During cell division, the full set of chromosomes needs to separate accurately at anaphase to maintain complete genomic information in each daughter cell. Failure of proper chromosome segregation at anaphase leads to biased genomic information in daughter cells, which causes developmental defect and contributes to tumor progression. Precise structural organization of mitotic chromosomes is a key element in maintaining integrity of chromosome segregation, which is regulated by multiple posttranslational protein modification systems. Both genetic and biochemical studies indicate that the posttranslational protein modification by Small Ubiquitin-like MOdifier (SUMO) modification pathway has an important role in normal progression of mitosis. I have found that mitotic specific SUMO-2 modification has a crucial role for completion of faithful chromosome segregation in anaphase in Xenopus egg extract assay system. This mitotic SUMO-2 modification specifically requires the activity of the PIASy protein that is a member of conserved PIAS family of E3. PIASy mediates SUMO-2 modification on multiple mitotic chromosomal proteins. Inhibition of SUMOylation in mitosis causes compromised chromosome segregation at anaphase. The major PIASy-mediated SUMO-2 substrate during mitosis is DNA topoisomerase II (TopoII), which is known to have an essential function in organizing mitotic chromosomes. Inhibition of SUMOylation alters chromosomal association status of TopoII. I hypothesize that the PIASy-mediated mitotic SUMOylation has an essential function in regulating the organization of mitotic chromosomes via its substrates, which plays a crucial role in chromosome segregation at anaphase. I propose to investigate a regulatory mechanism of PIASy specific SUMOylation pathway (Aim1) and a consequence of mitotic SUMOylation with determining the function of TopoII SUMOylation (Aim2) and determining the function of novel PIASy substrates, PARP1 (Aim3) by using Xenopus egg extracts as a model system. This investigation will demonstrate the function of SUMO-2 modification in organization of mitotic chromosomes in vertebrates. Determination of regulatory mechanisms of PIASy mediated SUMOylation will provide an invaluable information for understanding SUMOylation pathway that has an impact on diverse cellular physiological functions. Project Narrative Questions specifically related to separation of genomic DNA in mitosis are important both in understanding the fundamental biology of vertebrate cells and in addressing the molecular basis of human cancers caused by mis-regulation of genomic DNA separation resulting in genomic instability. My proposed research will provide insight into the consequences of protein modification by SUMO during mitosis and regulation of mitotic chromosomes and expected to provide the novel information for improvement of chemotherapeutic agents.
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