Fate of telomere entanglements is dictated by the timing of anaphase midregion nuclear envelope breakdown.

Persisting replication intermediates can confer mitotic catastrophe. Loss of the fission yeast telomere protein Taz1 (ortholog of mammalian TRF1/TRF2) causes telomeric replication fork (RF) stalling and consequently, telomere entanglements that stretch between segregating mitotic chromosomes. At ≤20 °C, these entanglements fail to resolve, resulting in lethality. Rif1, a conserved DNA replication/repair protein, hinders the resolution of telomere entanglements without affecting their formation. At mitosis, local nuclear envelope (NE) breakdown occurs in the cell's midregion. Here we demonstrate that entanglement resolution occurs in the cytoplasm following this NE breakdown. However, in response to taz1Δ telomeric entanglements, Rif1 delays midregion NE breakdown at ≤20 °C, in turn disfavoring entanglement resolution. Moreover, Rif1 overexpression in an otherwise wild-type setting causes cold-specific NE defects and lethality, which are rescued by membrane fluidization. Hence, NE properties confer the cold-specificity of taz1Δ lethality, which stems from postponement of NE breakdown. We propose that such postponement promotes clearance of simple stalled RFs, but resolution of complex entanglements (involving strand invasion between nonsister telomeres) requires rapid exposure to the cytoplasm.

Parallel genetic screens identify nuclear envelope homeostasis as a key determinant of telomere entanglement resolution in fission yeast.

In fission yeast lacking the telomere binding protein, Taz1, replication forks stall at telomeres, triggering deleterious downstream events. Strand invasion from one taz1Δ telomeric stalled fork to another on a separate (non-sister) chromosome leads to telomere entanglements, which are resolved in mitosis at 32°C; however, entanglement resolution fails at ≤20°C, leading to cold-specific lethality. Previously, we found that loss of the mitotic function of Rif1, a conserved DNA replication and repair factor, suppresses cold sensitivity by promoting resolution of entanglements without affecting entanglement formation. To understand the underlying pathways of mitotic entanglement resolution, we performed a series of genomewide synthetic genetic array screens to generate a comprehensive list of genetic interactors of taz1Δ and rif1Δ. We modified a previously described screening method to ensure that the queried cells were kept in log phase growth. In addition to recapitulating previously identified genetic interactions, we find that loss of genes encoding components of the nuclear pore complex (NPC) promotes telomere disentanglement and suppresses taz1Δ cold sensitivity. We attribute this to more rapid anaphase midregion nuclear envelope (NE) breakdown in the absence of these NPC components. Loss of genes involved in lipid metabolism reverses the ability of rif1+ deletion to suppress taz1Δ cold sensitivity, again pinpointing NE modulation. A rif1+ separation-of-function mutant that specifically loses Rif1's mitotic functions yields similar genetic interactions. Genes promoting membrane fluidity were enriched in a parallel taz1+ synthetic lethal screen at permissive temperature, cementing the idea that the cold specificity of taz1Δ lethality stems from altered NE homeostasis.

RNAi and Ino80 complex control rate limiting translocation step that moves rDNA to eroding telomeres.

The discovery of HAATIrDNA, a telomerase-negative survival mode in which canonical telomeres are replaced with ribosomal DNA (rDNA) repeats that acquire chromosome end-protection capability, raised crucial questions as to how rDNA tracts 'jump' to eroding chromosome ends. Here, we show that HAATIrDNA formation is initiated and limited by a single translocation that juxtaposes rDNA from Chromosome (Chr) III onto subtelomeric elements (STE) on Chr I or II; this rare reaction requires RNAi and the Ino80 nucleosome remodeling complex (Ino80C), thus defining an unforeseen relationship between these two machineries. The unique STE-rDNA junction created by this initial translocation is efficiently copied to the remaining STE chromosome ends, independently of RNAi or Ino80C. Intriguingly, both RNAi and Ino80C machineries contain a component that plays dual roles in HAATI subtype choice. Dcr1 of the RNAi pathway and Iec1 of Ino80C both promote HAATIrDNA formation as part of their respective canonical machineries, but both also inhibit formation of the exceedingly rare HAATISTE (where STE sequences mobilize throughout the genome and assume chromosome end protection capacity) in non-canonical, pathway-independent manners. This work provides a glimpse into a previously unrecognized crosstalk between RNAi and Ino80C in controlling unusual translocation reactions that establish telomere-free linear chromosome ends.

Centromeres are dismantled by foundational meiotic proteins Spo11 and Rec8.

Meiotic processes are potentially dangerous to genome stability and could be disastrous if activated in proliferative cells. Here we show that two key meiosis-defining proteins, the topoisomerase Spo11 (which forms double-strand breaks) and the meiotic cohesin Rec8, can dismantle centromeres. This dismantlement is normally observable only in mutant cells that lack the telomere bouquet, which provides a nuclear microdomain conducive to centromere reassembly1; however, overexpression of Spo11 or Rec8 leads to levels of centromere dismantlement that cannot be countered by the bouquet. Specific nucleosome remodelling factors mediate centromere dismantlement by Spo11 and Rec8. Ectopic expression of either protein in proliferating cells leads to the loss of mitotic kinetochores in both fission yeast and human cells. Hence, while centromeric chromatin has been characterized as extraordinarily stable, Spo11 and Rec8 challenge this stability and may jeopardize kinetochores in cancers that express meiotic proteins.

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Fission yeast telosomes: non-canonical histone-containing chromatin structures dependent on shelterin and RNA.

Despite the prime importance of telomeres in chromosome stability, significant mysteries surround the architecture of telomeric chromatin. Through micrococcal nuclease mapping, we show that fission yeast chromosome ends are assembled into distinct protected structures ('telosomes') encompassing the telomeric DNA repeats and over half a kilobase of subtelomeric DNA. Telosome formation depends on the conserved telomeric proteins Taz1 and Rap1, and surprisingly, RNA. Although yeast telomeres have long been thought to be free of histones, we show that this is not the case; telomere repeats contain histones. While telomeric histone H3 bears the heterochromatic lys9-methyl mark, we show that this mark is dispensable for telosome formation. Therefore, telomeric chromatin is organized at an architectural level, in which telomere-binding proteins and RNAs impose a unique nucleosome arrangement, and a second level, in which histone modifications are superimposed upon the higher order architecture.

Distinct 'safe zones' at the nuclear envelope ensure robust replication of heterochromatic chromosome regions.

Chromosome replication and transcription occur within a complex nuclear milieu whose functional subdomains are beginning to be mapped out. Here we delineate distinct domains of the fission yeast nuclear envelope (NE), focusing on regions enriched for the inner NE protein, Bqt4, or the lamin interacting domain protein, Lem2. Bqt4 is relatively mobile around the NE and acts in two capacities. First, Bqt4 tethers chromosome termini and the mat locus to the NE specifically while these regions are replicating. This positioning is required for accurate heterochromatin replication. Second, Bqt4 mobilizes a subset of Lem2 molecules around the NE to promote pericentric heterochromatin maintenance. Opposing Bqt4-dependent Lem2 mobility are factors that stabilize Lem2 beneath the centrosome, where Lem2 plays a crucial role in kinetochore maintenance. Our data prompt a model in which Bqt4-rich nuclear subdomains are 'safe zones' in which collisions between transcription and replication are averted and heterochromatin is reassembled faithfully.

RNAi drives nonreciprocal translocations at eroding chromosome ends to establish telomere-free linear chromosomes.

The identification of telomerase-negative HAATI (heterochromatin amplification-mediated and telomerase-independent) cells, in which telomeres are superseded by nontelomeric heterochromatin tracts, challenged the idea that canonical telomeres are essential for chromosome linearity and raised crucial questions as to how such tracts translocate to eroding chromosome ends and confer end protection. Here we show that HAATI arises when telomere loss triggers a newly recognized illegitimate translocation pathway that requires RNAi factors. While RNAi is necessary for the translocation events that mobilize ribosomal DNA (rDNA) tracts to all chromosome ends (forming "HAATIrDNA" chromosomes), it is dispensable for HAATIrDNA maintenance. Surprisingly, Dicer (Dcr1) plays a separate, RNAi-independent role in preventing formation of the rare HAATI subtype in which a different repetitive element (the subtelomeric element) replaces telomeres. Using genetics and fusions between shelterin components and rDNA-binding proteins, we mapped the mechanism by which rDNA loci engage crucial end protection factors-despite the absence of telomere repeats-and secure end protection. Sequence analysis of HAATIrDNA genomes allowed us to propose RNA and DNA polymerase template-switching models for the mechanism of RNAi-triggered rDNA translocations. Collectively, our results reveal unforeseen roles for noncoding RNAs (ncRNAs) in assembling a telomere-free chromosome end protection device.

Stretching, scrambling, piercing and entangling: Challenges for telomeres in mitotic and meiotic chromosome segregation.

The consequences of telomere loss or dysfunction become most prominent when cells enter the nuclear division stage of the cell cycle. At this climactic stage when chromosome segregation occurs, telomere fusions or entanglements can lead to chromosome breakage, wreaking havoc on genome stability. Here we review recent progress in understanding the mechanisms of detangling and breaking telomere associations at mitosis, as well as the unique ways in which telomeres are processed to allow regulated sister telomere separation. Moreover, we discuss unexpected roles for telomeres in orchestrating nuclear envelope breakdown and spindle formation, crucial processes for nuclear division. Finally, we discuss the discovery that telomeres create microdomains in the nucleus that are conducive to centromere assembly, cementing the unexpectedly influential role of telomeres in mitosis.

The functionally elusive RabI chromosome configuration directly regulates nuclear membrane remodeling at mitotic onset.

Despite its ubiquity in interphase eukaryotic nuclei, the functional significance of the RabI configuration, in which interphase centromeres are clustered at the nuclear envelope (NE) near the centrosome and telomeres localize at the opposite end of the nucleus, has remained mysterious. In a broad variety of organisms, including Schizosaccharomyces pombe, the RabI configuration is maintained throughout mitotic interphase. The fission yeast linker of nucleoskeleton and cytoskeleton (LINC) complex mediates this centromere association. The functional significance of centromere positioning during interphase has been recently revealed using a conditionally inactivated LINC allele that maintains LINC stability but releases interphase centromere-LINC contacts. Remarkably, this interphase release abolishes mitotic spindle formation. Here, we confirm these observations using an alternative strategy to explore the role of centromere-NE association without modifying the LINC complex. We analyze spindle dynamics in cells lacking Csi1, a stabilizer of centromere-LINC associations, and Lem2, a NE protein harboring lamin interacting domains. We recapitulate these observations and their implications for the functional significance of centromere positioning for cell cycle progression in fission yeast and most likely, a wide range of eukaryotes.

Chromosomes Orchestrate Their Own Liberation: Nuclear Envelope Disassembly.

The mammalian nuclear division cycle is coordinated with nuclear envelope breakdown (NEBD), in which the entire nuclear envelope (NE) is dissolved to allow chromosomes to access their segregation vehicle, the spindle. In other eukaryotes, complete NEBD is replaced by localized disassembly or remodeling of the NE. Although the molecular mechanisms controlling NE disassembly are incompletely understood, coordinated cycles of modification of specific NE components drive breakdown. Here, we review the current state of knowledge regarding NE disassembly and argue for a role of chromosome-NE contacts in triggering initiation of NE disassembly and thereby, cell division.

Life and cancer without telomerase: ALT and other strategies for making sure ends (don't) meet.

While most cancer cells rely on telomerase expression/re-activation for linear chromosome maintenance and sustained proliferation, a significant population of cancers (10-15%) employs telomerase-independent strategies, collectively dubbed Alternative Lengthening of Telomeres (ALT). Most ALT cells relax the usual role of telomeres as inhibitors of local homologous recombination while maintaining the ability of telomeres to prohibit local non-homologous end joining reactions. Here we review current concepts surrounding how ALT telomeres achieve this new balance via alterations in chromatin landscape, DNA damage repair processes and handling of telomeric transcription. We also discuss telomerase independent end maintenance strategies utilized by other organisms, including fruitflies and yeasts, to draw parallels and contrasts and highlight additional modes, beyond ALT, that may be available to telomerase-minus cancers. We conclude by commenting on promises and challenges in the development of effective anti-ALT cancer therapies.

Mitotic Nuclear Envelope Breakdown and Spindle Nucleation Are Controlled by Interphase Contacts between Centromeres and the Nuclear Envelope.

Faithful genome propagation requires coordination between nuclear envelope (NE) breakdown, spindle formation, and chromosomal events. The conserved linker of nucleoskeleton and cytoskeleton (LINC) complex connects fission yeast centromeres and the centrosome, across the NE, during interphase. During meiosis, LINC connects the centrosome with telomeres rather than centromeres. We previously showed that loss of telomere-LINC contacts compromises meiotic spindle formation. Here, we define the precise events regulated by telomere-LINC contacts and address the analogous possibility that centromeres regulate mitotic spindle formation. We develop conditionally inactivated LINC complexes in which the conserved SUN-domain protein Sad1 remains stable but severs interphase centromere-LINC contacts. Strikingly, the loss of such contacts abolishes spindle formation. We pinpoint the defect to a failure in the partial NE breakdown required for centrosome insertion into the NE, a step analogous to mammalian NE breakdown. Thus, interphase chromosome-LINC contacts constitute a cell-cycle control device linking nucleoplasmic and cytoplasmic events.

Rif1 Regulates the Fate of DNA Entanglements during Mitosis.

Clearance of entangled DNA from the anaphase mid-region must accurately proceed in order for chromosomes to segregate with high fidelity. Loss of Taz1 (fission yeast ortholog of human TRF1/TRF2) leads to stalled telomeric replication forks that trigger telomeric entanglements; the resolution of these entanglements fails at ≤20°C. Here, we investigate these entanglements and their promotion by the conserved replication/repair protein Rif1. Rif1 plays no role in taz1Δ fork stalling. Rather, Rif1 localizes to the anaphase mid-region and regulates the resolution of persisting DNA structures. This anaphase role for Rif1 is genetically separate from the role of Rif1 in S/G2, though both roles require binding to PP1 phosphatase, implying spatially and temporally distinct Rif1-regulated phosphatase substrates. Rif1 thus acts as a double-edged sword. Although it inhibits the resolution of taz1Δ telomere entanglements, it promotes the resolution of non-telomeric ultrafine anaphase bridges at ≤20°C. We suggest a unifying model for Rif1's seemingly diverse roles in chromosome segregation in eukaryotes.

Finding a place in the SUN: telomere maintenance in a diverse nuclear landscape.

Telomeres function in the context of a complex nuclear milieu in which telomeres tend to occupy distinct subnuclear regions. Indeed, regulation of the subnuclear positioning of telomeres is conserved from yeast to human, raising the age-old question: to what extent is location important for function? In mitotically dividing cells, the positioning of telomeres affects their epigenetic state and influences telomere processing and synthesis. In meiotic cells, telomere location is important for homologue pairing, centromere assembly and spindle formation. Here we focus on recent insights into the functions of telomere positioning in maintaining genome integrity.

The telomere bouquet regulates meiotic centromere assembly.

The role of the conserved meiotic telomere bouquet has been enigmatic for over a century. We showed previously that disruption of the fission yeast bouquet impairs spindle formation in approximately half of meiotic cells. Surprisingly, bouquet-deficient meiocytes with functional spindles harbour chromosomes that fail to achieve spindle attachment. Kinetochore proteins and the centromeric histone H3 variant Cnp1 fail to localize to those centromeres that exhibit spindle attachment defects in the bouquet's absence. The HP1 orthologue Swi6 also fails to bind these centromeres, suggesting that compromised pericentromeric heterochromatin underlies the kinetochore defects. We find that centromeres are prone to disassembly during meiosis, but this is reversed by localization of centromeres to the telomere-proximal microenvironment, which is conducive to heterochromatin formation and centromere reassembly. Accordingly, artificially tethering a centromere to a telomere rescues the tethered centromere but not other centromeres. These results reveal an unanticipated level of control of centromeres by telomeres.

Telomeres and centromeres have interchangeable roles in promoting meiotic spindle formation.

Telomeres and centromeres have traditionally been considered to perform distinct roles. During meiotic prophase, in a conserved chromosomal configuration called the bouquet, telomeres gather to the nuclear membrane (NM), often near centrosomes. We found previously that upon disruption of the fission yeast bouquet, centrosomes failed to insert into the NM at meiosis I and nucleate bipolar spindles. Hence, the trans-NM association of telomeres with centrosomes during prophase is crucial for efficient spindle formation. Nonetheless, in approximately half of bouquet-deficient meiocytes, spindles form properly. Here, we show that bouquet-deficient cells can successfully undergo meiosis using centromere-centrosome contact instead of telomere-centrosome contact to generate spindle formation. Accordingly, forced association between centromeres and centrosomes fully rescued the spindle defects incurred by bouquet disruption. Telomeres and centromeres both stimulate focal accumulation of the SUN domain protein Sad1 beneath the centrosome, suggesting a molecular underpinning for their shared spindle-generating ability. Our observations demonstrate an unanticipated level of interchangeability between the two most prominent chromosomal landmarks.

The Chromosomal Courtship Dance-homolog pairing in early meiosis.

The intermingling of genomes that characterizes sexual reproduction requires haploid gametes in which parental homologs have recombined. For this, homologs must pair during meiosis. In a crowded nucleus where sequence homology is obscured by the enormous scale and packaging of the genome, partner alignment is no small task. Here we review the early stages of this process. Chromosomes first establish an initial docking site, usually at telomeres or centromeres. The acquisition of chromosome-specific patterns of binding factors facilitates homolog recognition. Chromosomes are then tethered to the nuclear envelope (NE) and subjected to nuclear movements that 'shake off' inappropriate contacts while consolidating homolog associations. Thereafter, homolog connections are stabilized by building the synaptonemal complex or its equivalent and creating genetic crossovers. Recent perspectives on the roles of these stages will be discussed.

A single internal telomere tract ensures meiotic spindle formation.

Contact between telomeres and the fission yeast spindle pole body during meiotic prophase is crucial for subsequent spindle assembly, but the feature of telomeres that confers their ability to promote spindle formation remains mysterious. Here we show that while strains harbouring circular chromosomes devoid of telomere repeat tracts undergo aberrant meiosis with defective spindles, the insertion of a single internal telomere repeat stretch rescues the spindle defects. Moreover, the telomeric overhang-binding protein Pot1 is dispensable for rescue of spindle formation. Hence, an inherent feature of the double-strand telomeric region endows telomeres with the capacity to promote spindle formation.

Myb-domain protein Teb1 controls histone levels and centromere assembly in fission yeast.

The TTAGGG motif is common to two seemingly unrelated dimensions of chromatin function-the vertebrate telomere repeat and the promoter regions of many Schizosaccharomyces pombe genes, including all of those encoding canonical histones. The essential S. pombe protein Teb1 contains two Myb-like DNA binding domains related to those found in telomere proteins and binds the human telomere repeat sequence TTAGGG. Here, we analyse Teb1 binding throughout the genome and the consequences of reduced Teb1 function. Chromatin immunoprecipitation (ChIP)-on-chip analysis reveals robust Teb1 binding at many promoters, notably including all of those controlling canonical histone gene expression. A hypomorphic allele, teb1-1, confers reduced binding and reduced levels of histone transcripts. Prompted by previously suggested connections between histone expression and centromere identity, we examined localization of the centromeric histone H3 variant Cnp1 and found reduced centromeric binding along with reduced centromeric silencing. These data identify Teb1 as a master regulator of histone levels and centromere identity.