SUMOylation at the centromere: The role of SUMOylation of the DNA topoisomerase IIα C-terminal domain in the regulation of mitotic kinases in cell cycle progression.

Abstract

In many model systems, SUMOylation is required for proper mitosis; in particular, chromosome segregation during anaphase. It was previously shown that interruption of SUMOylation through the addition of the dominant negative E2 SUMO conjugating enzyme Ubc9 in mitosis causes abnormal chromosome segregation in Xenopus laevis egg extract (XEE) cell-free assays, and DNA topoisomerase IIα (TOP2A) was identified as a substrate for SUMOylation at the mitotic centromeres. TOP2A is SUMOylated at K660 and multiple sites in the C-terminal domain (CTD). We sought to understand the role of TOP2A SUMOylation at the mitotic centromeres by identifying specific binding proteins for SUMOylated TOP2A CTD. Through affinity isolation, we have identified Haspin, a histone H3 threonine 3 (H3T3) kinase, as a SUMOylated TOP2A CTD binding protein. Haspin is important for phosphorylating H3T3 at the centromeres during M phase, which is essential for the recruitment of chromosomal passenger complex (CPC) to the centromere of chromatin for the proper progression of mitosis. However, the mechanism of Haspin localization on the centromere to target centromeric H3T3 was not clearly understood. We determined that Haspin is enriched at the centromeres of sister chromatids with TOP2A in a SUMO-dependent manner in mitotic XEE as interruption of SUMOylation caused a reduction in Haspin’s centromeric localization as well as in centromeric H3T3 phosphorylation (H3T3p). Mutations in TOP2A CTD SUMOylation sites or Haspin’s SUMO-interacting motif (SIM) reduced the binding interaction between TOP2A and Haspin on chromosomes. Haspin and SUMOylated TOP2A CTD interaction is also specific to mitotic XEE. A characteristic of Haspin during mitosis is that Haspin is hyperphosphorylated. We have discovered that T206 phosphorylation regulates TOP2A and Haspin binding interaction as a T206A mutant had a reduction in binding, and the T206A SIM mutant caused a greater binding reduction. This dissertation shows a dual post-translational modification regulation of TOP2A-Haspin interaction that regulates Aurora B at the centromere. Having multiple means in regulating Haspin localization and activity allows for the cell to ensure proper timing in CPC localization for the progression of mitosis. Furthermore, TOP2 inhibitors caused an increase in the SUMOylation of TOP2A as well as a upregulation in the SUMO-binding proteins and Aurora B. Determining the role of TOP2A SUMOylation will help in understanding the mechanism for proper chromosome segregation during mitosis.