The termini of eukaryotic chromosomes are potentially dangerous sites, as their resemblance to damage-induced DNA breaks makes them vulnerable to degradation and end-joining pathways that provoke cancer. Telomeres protect chromosome ends from these hazards. Conversely, we are increasingly recognizing that telomeres provide regulatory and choreography-related opportunities to the cell - for instance, their heterochromatic nature along with their tendency to cluster together allows telomeres to create subnuclear 'micro environments' that can be exploited to promote crucial nuclear activities like centromere assembly and the regulation of local nuclear envelope breakdown. Fission yeast telomeres are remarkably similar to those of human but present substantial experimental benefits, like precise genetic manipulability. The components of human 'shelterin' are also found in fission yeast and we are building an integrated picture of how these proteins interact to protect chromosome ends. We have also identified unforeseen additional functions of telomeres that are likely to be widely conserved. Advances over the past year include: 1. Previously we found that stalled telomeric replication forks trigger chromosome entanglement in a manner dependent on the widely conserved protein, Rif1; these entanglements control the final segregation of chromosomes at anaphase. We find that this activity of Rif1 is separable from its described roles in controlling replication initiation and the resection of broken chromosome ends. Moreover, we have found that Rif1 acts as a 'double edged sword', blocking telomeric entanglement resolution while promoting centromeric entanglement resolution. We carried out a genetic screen that uncovered a key part of the mechanism by which Rif1 controls chromosome derangement at anaphase, finding that Rif1 mediates enhanced spindle persistence in response to unsegregated chromosomes stretching across the cell mid zone. Spindle persistence is in turn good for centromere derangement but bad for telomere derangement. We find that the relevant difference between the telomeric and centromeric entanglements is the presence of nonsister associations among the telomeric variety. 2. Survival without telomeres - Cells can occasionally survive the absence of telomerase, by maintaining telomeres via recombination or by circularizing their chromosomes. We had identified a third mode of telomerase-minus survival in which linear chromosomes are maintained using a strategy we dubbed 'HAATI' (heterochromatin amplification-mediated and telomerase independent). In HAATI cells, telomere repeats are absent but tracts of 'generic' heterochromatin jump to each chromosome end. This heterochromatin, along with a non-telomeric terminal 3'-overhang, recruits Pot1, which is essential for HAATI chromosome linearity. This discovery revealed an alternative mode by which cancer cells might survive without telomerase activation. We have now found that HAATI formation is limited only by the chromosome rearrangements that place generic heterochromatin at HAATI chromosome ends - once this 'jumping' of DNA sequences occurs, the cell has no problem in engaging chromosome end-protection at the non-telomeric chromosome termini. The sequence jumping involves non homologous DNA translocations by an as-yet-undefined mechanism we are studying. We know the translocations are controlled by the RNAi pathway in both a canonical and a non-canonical capacity. We have also uncovered a role for the chromatin remodeling Ino80 complex in maintaining an unusual form of HAATI, providing the first foothold into understanding this genome-disrupting mode. 3. The spindles that form at mitosis and meiosis are often thought of as semi-autonomous architectural structures that control the movement of chromosomes. Our recent findings overturned this notion by revealing that telomeres, which gather together near the centrosome in early stages of meiosis to form the highlyconserved 'bouquet', regulate the formation of meiotic spindles. These observations raise exciting and novel questions about mechanisms by which chromosomes control cell cycle progression during both meiosis and mitosis. Moreover, we find that while chromatin contact is not required for the duplication of the spindle pole body (SPB), it is required for the partial nuclear envelope breakdown (NEBD) necessary for the SPB to initiate spindle formation - this step is analogous to controlling initiation of the complete NEBD that occurs upon mitotic and meiotic onset in mammalian cells. Excitingly, we find that the centromere controls this step in mitotic cell cycles, uncovering a newly recognized layer of chromosomal control of cell cycle progression. We are investigating what biophysical and biochemical properties of telomeres and centromeres endows them with NEBD-controlling activity. We have data implicating phase separation properties in conferring this NEBD control by telomeres and centromeres. 4. In bouquet-deficient cells forming proper meiotic spindles, the attachment of chromosomes, via their centromeres, to those spindles often fails. We find that the domain surrounding the telomere bouquet constitutes a nuclear microenvironment conducive to centromere assembly. Moreover, we find that centromeres are prone to dismantling during meiosis, making this telomeric environment particularly crucial as centromeres need to be reassembled. We have uncovered key features of the mechanism by which centromeres become dismantled upon meiotic entry; it involves the key meiotic proteins, the meiotic cohesin and DNA break-inducing protein required for meiotic homologous recombination. These proteins evict centromeric nucleosomes and moreover, their ectopic expression in proliferating cells also evicts centromeric nucleosomes. We have extended this study to human cells as cancer cells often misexpress these proteins. We are also defining exactly how telomeres promote the reassembly of compromised centromeres; this pathway involves telomeric heterochromatin and its ability to recruit Aurora B kinases. 5. We find a discrete noncanonical chromatin footprint at telomeres and have defined its genetic determinants; this is accompanied by an altered torsional state at telomeres. We are pursuing the biophysical basis for this pattern and its universality among specialized chromatin regions.
Showing the most recent 10 out of 19 publications
