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. On the other hand, 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 spindle formation. 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. Having shown previously that stalled telomeric replication forks trigger chromosome entanglement, we have now found a role for a widely conserved protein, Rif1, in controlling the final resolution of such entanglements at anaphase, thus uncovering an unanticipated facet (and time of action) of Rif1's activities and illuminating a final, regulated step of chromosome segregation. We find that this activity of Rif1 is separable from its described roles in controlling replication initiation and the resection of broken chromosome ends, being controlled independently of protein phosphatase binding. Moreover, we have defined several features of the chromosome 'bridges' whose resolution is blocked by Rif1 (e.g., the persistence of replicative DNA polymerases on these bridges), bringing us closer to defining the molecular mechanism of entanglement and its resolution. We have implicated these processes in the resolution of non-telomeric 'ultra-fine anaphase bridges' as well. 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 details of this end-protection are under intense investigation. The sequence jumping is itself 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. In a screen for factors required for HAATI maintenance, we uncovered a role for specific subnuclear domain positioning factor. We have characterized the role of this factor in wild type cells - it is required for tethering heterochromatic domains to the nuclear membrane specifically during the period in which these domains undergo DNA replication. In turn, this positioning delimits the reassembly of heterochromatin. We find that the nuclear membrane is nota homogeneous environment - different domains at the nuclear periphery have different properties and abilities to support chromatin reassembly; we have begun to define how these different domains are organized at the molecular level. 4. 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 highly conserved '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. We now find that centromeres and telomeres share the ability to control spindle formation. We have also begun to define the means by which these specialized chromosome regions regulate spindle formation - while chromatin contact is not required for the duplication of the spindle pole body (SPB), it is required for the nuclear membrane breakdown necessary for the SPB to initiate spindle formation - this step is analogous to controlling nuclear membrane breakdown 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. 5. 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 find interesting dependencies of CDK kinase localization on the bouquet as well as features that are transferrable from telomeres to centromeres. This work in progress aims to define what cell conditions cause centromeres to fall apart, and what environmental factors reverse this. 6. We find a discrete noncanonical chromatin footprint at telomeres and have defined its genetic determinants. We are pursuing the biophysical basis for this pattern and its universality among specialized chromatin regions.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011519-05
Application #
9556618
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Hou, Haitong; Cooper, Julia Promisel (2018) Stretching, scrambling, piercing and entangling: Challenges for telomeres in mitotic and meiotic chromosome segregation. Differentiation 100:12-20
Ebrahimi, Hani; Masuda, Hirohisa; Jain, Devanshi et al. (2018) Distinct 'safe zones' at the nuclear envelope ensure robust replication of heterochromatic chromosome regions. Elife 7:
Begnis, Martina; Apte, Manasi S; Masuda, Hirohisa et al. (2018) RNAi drives nonreciprocal translocations at eroding chromosome ends to establish telomere-free linear chromosomes. Genes Dev 32:537-554
Greenwood, Jessica; Patel, Harshil; Cech, Thomas R et al. (2018) Fission yeast telosomes: non-canonical histone-containing chromatin structures dependent on shelterin and RNA. Nucleic Acids Res :
Fernández-Álvarez, Alfonso; Cooper, Julia Promisel (2017) Chromosomes Orchestrate Their Own Liberation: Nuclear Envelope Disassembly. Trends Cell Biol 27:255-265
Apte, Manasi S; Cooper, Julia Promisel (2017) Life and cancer without telomerase: ALT and other strategies for making sure ends (don't) meet. Crit Rev Biochem Mol Biol 52:57-73
Fernández-Álvarez, Alfonso; Cooper, Julia Promisel (2017) The functionally elusive RabI chromosome configuration directly regulates nuclear membrane remodeling at mitotic onset. Cell Cycle 16:1392-1396
Fernández-Álvarez, Alfonso; Bez, Cécile; O'Toole, Eileen T et al. (2016) Mitotic Nuclear Envelope Breakdown and Spindle Nucleation Are Controlled by Interphase Contacts between Centromeres and the Nuclear Envelope. Dev Cell 39:544-559
Zaaijer, Sophie; Shaikh, Nadeem; Nageshan, Rishi Kumar et al. (2016) Rif1 Regulates the Fate of DNA Entanglements during Mitosis. Cell Rep 16:148-60
Ebrahimi, Hani; Cooper, Julia Promisel (2016) Finding a place in the SUN: telomere maintenance in a diverse nuclear landscape. Curr Opin Cell Biol 40:145-52

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