An Inducible System for Silencing Establishment Reveals a Stepwise Mechanism in Which Anchoring at the Nuclear Periphery Precedes Heterochromatin Formation.

In eukaryotic cells, silent chromatin is mainly found at the nuclear periphery forming subnuclear compartments that favor silencing establishment. Here, we set up an inducible system to monitor silencing establishment at an ectopic locus in relation with its subnuclear localization in budding yeast. We previously showed that introducing LacI bound lacO arrays in proximity to gene flanked by HML silencers favors the recruitment of the yeast silencing complex SIR at this locus, leading to its silencing and anchoring at the nuclear periphery. Using an inducible version of this system, we show that silencing establishment is a stepwise process occurring over several cell cycles, with the progressive recruitment of the SIR complex. In contrast, we observed a rapid, SIR-independent perinuclear anchoring, induced by the high amount of LacI binding at the lacO array leading to nucleosome eviction at this array and to the phosphorylation of H2A in the neighboring nucleosomes by Mec1 kinase. While the initial phosphorylation of H2A (H2A-P) and perinuclear anchoring are independent of the SIR complex, its latter recruitment stabilizes H2A-P and reinforces the perinuclear anchoring. Finally, we showed that Sir3 spreading stabilizes nucleosomes and limits the access of specific DNA-binding protein to DNA.

Co-translational assembly and localized translation of nucleoporins in nuclear pore complex biogenesis.

mRNA translation is coupled to multiprotein complex assembly in the cytoplasm or to protein delivery into intracellular compartments. Here, by combining systematic RNA immunoprecipitation and single-molecule RNA imaging in yeast, we have provided a complete depiction of the co-translational events involved in the biogenesis of a large multiprotein assembly, the nuclear pore complex (NPC). We report that binary interactions between NPC subunits can be established during translation, in the cytoplasm. Strikingly, the nucleoporins Nup1/Nup2, together with a number of nuclear proteins, are instead translated at nuclear pores, through a mechanism involving interactions between their nascent N-termini and nuclear transport receptors. Uncoupling this co-translational recruitment further triggers the formation of cytoplasmic foci of unassembled polypeptides. Altogether, our data reveal that distinct, spatially segregated modes of co-translational interactions foster the ordered assembly of NPC subunits and that localized translation can ensure the proper delivery of proteins to the pore and the nucleus.

Sir3 mediates long-range chromosome interactions in budding yeast.

Physical contacts between distant loci contribute to regulate genome function. However, the molecular mechanisms responsible for settling and maintaining such interactions remain poorly understood. Here, we investigate the well-conserved interactions between heterochromatin loci. In budding yeast, the 32 telomeres cluster in 3-5 foci in exponentially growing cells. This clustering is functionally linked to the formation of heterochromatin in subtelomeric regions through the recruitment of the silencing SIR complex composed of Sir2/3/4. Combining microscopy and Hi-C on strains expressing different alleles of SIR3, we show that the binding of Sir3 directly promotes long-range contacts between distant regions, including the rDNA, telomeres, and internal Sir3-bound sites. Furthermore, we unveil a new property of Sir3 in promoting rDNA compaction. Finally, using a synthetic approach, we demonstrate that Sir3 can bond loci belonging to different chromosomes together, when targeted to these loci, independently of its interaction with its known partners (Rap1, Sir4), Sir2 activity, or chromosome context. Altogether, these data suggest that Sir3 acts as a molecular bridge that stabilizes long-range interactions.

Quantifying Tubulin Concentration and Microtubule Number Throughout the Fission Yeast Cell Cycle.

The fission yeast Schizosaccharomycespombe serves as a good genetic model organism for the molecular dissection of the microtubule (MT) cytoskeleton. However, analysis of the number and distribution of individual MTs throughout the cell cycle, particularly during mitosis, in living cells is still lacking, making quantitative modelling imprecise. We use quantitative fluorescent imaging and analysis to measure the changes in tubulin concentration and MT number and distribution throughout the cell cycle at a single MT resolution in living cells. In the wild-type cell, both mother and daughter spindle pole body (SPB) nucleate a maximum of 23 ± 6 MTs at the onset of mitosis, which decreases to a minimum of 4 ± 1 MTs at spindle break down. Interphase MT bundles, astral MT bundles, and the post anaphase array (PAA) microtubules are composed primarily of 1 ± 1 individual MT along their lengths. We measure the cellular concentration of αß-tubulin subunits to be ~5 µM throughout the cell cycle, of which one-third is in polymer form during interphase and one-quarter is in polymer form during mitosis. This analysis provides a definitive characterization of αß-tubulin concentration and MT number and distribution in fission yeast and establishes a foundation for future quantitative comparison of mutants defective in MTs.

Spatial reorganization of telomeres in long-lived quiescent cells.

BACKGROUND: The spatiotemporal behavior of chromatin is an important control mechanism of genomic function. Studies in Saccharomyces cerevisiae have broadly contributed to demonstrate the functional importance of nuclear organization. Although in the wild yeast survival depends on their ability to withstand adverse conditions, most of these studies were conducted on cells undergoing exponential growth. In these conditions, as in most eukaryotic cells, silent chromatin that is mainly found at the 32 telomeres accumulates at the nuclear envelope, forming three to five foci. RESULTS: Here, combining live microscopy, DNA FISH and chromosome conformation capture (HiC) techniques, we report that chromosomes adopt distinct organizations according to the metabolic status of the cell. In particular, following carbon source exhaustion the genome of long-lived quiescent cells undergoes a major spatial re-organization driven by the grouping of telomeres into a unique focus or hypercluster localized in the center of the nucleus. This change in genome conformation is specific to quiescent cells able to sustain long-term viability. We further show that reactive oxygen species produced by mitochondrial activity during respiration commit the cell to form a hypercluster upon starvation. Importantly, deleting the gene encoding telomere associated silencing factor SIR3 abolishes telomere grouping and decreases longevity, a defect that is rescued by expressing a silencing defective SIR3 allele competent for hypercluster formation. CONCLUSIONS: Our data show that mitochondrial activity primes cells to group their telomeres into a hypercluster upon starvation, reshaping the genome architecture into a conformation that may contribute to maintain longevity of quiescent cells.

The SENP7 SUMO-Protease Presents a Module of Two HP1 Interaction Motifs that Locks HP1 Protein at Pericentric Heterochromatin.

HP1 enrichment at pericentric heterochromatin is essential for proper chromosome segregation. While H3K9me3 is thought to be a major contributor to HP1 enrichment at pericentric domains, in mouse cells, the SUMO-protease SENP7 is required in addition to H3K9me3. How this is achieved remains elusive. Here, we find that loss of SENP7 leads to an increased time spent in mitosis. Furthermore, we reveal that a short module comprising two consecutive HP1 interaction motifs on SENP7 is the determinant for HP1 enrichment and acts by restricting HP1 mobility at pericentric domains. We propose a mechanism for maintenance of HP1 enrichment in which this module functions on top of H3K9me3 to lock contiguous HP1 molecules already docked on H3K9me3-modified nucleosomes. H3K9me3 would thus promote HP1 enrichment only if a locking system is in place. This mechanism may apply to other nuclear domains to contribute to the control of genome plasticity and integrity.

Scoring and manipulating gene position and dynamics using FROS in budding yeast.

The spatial organization of the genome within the nucleus is now seen as a key contributor to genome function. Studying chromatin dynamics in living cells has been rendered possible by the development of fast microscopy coupled with fluorescent repressor operator systems (FROS). In these systems, arrays of protein-binding sites integrated at specific loci by homologous recombination are monitored through the fluorescence of tagged DNA-binding proteins. In the budding yeast, where homologous recombination is efficient, this technique, combined with targeting assay and genetic analysis, has been extremely powerful for studying the determinants and function of chromatin dynamics in living cells. However, issues have been recurrently raised in different species regarding the use of these systems. Here we discuss the different uses of gene tagging with FROS and their limitations, focusing in budding yeast as a model organism.

Tight protein-DNA interactions favor gene silencing.

The heterochromatin-like structure formed by the yeast silent information regulator complex (SIR) represses transcription at the silent mating type loci and telomeres. Here, we report that tight protein-DNA complexes induce ectopic recruitment of the SIR complex, promoting gene silencing and changes in subnuclear localization when cis-acting elements are nearby. Importantly, lack of the replication fork-associated helicase Rrm3 enhances this induced gene repression. Additionally, Sir3 and Sir4 are enriched genome-wide at natural replication pause sites, including tRNA genes. Consistently, inserting a tRNA gene promotes SIR-mediated silencing of a nearby gene. These results reveal that replication stress arising from tight DNA-protein interactions favors heterochromatin formation.

Clustering heterochromatin: Sir3 promotes telomere clustering independently of silencing in yeast.

A general feature of the nucleus is the organization of repetitive deoxyribonucleic acid sequences in clusters concentrating silencing factors. In budding yeast, we investigated how telomeres cluster in perinuclear foci associated with the silencing complex Sir2-Sir3-Sir4 and found that Sir3 is limiting for telomere clustering. Sir3 overexpression triggers the grouping of telomeric foci into larger foci that relocalize to the nuclear interior and correlate with more stable silencing in subtelomeric regions. Furthermore, we show that Sir3's ability to mediate telomere clustering can be separated from its role in silencing. Indeed, nonacetylable Sir3, which is unable to spread into subtelomeric regions, can mediate telomere clustering independently of Sir2-Sir4 as long as it is targeted to telomeres by the Rap1 protein. Thus, arrays of Sir3 binding sites at telomeres appeared as the sole requirement to promote trans-interactions between telomeres. We propose that similar mechanisms involving proteins able to oligomerize account for long-range interactions that impact genomic functions in many organisms.

Phospho-regulated interaction between kinesin-6 Klp9p and microtubule bundler Ase1p promotes spindle elongation.

The spindle midzone-composed of antiparallel microtubules, microtubule-associated proteins (MAPs), and motors-is the structure responsible for microtubule organization and sliding during anaphase B. In general, MAPs and motors stabilize the midzone and motors produce sliding. We show that fission yeast kinesin-6 motor klp9p binds to the microtubule antiparallel bundler ase1p at the midzone at anaphase B onset. This interaction depends upon the phosphorylation states of klp9p and ase1p. The cyclin-dependent kinase cdc2p phosphorylates and its antagonist phosphatase clp1p dephosphorylates klp9p and ase1p to control the position and timing of klp9p-ase1p interaction. Failure of klp9p-ase1p binding leads to decreased spindle elongation velocity. The ase1p-mediated recruitment of klp9p to the midzone accelerates pole separation, as suggested by computer simulation. Our findings indicate that a phosphorylation switch controls the spatial-temporal interactions of motors and MAPs for proper anaphase B, and suggest a mechanism whereby a specific motor-MAP conformation enables efficient microtubule sliding.

Crosslinkers and motors organize dynamic microtubules to form stable bipolar arrays in fission yeast.

Microtubule (MT) nucleation not only occurs from centrosomes, but also in large part from dispersed nucleation sites. The subsequent sorting of short MTs into networks like the mitotic spindle requires molecular motors that laterally slide overlapping MTs and bundling proteins that statically connect MTs. How bundling proteins interfere with MT sliding is unclear. In bipolar MT bundles in fission yeast, we found that the bundler ase1p localized all along the length of antiparallel MTs, whereas the motor klp2p (kinesin-14) accumulated only at MT plus ends. Consequently, sliding forces could only overcome resistant bundling forces for short, newly nucleated MTs, which were transported to their correct position within bundles. Ase1p thus regulated sliding forces based on polarity and overlap length, and computer simulations showed these mechanisms to be sufficient to generate stable bipolar bundles. By combining motor and bundling proteins, cells can thus dynamically organize stable regions of overlap between cytoskeletal filaments.

Ase1p organizes antiparallel microtubule arrays during interphase and mitosis in fission yeast.

Proper microtubule organization is essential for cellular processes such as organelle positioning during interphase and spindle formation during mitosis. The fission yeast Schizosaccharomyces pombe presents a good model for understanding microtubule organization. We identify fission yeast ase1p, a member of the conserved ASE1/PRC1/MAP65 family of microtubule bundling proteins, which functions in organizing the spindle midzone during mitosis. Using fluorescence live cell imaging, we show that ase1p localizes to sites of microtubule overlaps associated with microtubule organizing centers at both interphase and mitosis. ase1Delta mutants fail to form overlapping antiparallel microtubule bundles, leading to interphase nuclear positioning defects, and premature mitotic spindle collapse. FRAP analysis revealed that interphase ase1p at overlapping microtubule minus ends is highly dynamic. In contrast, mitotic ase1p at microtubule plus ends at the spindle midzone is more stable. We propose that ase1p functions to organize microtubules into overlapping antiparallel bundles both in interphase and mitosis and that ase1p may be differentially regulated through the cell cycle.

The entire Nup107-160 complex, including three new members, is targeted as one entity to kinetochores in mitosis.

In eukaryotes, bidirectional transport of macromolecules between the cytoplasm and the nucleus occurs through elaborate supramolecular structures embedded in the nuclear envelope, the nuclear pore complexes (NPCs). NPCs are composed of multiple copies of approximately 30 different proteins termed nucleoporins, of which several can be biochemically isolated as subcomplexes. One such building block of the NPC, termed the Nup107-160 complex in vertebrates, was so far demonstrated to be composed of six different nucleoporins. Here, we identify three WD (Trp-Asp)-repeat nucleoporins as new members of this complex, two of which, Nup37 and Nup43, are specific to higher eukaryotes. The third new member Seh1 is more loosely associated with the Nup107-160 complex biochemically, but its depletion by RNA interference leads to phenotypes similar to knock down of other constituents of this complex. By combining green fluorescent protein-tagged nucleoporins and specific antibodies, we show that all the constituents of this complex, including Nup37, Nup43, Seh1, and Sec13, are targeted to kinetochores from prophase to anaphase of mitosis. Together, our results indicate that the entire Nup107-160 complex, which comprises nearly one-third of the so-far identified nucleoporins, specifically localizes to kinetochores in mitosis.

The conserved Nup107-160 complex is critical for nuclear pore complex assembly.

Nuclear pore complexes (NPCs) are large multiprotein assemblies that allow traffic between the cytoplasm and the nucleus. During mitosis in higher eukaryotes, the Nuclear Envelope (NE) breaks down and NPCs disassemble. How NPCs reassemble and incorporate into the NE upon mitotic exit is poorly understood. We demonstrate a function for the conserved Nup107-160 complex in this process. Partial in vivo depletion of Nup133 or Nup107 via RNAi in HeLa cells resulted in reduced levels of multiple nucleoporins and decreased NPC density in the NE. Immunodepletion of the entire Nup107-160 complex from in vitro nuclear assembly reactions produced nuclei with a continuous NE but no NPCs. This phenotype was reversible only if Nup107-160 complex was readded before closed NE formation. Depletion also prevented association of FG-repeat nucleoporins with chromatin. We propose a stepwise model in which postmitotic NPC assembly initiates on chromatin via early recruitment of the Nup107-160 complex.