How studying the interaction of Sir4 with a subset of Nups uncovered a new chromatin bound complex.

  • Author / Creator
    Lapetina, Diego Lomnitzer
  • The nucleus is the defining feature of eukaryotic cells and its major function is to house the cell’s genome and segregate it from the surrounding cytoplasm. The membranes encapsulating the nucleus constitute the nuclear envelope (NE). The interactions between the NE and chromatin facilitate the spatial organization of chromosomes. Moreover, chromatin positioning inside the nucleus can modulate transcriptional status. The peripheral localization of chromatin is usually associated with gene repression (heterochromatin), and the central regions of the nucleus are often populated by transcriptionally active genes (euchromatin). Yeast have proven to be an important model system for studying the interactions of heterochromatin with the NE membrane, specifically the interaction of telomeres with the inner nuclear membrane proteins. In this study, we expanded on previous observations that nuclear pore complexes (NPCs) are required for telomere tethering and gene silencing. We showed that four proteins involved in chromatin regulation (Nup170, Siz2, Sir4, and Esc1) physically and functionally interact with one another. Furthermore, these proteins also physically interact with nucleoporins present at the NPC core scaffold. Importantly, the structure formed by the NPC core scaffold, Sir4, Esc1 and Siz2 lacks Nups present in other NPC sub-complexes. We termed this structure as the Sir4 associated Nups complex (Snups). The detection of proteins involved in telomere positioning suggested that Snup complexes function in the tethering of telomeres to the NE. In our analysis of this complex, we observed that SUMO ligase, Siz2, was bound to proteins from the Snup complex. This observation was intriguing as SUMOylation, and specifically Siz2, has been implicated in tethering telomeres to the NE. Given the importance of Siz2 in telomere tethering, we decided to better characterize its cellular distribution and protein stability. Previous observations suggested that Siz2 is present through out the nucleoplasm. However, we observed that Siz2 was also detectable at the NE, consistent with the physical association we observed between Siz2 and proteins from the Snup complex. We also observed a robust recruitment of Siz2 to the NE during mitosis, coinciding with its phosphorylation. We identified the sites of phosphorylation in Siz2 and constructed point mutants that block Siz2 phosphorylation. Using these mutants, we show that the recruitment of Siz2 to the NE during mitosis is required for the proper association of telomeres with the NE.

  • Subjects / Keywords
  • Graduation date
    Fall 2017
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
  • Supervisor / co-supervisor and their department(s)
  • Examining committee members and their departments
    • Schultz, Michael (Biochemistry)
    • LaPointe, Paul (Cell Biology)
    • Wozniak, Richard W. (Cell Biology)
    • Fabre, Emmanuelle (Jean Bernard - University of Paris)
    • Underhill, Alan (Oncology)