Telomere anchoring in S phase
Over the last decade the research on telomere anchoring has led to a picture in which many components are in redundant use. In S phase, yKu anchoring might at least partly function through its interaction with Sir4, which itself binds to Esc1 (Figure 3). Nevertheless it is known that yKu can recruit chromatin to the NE in the absence of either Sir4 or Esc1 (Taddei et al., 2004), strongly suggesting another binding partner for yKu at the NE. From my research presented here in the 2nd result chapter, I argue for Mps3 as binding partner for yKu at the NE. The picture that emerges from this study is that yKu which is loaded on TLC1 recruits telomeres to the periphery specifically in S phase. Est2 itself possesses the ability to tether chromatin to the NE and this ability is impaired by the expression of a dominant negative allele of Mps3 that is comprised only of its N’ terminus but lacks the transmembrane domain and is thus diffusively distributed in the nucleus.
Furthermore, I could show that the interaction of yKu with TLC1 is crucial for its ability to recruit chromatin to the NE in S phase. The finding that Mps3 is needed for telomere anchoring in S phase has recently been published by Bupp and colleagues (Bupp et al., 2007) and in particular the study shows that amino acids 75-150 of Mps3 bind to a domain in the C’ terminus of Sir4 (residues 839-1150), partly overlapping with the “partitioning and anchoring domain” of Sir4 (Sir4-PAD, residues 950 to 1262 (Andrulis et al., 2002), which binds to Esc1 (Taddei et al., 2004).
So Sir4 alone has at least two different binding partners at the NE.
Furthermore, Sir4 binds the yKu complex (Taddei et al., 2004), hence yKu can always exploit this interaction in order to recruit chromatin to the NE (Figure 3). Thus far one important question that remains open is whether yKu and Est2 bind directly to Mps3 or if adaptors are required. Also, it is not clear if Est2 mediated anchoring requires Sir4. From my experiments I could show that Est2 can still be recruited to the NE in the absence Esc1, eliminating the Sir4-Esc1 anchoring pathway. Yet since it has become clear that Sir4 can also bind to Mps3 at the NE, and the results of my study suggest that Est2 recruits chromatin to the NE by interacting with Mps3, a simple model for Est2 mediated recruitment would be that Est2 binds to Sir4. Since it has been shown that Mps3 can also interact with Est1 (Antoniacci et al., 2007; Uetz et al., 2000), I propose that the telomerase core complex is recruited to the NE via this Est1 – Mps3 interaction.
How is telomere anchoring mediated in G1?
Myself as well as others have found that telomeres utilize redundant pathways (Sir4 – Esc1, Sir4 – Mps3, telomerase complex - Mps3, see previous section) to localize to the NE in S phase. Other pathways in G1 are likely to rely on as yet unidentified protein-protein interactions. My results show that Est2 is able to recruit chromatin to the NE in G1- as well as in S phase. However, since the presence of Est1 is cell cycle restricted and has been shown to be absent in G1 (Osterhage et al., 2006), Est2 cannot anchor chromatin through Est1 to Mps3. This means that either Est2 binds to Mps3 directly or though another adaptor or that Est2 may bind yet another peripheral protein in G1.
These interactions may depend on proper cohesion loading as suggested by the study of Hiraga and colleagues (Hiraga et al., 2006). After the identification of Mps3 as physical anchor for telomeres that acts predominantly in S phase the individual protein components that anchor in G1 remain unidentified. Maybe cohesin, which is loaded onto chromatin in G1, indeed plays a direct role in this process. In this scenario it binds to a peripherally localized protein and possibly an S phase specific phosphorylation event releases cohesin mediated telomeric anchoring.
Further experiments are needed in order to identify the G1 anchor. I note that it has been shown that deletion of both yku70 and sir4 leads to a complete loss of telomeric anchoring (Hediger et al., 2002) and hence either of the two pathways must be involved in the cohesin pathway of anchoring.
In another set of experiments I set out to visualize elongating telomeres. Monitoring single telomeres while they are elongated suggested
that telomerase action takes place just before mitosis and antagonizes anchoring. In this light, another interpretation of my finding that elongating telomeres shift to a more interior position in the nucleus would be that short telomeres are replicated early in S phase at the NE and that the movement away from the periphery is a means of stopping telomerase from being active.
This model would be consistent with the findings of Bianchi et al (Bianchi and Shore, 2007), who demonstrated that one single telomere that is shortened by forced excision of telomeric repeats shifts its replication from late to early. In this light it seems plausible that at the time when I see the elongating telomere move inwards into the nucleus, replication and telomerase action have already taken place. However, early replication does not necessarily mean that telomerase itself is also active earlier in S phase on these short telomeres. And furthermore, in the system used in my study, the telomerase recipient cell has all of its telomeres critically short due to the absence of telomerase activity. So the zygote which I monitored contains 50% short telomeres (c.f. ~ 3% which is one out of 32 telomeres in a haploid cell), and it is unclear whether 50% of the telomeres in this zygote shift to early replication.
Another difference is the absence of telomerase inhibitor rif1 in my study presented here, which was a necessity to achieve a high level of activity in the first round of replication.
My study on the anchoring of telomeres to the NE contributes to the growing knowledge of telomere biology. In this field it has become evident that telomerase capping and length regulation is regulated on multiple levels as well as temporarily as spatially and my study emphasizes the requirement
of correctly localized core components of telomerase in order to maintain the ends of the linear chromosomes stably across generation of cell division and cell growth.
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Above all I would like to thank my Ph.D. supervisor Susan Gasser. Her passion for science is contagious. Logically, after 5 years of exposure I am infected with this passion, and I have enjoyed very much to be part of her lab.
During this time I have profited from the many benefits that come with working in Susan’s laboratory, which means I had a lot of freedom, the possibility to attend conferences around the world, and if the arguments had rhyme and reason, to carry out the experiments of my choice. Thank you Susan, I really grew up during the time I spend in your lab.
Next, I would like to thank David Shore for being my official Thesis Director and for his unconventional administrational help. Next I would like to thank my thesis committee members; David Shore and Joachim Lingner for having taken the time to read and evaluate my thesis and for giving me the possibility to defend my thesis in Basel.
I was very happy to have the possibility to work with Véronique Kalc.
She does a great job. Busy as a bee she was always there to help me wherever she could: Merci beaucoup Véro, tu es terrible!
Another important person to whom my education owes a lot during the last five years is Angela Taddei. As bench and desk mate, she was always there to listen to my problems and always came up with approaches of how to tackle them. Furthermore she always reviewed my experiments and adverted to me to critical controls. Merci Angela, j’ai appris beaucoup de toi.
A big part of my research I carried out in the last 5 years is based on microscopy and imaging. I enjoyed very much to get my first lessons in this
Thierry! Later on I would have been lost without the advice and help from Peter Meister, thank you very much!
A big “thank you!” goes to Brietta Pike for critically reading and correcting my thesis (between Christmas and New Year!!), your help was indispensable and I am very grateful.
Obviously I would like to thank the lab members in Geneva and later in Basel for the good and inspiring atmosphere, it was great working with you!
Natürlich wäre nichts von all dem möglich gewesen, wenn meine lieben Eltern, Kurt und Ellen Schober, mich nicht immer und überall mit voller Unterstützung bedacht hätten. Tausend Dank, ihr seit toll.