Telomere interactions and signaling during the cell division cycle

Telomeres are formed by specific DNA sequences at the ends of the chromosomes and their shortening during cell division plays an important role in limiting proliferation. Recently, it has become apparent that telomere signaling can also be induced in cancer cells delayed in mitosis, leading to activation of the ATM kinase and subsequent activation of the p53 tumor suppressor (Hayashi et al, 2012). This response can be induced by drugs such as taxol (paclitaxel) that inhibit progression through mitosis. This response may be very important in determining the balance between cell survival and cell death in response to taxol (Colin et al).

 A key question is what are the molecular mechanisms that regulate signaling at telomeres following mitotic arrest and how is the cellular response controlled. To investigate the molecular interactions of human telomeres during mitotic exit and early G1 we will employ the BioID method described by Roux et al. (Roux et al., 2012) BioID takes advantage of BirA*, a promiscuous mutant of the E. coli protein biotin ligase, BirA. When expressed in mammalian cells growing in medium supplemented with 50µM biotin, BirA* will biotinylate neighbouring proteins. These proteins may then be recovered on magnetic beads conjugated with streptavidin and identified by mass spectrometry. To examine potential protein-protein interactions, BirA* may be fused to any protein of interest (a bait protein). Expression of the fusion protein should then yield a population of biotinylated proteins that are enriched in those that are either proximal to or interact with the bait in vivo. This method has been applied successfully in several systems to identify proteins that associate with A-type lamins (Roux et al., 2012), the junctional protein ZO-1 (Van Itallie et al., 2013) and cytoskeletal components in Trypanosoma brucei (Morriswood et al., 2013).

One of us (Oliver Dreesen, IMB, Singapore) recently constructed cell lines expressing a doxycycline-inducible BirA*-TRF1 fusion protein. TRF1 is a telomere repeat-binding factor that is a component of the shelterin complex (De Lange, 2005). Like wild-type TRF1 the BirA*-TRF1 fusion protein localizes precisely to telomeres. This cell line will, therefore, provide us with a unique tool with which we can examine the changing interactions of telomeres during different stages of the cell cycle.

References

  1. Hayashi MT, Cesare AJ, Fitzpatrick JA, Lazzerini-Denchi E, and Karlseder J. 2012 A telomere-dependent DNA damage checkpoint induced by prolonged mitotic arrest. Nat Struct Mol Biol. 2012 19:387-394
  2. Colin DJ, Allan LA and Clarke PR. Cellular responses to microtubule poisons are determined by DNA damage signalling initiated at telomeres during delayed mitosis (unpublished).
  3. De Lange T. 2005. Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev. 19. 2100-2110.
  4. Morriswood B, Havlicek K, Demmel L, Yavuz S, Sealey-Cardona M, Vidilaseris K, Anrather D, Kostan J, Djinovic-Carugo K, Roux K, and Warren G. 2013. Novel Bilobe Components in Trypanosoma brucei Identified Using Proximity-Dependent Biotinylation. Eukaryotic Cell. 12:356-367.
  5. Roux KJ, Kim DI, Raida M, and Burke B. 2012. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol. 196, 801-810.
  6. Van Itallie CM, Aponte A, Tietgens AJ, Gucek M, Fredriksson K, and Anderson JM. 2013. The N- and C- termini of ZO-1 are surrounded by distinct proteins and functional protein networks. J Biol Chem. 288, 13775-13788.
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Professor Paul Clarke
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Eligible PhD students from the University of Dundee will spend 1 to 2 years of their PhD at an A*STAR research institute under the joint supervision of staff at the University of Dundee and an A*STAR research institute.