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 by either addition of mutant form of Ubc9, SUMO E2, or anti-PIASy neutralizing antibodies causes compromised chromosome segregation at anaphase. The major PIASy-mediated SUMO-2 substrate during mitosis is DMA topoisomerase II (Topoll), which is known to have an essential function in organizing mitotic chromosomes. Inhibition of SUMOylation alters the chromosomal association status of Topoll. 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 Topoll SUMOylation (Aim2) and identifying novel PIASy substrates (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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Special Emphasis Panel (ZRG1-NDT-K (01))
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Hamlet, Michelle R
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University of Kansas Lawrence
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Kaur, Kawaljit; Park, Hyewon; Pandey, Nootan et al. (2017) Identification of a new small ubiquitin-like modifier (SUMO)-interacting motif in the E3 ligase PIASy. J Biol Chem 292:10230-10238
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Sridharan, Vinidhra; Park, Hyewon; Ryu, Hyunju et al. (2015) SUMOylation regulates polo-like kinase 1-interacting checkpoint helicase (PICH) during mitosis. J Biol Chem 290:3269-76
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Ryu, Hyunju; Gygi, Steven P; Azuma, Yoshiaki et al. (2014) SUMOylation of Psmd1 controls Adrm1 interaction with the proteasome. Cell Rep 7:1842-8
Al-Ani, Gada; Briggs, Koan; Malik, Shuja Shafi et al. (2014) Quantitative determination of binding of ISWI to nucleosomes and DNA shows allosteric regulation of DNA binding by nucleotides. Biochemistry 53:4334-45
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Ryu, Hyunju; Azuma, Yoshiaki (2010) Rod/Zw10 complex is required for PIASy-dependent centromeric SUMOylation. J Biol Chem 285:32576-85
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Ryu, Hyunju; Al-Ani, Gada; Deckert, Katelyn et al. (2010) PIASy mediates SUMO-2/3 conjugation of poly(ADP-ribose) polymerase 1 (PARP1) on mitotic chromosomes. J Biol Chem 285:14415-23

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