Regulation of accurate chromosome segregation by epigenetic cues

Abstract

Post DNA replication, owing to the nature of DNA itself, the replicated genome exists in a highly catenated state. The complete and flawless resolution of the catenated genome, followed by its meticulous partitioning during chromosome segregation in mitosis is essential for the maintenance of genomic stability and thereby, the prevention of tumorigenesis. Topoisomerase II (TopoII) is a key enzyme involved in the resolution of the catenated genome; in particular TopoIIα is indispensable during mitosis. There is a plethora of knowledge in the field regarding the presence of TopoIIα on the mitotic chromosome and its importance during chromosome segregation. However, we do not know yet, how exactly TopoIIα (in contrast to its sister isoform, TopoIIβ) associates with the mitotic chromatin and what factors control its dynamic association with chromosomes. In pursuit of this, we have unraveled a novel mechanism of TopoIIα association with the mitotic chromosomes, dictated by epigenetic cues. We have found that the histone tail posttranslational modification status, particularly that of Histone H3 lysine 27 tri-methylation (H3K27me3) plays a critical role in TopoIIα recruitment to the chromosome to facilitate complete catenated genome resolution. We have also shown that the TopoIIα -H3K27me3 interaction is mediated by TopoIIα ‘s Chromatin Tether (αChT) domain, consistent with previous findings. Through this work, we have demonstrated that mutants of TopoIIα or TopoIIβ, that fail to bind to H3K27me3 on chromatin in vitro, lose their ability to completely resolve the catenated genome, as indicated by the appearance of an abnormally high number of PICH coated Ultra- fine bridges (UFBs) in anaphase. Such mutants with reduced or no H3K27me3 binding capacity also show reduced association with mitotic chromosomes, indicating the importance of this interaction in the proper regulation of TopoIIα dynamics on the chromosome. The relevance of H3K27me3, in particular, was confirmed in our work, through the use of an inhibitor against the H3K27 methyl transferase (GSK- 343), thereby eliminating this epigenetic cue. We noted a dosage-dependent increase in the number of UFBs/cell, indicating remaining catenations in the genome, and we observed no synergistic increase in the number of UFBs/cell with the inhibitor when the cells were replaced with a H3K27me3-binding deficit mutants. Further, there was a decrease in the intensity of centromeric TopoIIα on cells treated with GSK-343. Taken together, these results point to H3K27me3 as a key epigenetic cue that regulates TopoIIα binding through its ChT domain to ensure complete resolution of the catenated genome and thereby genomic stability in a system.In addition, in this work, we have established that the isoforms of Heterochromatin Protein1 (HP1), which are readers of epigenetic information, respond differentially to inhibition of mitotic SUMOylation in the Xenopus Egg Extract (XEE) system. Although it has been established previously that both HP1α and HP1γ are essential for proper mitotic progression and that there are several documented differences observed in their localization patterns, we are the first to establish a relationship between mitotic SUMOylation and isoform specific HP1γ recruitment. HP1α does not require mitotic SUMOylation for its centromeric recruitment on metaphase chromosomes isolated from XEE, but HP1γ does. We also found that this requirement of SUMOylation by HP1γ for its recruitment is conferred to it by its chromodomain and hinge regions. Following this, CRISPR-Cas9 mediated genome edited DLD-1 cell lines were also established in which the HP1 isoforms were independently tagged with mNeon to be able to visualize them. Using these cell lines, it will be possible to extend this work, in terms of understanding the localization differences between the isoforms as well as determining whether the SUMOylation dependency of HP1γ is conserved across species. This work, therefore, sheds light on how the regulation of some of the factors that are essential for proper mitosis is facilitated by a suitably conducive epigenetic environment.