LY2835219 and trabectedin, compounds previously reported to affect EWS::FLI1 function, disrupt and mislocalize endogenous EWS::FLI1 hubs, respectively, highlighting their therapeutic potential for treating Ewing sarcoma. Together, our results reveal new insights into the assembly and regulation of endogenous EWS::FLI1 hubs at an unprecedented resolution and establish a framework for biophysical and functional characterization of other TF hubs in the future.

Eukaryotic transcription factors (TFs) form local, high-concentration hubs at specific genomic loci through dynamic, multivalent protein-protein interactions mediated by their low-complexity domains. The hub formation behavior plays an essential role in TFs’ transcriptional activation activities. Characterizing the dimensions, dynamics, and regulation of TF hubs requires high-resolution imaging of TFs in their native cellular environment, but much of such biophysical characterization remains missing. Here, we combined CRISPR/Cas9-mediated genome editing and advanced quantitative cell imaging, including single-molecule microscopy, to investigate the dynamic behaviors of the endogenous oncogenic fusion TF EWS::FLI1 in Ewing sarcoma cells. We found that endogenous EWS::FLI1 forms dynamic, sub-diffraction-limit hubs with mechanisms of dissolution that prevent the hubs from achieving macroscopic liquid-liquid phase separation. Hub formation is a neomorphic behavior of EWS::FLI1 that is not directly conferred by its parental proteins, EWSR1 and FLI1. We found that during mitosis, EWS::FLI1 hubs dissolve, but EWS::FLI1 molecules continue to dynamically bind and unbind mitotic chromosomes, revealing a role of EWS::FLI1 in mitotic bookmarking. Nascent RNA destabilizes EWS::FLI1 hubs on chromatin, but it does not affect the dimensions of the hubs. Finally, we visualized endogenous EWS::FLI1 hubs upon treatment with various compounds that were previously indicated to affect EWS::FLI1 function. We found that LY2835219 and trabectedin significantly alter the nuclear distribution of endogenous EWS::FLI1, disrupting and mislocalizing EWS::FLI1 hubs, respectively. This finding highlights the therapeutic potential of both compounds for Ewing sarcoma. Together, our results reveal new insights into the assembly and regulation of endogenous EWS::FLI1 hubs at an unprecedented resolution. The methodology developed here will be useful for characterizing the functional hubs of many regular and pathological TFs in the future.

Metazoan genomes are duplicated by the coordinated activation of clusters of replication origins at different times during S phase, but the underlying mechanisms of this temporal program remain unclear during early development. Rif1, a key replication timing factor, inhibits origin firing by recruiting protein phosphatase 1 (PP1) to chromatin counteracting S phase kinases. We have previously described that Rif1 depletion accelerates early Xenopus laevis embryonic cell cycles. Here, we find that in the absence of Rif1, patterns of replication foci change along with the acceleration of replication cluster activation. However, initiations increase only moderately inside active clusters. Our numerical simulations suggest that the absence of Rif1 compresses the temporal program towards more homogeneity and increases the availability of limiting initiation factors. We experimentally demonstrate that Rif1 depletion increases the chromatin-binding of the S phase kinase Cdc7/Drf1, the firing factors Treslin, MTBP, Cdc45, RecQL4, and the phosphorylation of both Treslin and MTBP. We show that Rif1 globally, but not locally, restrains the replication program in early embryos, possibly by inhibiting or excluding replication factors from chromatin.

CoAlation of the kinase Aurora A induces an open dimer conformation that facilitates its activation.

Besides TopBP1, ETAA1 has been identified more recently as an activator of the ATR-ATRIP complex in human cells. We have examined the role of ETAA1 in the Xenopus egg-extract system, which has been instrumental in the study of ATR-ATRIP. Depletion of ETAA1 from egg extracts did not noticeably reduce the activation of ATR-ATRIP in response to replication stress, as monitored by the ATR-dependent phosphorylation of Chk1 and RPA. Moreover, lack of ETAA1 did not appear to affect DNA replication during an unperturbed S-phase. Significantly, we find that TopBP1 is considerably more abundant than ETAA1 in egg extracts. We proceeded to show that ETAA1 could support the activation of ATR-ATRIP in response to replication stress if we increased its concentration in egg extracts by adding extra full-length recombinant ETAA1. Thus, TopBP1 appears to be the predominant activator of ATR-ATRIP in response to replication stress in this system. We have also explored the biochemical mechanism by which ETAA1 activates ATR-ATRIP. We have developed an in vitro system in which full-length recombinant ETAA1 supports activation of ATR-ATRIP in the presence of defined components. We find that binding of ETAA1 to RPA associated with single-stranded DNA (ssDNA) greatly stimulates its ability to activate ATR-ATRIP. Thus, RPA-coated ssDNA serves as a direct positive effector in the ETAA1-mediated activation of ATR-ATRIP.

DNA topoisomerase II (TopoII) regulates DNA topology by its strand passaging reaction, which is required for genome maintenance by resolving tangled genomic DNA. In addition, TopoII contributes to the structural integrity of mitotic chromosomes and to the activation of cell cycle checkpoints in mitosis. Post-translational modification of TopoII is one of the key mechanisms by which its broad functions are regulated during mitosis. SUMOylation of TopoII is conserved in eukaryotes and plays a critical role in chromosome segregation. Using Xenopus laevis egg extract, we demonstrated previously that TopoIIα is modified by SUMO on mitotic chromosomes and that its activity is modulated via SUMOylation of its lysine at 660. However, both biochemical and genetic analyses indicated that TopoII has multiple SUMOylation sites in addition to Lys660, and the functions of the other SUMOylation sites were not clearly determined. In this study, we identified the SUMOylation sites on the C-terminal domain (CTD) of TopoIIα. CTD SUMOylation did not affect TopoIIα activity, indicating that its function is distinct from that of Lys660 SUMOylation. We found that CTD SUMOylation promotes protein binding and that Claspin, a well-established cell cycle checkpoint mediator, is one of the SUMOylation-dependent binding proteins. Claspin harbors 2 SUMO-interacting motifs (SIMs), and its robust association to mitotic chromosomes requires both the SIMs and TopoIIα-CTD SUMOylation. Claspin localizes to the mitotic centromeres depending on mitotic SUMOylation, suggesting that TopoIIα-CTD SUMOylation regulates the centromeric localization of Claspin. Our findings provide a novel mechanistic insight regarding how TopoIIα-CTD SUMOylation contributes to mitotic centromere activity.

Wee1 inactivates the Cdc2–cyclin B complex during interphase by phosphorylating Cdc2 on Tyr-15. The activity of Wee1 is highly regulated during the cell cycle. In frog egg extracts, it has been established previously that Xenopus Wee1 (Xwee1) is present in a hypophosphorylated, active form during interphase and undergoes down-regulation by extensive phosphorylation at M-phase. We report that Xwee1 is also regulated by association with 14-3-3 proteins. Binding of 14-3-3 to Xwee1 occurs during interphase, but not M-phase, and requires phosphorylation of Xwee1 on Ser-549. A mutant of Xwee1 (S549A) that cannot bind 14-3-3 is substantially less active than wild-type Xwee1 in its ability to phosphorylate Cdc2. This mutation also affects the intranuclear distribution of Xwee1. In cell-free kinase assays, Xchk1 phosphorylates Xwee1 on Ser-549. The results of experiments in which Xwee1, Xchk1, or both were immunodepleted fromXenopus egg extracts suggested that these two enzymes are involved in a common pathway in the DNA replication checkpoint response. Replacement of endogenous Xwee1 with recombinant Xwee1-S549A in egg extracts attenuated the cell cycle delay induced by addition of excess recombinant Xchk1. Taken together, these results suggest that Xchk1 and 14-3-3 proteins act together as positive regulators of Xwee1.