SUMOylation in centromere organization: Regulation of a putative chromatin remodeler, Polo-like kinase 1 interacting checkpoint helicase (PICH) by mitotic SUMOylation

SUMOylation is a posttranslational modification that can mediate diverse cellular processes such as DNA replication, transcription, DNA repair, cell cycle regulation, signal transduction and cell death. Similar to ubiquitination, SUMOylation requires a cascade of enzymes in its conjugation pathway: a SUMO activating E1 enzyme, a SUMO conjugating E2 enzyme and a SUMO E3 ligase. This process of SUMO modification of proteins is reversible and SUMO can be cleaved from its substrate by deSUMOylating enzymes. Mitotic SUMOylation has an essential role in faithful chromosome segregation in eukaryotes, although its molecular consequences are not yet fully understood. Previously, we have identified two mitotic SUMO substrates: Topoisomerase IIalpha and Poly (ADP-ribose) polymerase 1, PARP1 using Xenopus egg extract. In Xenopus egg extract assays, we showed that poly (ADP-ribose) polymerase 1 (PARP1) is modified by SUMO2/3 at mitotic centromeres and that SUMOylation could regulate its enzymatic activity, most likely by mediating substrate specificity. To determine the molecular consequence of mitotic SUMOylation, we analyzed SUMOylated PARP1-specific binding proteins. We identified Polo-like kinase 1- interacting checkpoint helicase (PICH) as an interaction partner of SUMOylated PARP1 in Xenopus egg extract. Interestingly, PICH also bound to SUMOylated Topoisomerase IIalpha (TopoIIalpha), a major centromeric SUMO substrate. Purified recombinant human PICH interacted with SUMOylated substrates, indicating that PICH directly interacts with SUMO, and this interaction is conserved among species. Polo-like kinase 1 (Plk1)-interacting checkpoint helicase (PICH) localizes at the centromere and is critical for proper chromosome segregation during mitosis. However, the precise molecular mechanism of PICH's centromeric localization and function at the centromere is not yet fully understood. We found that PICH is modified by SUMO2/3 on mitotic chromosomes prepared by Xenopus egg extract and in vitro. PICH SUMOylation is highly dependent on PIASy, consistent with other mitotic chromosomal SUMO substrates. Finally, the SUMOylation of PICH significantly reduced its DNA-binding capability, implicating that SUMOylation might regulate its DNA- dependent ATPase activity. To further determine the molecular consequence of PICH/SUMO interaction on PICH function, we identified three SUMO-interacting motifs (SIMs) on PICH and generated a SIM-deficient PICH mutant. Mutation of SIMs on PICH drastically reduced PICH binding to SUMOylated substrate. To understand how SIM on PICH is important during mitosis, we established TET-ON mediated conditional ectopic expression of PICH in HeLa cells. Using the conditional expression of PICH in cells, we found distinct roles of PICH SIMs during mitosis. Although all SIMs are dispensable for PICH's localization on ultrafine anaphase DNA bridges, only SIM3 is critical for its centromeric localization. Intriguingly, the other two SIMs function in chromatin bridge prevention. Currently, using the combination of siRNA and inducible expression of different PICH mutant constructs, we are examining how SUMOylation is spatially and temporally regulating PICH's localization and elucidating its role on centromeric nucleosomes of mitotic chromosomes. With these results, we propose a novel SUMO-dependent regulation of PICH's function on mitotic centromeres. In parallel, we observed SUMO- dependent localization defects on mitotic centromere markers/histones (HP1gamma and H2A.Z). Collectively, our findings suggest SUMOylation dependent centromere organization and a novel SUMO-mediated regulation of PICH's function at mitotic centromeres, which are required for faithful chromosome segregation in mitosis.