Alterations in O-GlcNAc cycling, the addition and removal of O-GlcNAc, lead to mitotic defects and increased aneuploidy. Herein, we generated stable O-GlcNAcase (OGA, the enzyme that removes OGlcNAc) knockdown HeLa cell lines and characterized the effect of the reduction in OGA activity on cell cycle progression. After release from G 1 /S, the OGA knockdown cells progressed normally through S phase but demonstrated mitotic exit defects. Cyclin A was increased in the knockdown cells while Cyclin B and D expression was reduced. Retinoblastoma protein (RB) phosphorylation was also increased in the knockdown compared to control. At M phase, the knockdown cells showed more compact spindle chromatids than control cells and had a greater percentage of cells with multipolar spindles. Furthermore, the timing of the inhibitory tyrosine phosphorylation of Cyclin Dependent Kinase 1 (CDK1) was altered in the OGA knockdown cells. Although expression and localization of the chromosomal passenger protein complex (CPC) was unchanged, histone H3 threonine 3 phosphorylation was decreased in one of the OGA knockdown cell lines. The Ewing Sarcoma Breakpoint Region 1 Protein (EWS) participates in organizing the CPC at the spindle and is a known substrate for O-GlcNAc transferase (OGT, the enzyme that adds OGlcNAc). EWS O-GlcNAcylation was significantly increased in the OGA knockdown cells promoting uneven localization of the mitotic midzone. Our data suggests that O-GlcNAc cycling is an essential mechanism for proper mitotic signaling and spindle formation, and alterations in the rate of O-GlcNAc cycling produces aberrant spindles and promotes aneuploidy.

Regulated mitotic progression coupled with high‐fidelity chromosome segregation is crucial for normal cellular division. Phosphorylation. Although, phosphorylation plays a critical role regulating mitosis, it alone cannot account for all the complexities of mitosis. We have demonstrated that O‐GlcNAcylation is critical for proper mitotic regulation and disruptions in O‐GlcNAc signaling lead to aberrant cell division. O‐GlcNAc (b‐N‐acetylglucosamine) is an ubiquitous protein modification consisting of a single N‐acetylglucosamine residue attached to Ser or Thr in cytoplasmic and nuclear proteins in response to changes in the cellular environment. Alterations to the proper rate of O‐GlcNAc cycling lead to mitotic defects. The need for detailed O‐GlcNAc site‐mapping information is paramount for our understanding of the role of O‐GlcNAc in regulating mitotic progression. We have developed a combination of tools and methods to site map O‐GlcNAc using a Q Exactive hybrid Quadrupole‐Orbitrap Mass Spectrometer. Interestingly, we have found the Repo‐Man protein to be modified by O‐GlcNAc. Repo‐man is a Protein Phosphatase 1γ binding protein that recruits the phosphatase to the spindle in order to antagonize the function of the spindle kinase Aurora B (AurB). Repo‐man is critical for proper spindle development; loss of Repo‐man expression is lethal. Together, our data demonstrate an efficient and robust method to map O‐GlcNAc sites. The identification of O‐GlcNAc modified mitotic proteins will provide new context as to how O‐GlcNAc cycling regulates mitotic progression and spindle development. NIH‐NHLBI Contract No. HHSN268201000031C, NIH‐GM grant P41 GM104603 and DK100595