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Accurate chromosome segregation during meiosis is essential for reproductive success. Yet, many fundamental aspects of meiosis remain unclear, including the mechanisms regulating homolog pairing across species. This gap is partially due to our inability to visualize individual chromosomes during meiosis. Here, we employ Oligopaint FISH to investigate homolog pairing and compaction of meiotic chromosomes and resurrect a classical model system, the silkworm Bombyx mori. Our Oligopaint design combines multiplexed barcoding with secondary oligo labeling for high flexibility and low cost. These studies illustrate that Oligopaints are highly specific in whole-mount gonads and on meiotic squashes. We show that meiotic pairing is robust in both males and females and that pairing can occur through numerous partially paired intermediate structures. We also show that pairing in male meiosis occurs asynchronously and seemingly in a transcription-biased manner. Further, we reveal that meiotic bivalent formation in B. mori males is highly similar to bivalent formation in C. elegans, with both of these pathways ultimately resulting in the pairing of chromosome ends with non-paired ends facing the spindle pole. Additionally, microtubule recruitment in both C. elegans and B. mori is likely dependent on kinetochore proteins but independent of the centromere-specifying histone CENP-A. Finally, using super-resolution microscopy in the female germline, we show that homologous chromosomes remain associated at telomere domains in the absence of chiasma and after breakdown and modification to the synaptonemal complex in pachytene. These studies reveal novel insights into mechanisms of meiotic homolog pairing both with or without recombination., Author summary Meiosis is the specialized cell division occurring exclusively in ovaries and testes to produce egg and sperm cells, respectively. The accurate distribution of chromosomes (the genetic material) during this process is essential to prevent infertility/sterility and developmental disorders in offspring. As researchers are specifically unable to study the mechanisms regulating meiosis in depth in humans, identifying broadly conserved aspects of meiotic chromosome segregation is essential for making accurate inferences about human biology. Here, we use a sophisticated chromosome painting approach called Oligopaints to visualize and study chromosomes during meiosis in the silkworm, Bombyx mori. Using this method, we show that chromosome ends (telomeres) play a central role in regulating interactions between maternal and paternal chromosome copies during both sperm and egg development.

Despite their essentiality for chromosome segregation, centromeres are diverse among eukaryotes and embody two main configurations: mono- and holocentromeres, referring respectively to a localized or unrestricted distribution of centromeric activity. Previous studies revealed that holocentricity in many insects coincides with the loss of the otherwise essential centromere component CenH3 (CENP-A), suggesting a molecular link between the two events. In this study, we leveraged recently-identified centromere components to map and characterize the centromeres of Bombyx mori. This uncovered a robust correlation between centromere profiles and regions of low chromatin dynamics. Transcriptional perturbation experiments showed that low chromatin activity is crucial for centromere formation in B. mori. Our study points to a novel mechanism of centromere formation that occurs in a manner recessive to the chromosome-wide chromatin landscape. Based on similar profiles in additional Lepidoptera, we propose an evolutionarily conserved mechanism that underlies the establishment of holocentromeres through loss of centromere specificity.

RNA interference (RNAi) is a gene-silencing pathway that can play roles in viral defense, transposon silencing, heterochromatin formation and post-transcriptional gene silencing. Although absent from Saccharomyces cerevisiae, RNAi is present in other budding-yeast species, including Naumovozyma castellii, which have an unusual Dicer and a conventional Argonaute that are both required for gene silencing. To identify other factors that act in the budding-yeast pathway, we performed an unbiased genetic selection. This selection identified Xrn1p, the cytoplasmic 5′-to-3′ exoribonuclease, as a cofactor of RNAi in budding yeast. Deletion of XRN1 impaired gene silencing in N. castellii, and this impaired silencing was attributable to multiple functions of Xrn1p, including affecting the composition of siRNA species in the cell, influencing the efficiency of siRNA loading into Argonaute, degradation of cleaved passenger strand and degradation of sliced target RNA.

Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. In most eukaryotes, kinetochore assembly is primed by the histone H3 variant CenH3, which physically interacts with components of the inner kinetochore constitutive-centromere-associated-network (CCAN). Unexpected to its critical function, previous work identified that select eukaryotic lineages, including several insects, have lost CenH3, while having retained homologs of the CCAN. These findings imply alternative CCAN assembly pathways in these organisms that function in CenH3-independent manners. Here, we study the composition and assembly of CenH3-deficient kinetochores of Lepidoptera (butterflies and moths). We show that lepidopteran kinetochores consist of previously identified CCAN homologs as well as additional components including a divergent CENP-T homolog, which are required for accurate mitotic progression. Our study focuses on CENP-T that we find both necessary and sufficient to recruit the Mis12 outer kinetochore complex. In addition, CRISPR-mediated gene editing inBombyx moriestablishes an essential function of CENP-Tin vivo. Finally, the retention of CENP-T homologs in other independently-derived CenH3-deficient insects indicates a conserved mechanism of kinetochore assembly between these lineages. Our study provides the first functional insights into CCAN-based kinetochore assembly pathways that function independently of CenH3, thus contributing to the emerging picture of an unexpected plasticity to build a kinetochore.

International audience; The development of new technologies and experimental techniques is enabling researchers to see what was once unable to be seen. For example, the centromere was first seen as the mediator between spindle fiber and chromosome during mitosis and meiosis. Although this continues to be its most prominent role, we now know that the centromere functions beyond cellular division with important roles in genome organization and chromatin regulation. Here we aim to share the structures and functions of centromeres in various organisms beginning with the diversity of their DNA sequence anatomies. We zoom out to describe their position in the nucleus and ultimately detail the different ways they contribute to genome organization and regulation at the spatial level.

Senaratne and Drinnenberg discuss the potential universality of eukaryotic kinetochore proteins based on new work by D’Archivio and Wickstead., The kinetochore drives faithful chromosome segregation in all eukaryotes, yet the underlying machinery is diverse across species. D’Archivio and Wickstead (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201608043) apply sensitive homology predictions to identify proteins in kinetoplastids with similarity to canonical outer kinetochore proteins, suggesting some degree of universality in the eukaryotic kinetochore.

Faithful chromosome segregation in all eukaryotes relies on centromeres, the chromosomal sites that recruit kinetochore proteins and mediate spindle attachment during cell division. The centromeric histone H3 variant, CenH3, is the defining chromatin component of centromeres in most eukaryotes, including animals, fungi, plants, and protists. In this study, using detailed genomic and transcriptome analyses, we show that CenH3 was lost independently in at least four lineages of insects. Each of these lineages represents an independent transition from monocentricity (centromeric determinants localized to a single chromosomal region) to holocentricity (centromeric determinants extended over the entire chromosomal length) as ancient as 300 million years ago. Holocentric insects therefore contain a CenH3-independent centromere, different from almost all the other eukaryotes. We propose that ancient transitions to holocentricity in insects obviated the need to maintain CenH3, which is otherwise essential in most eukaryotes, including other holocentrics. DOI: http://dx.doi.org/10.7554/eLife.03676.001, eLife digest Cell division is a fundamentally important process for living organisms. In eukaryotes, such as plants and animals, genomic DNA is tightly packaged into chromosomes, which needs to be copied and faithfully divided into daughter cells. Segregating the chromosomes is accomplished by the kinetochore, a protein complex that assembles on the chromosome and forms attachments to the machinery that provides the force for chromosome segregation. Kinetochores assemble on specialized chromosomal regions called centromeres. In most eukaryotes, kinetochore assembly relies on a centromeric protein called CenH3 that is essential for the process of chromosome segregation. Most animal and plant species are monocentric—one part of the chromosome is dedicated to CenH3 loading and centromere function and paired chromosomes appear to be joined at a single point, or primary constriction. In contrast, holocentric species instead have centromeric activity distributed along the entire length of the paired chromosomes. How holocentricity arose from monocentricity over the course of evolution remains unclear. Drinnenberg et al. took advantage of the fact that insects represent at least four independent transitions from monocentric to holocentric chromosomes. Several species of insects are holocentric—including butterflies and moths, bugs and lice, earwigs, and dragonflies—while others are monocentric—such as flies, bees, and beetles. Drinnenberg et al. compared the repertoire of kinetochore proteins from each of these insect lineages and found that CenH3 was absent in all the holocentric insects examined but present in all the monocentric insect species. Despite the loss of CenH3 in the holocentric insects, they still had many of the kinetochore proteins—particularly, those proteins that attach to the machinery that forces chromosomes apart. Based on an evolutionary reconstruction, Drinnenberg et al. infer that each independent transition to holocentricity in insects likely introduced changes to the centromere that eliminated the need for the otherwise essential CenH3 protein. This study challenges the notion that CenH3 is essential in all eukaryotes. Indeed, holocentric insects, which make-up 16% of the biodiversity of the currently known eukaryote species, appear to have evolved a completely novel way to define their centromeres, distinct from all the other eukaryotes. DOI: http://dx.doi.org/10.7554/eLife.03676.002

The generation of mature functional RNAs from nascent transcripts requires the precise and coordinated action of numerous RNAs and proteins. One such protein family, the ribonuclease III (RNase III) endonucleases, includes Rnt1, which functions in fungal ribosome and spliceosome biogenesis, and Dicer, which generates the siRNAs of the RNAi pathway. The recent discovery of small RNAs in Candida albicans led us to investigate the function of C. albicans Dicer (CaDcr1). CaDcr1 is capable of generating siRNAs in vitro and is required for siRNA generation in vivo. In addition, CaDCR1 complements a Dicer knockout in Saccharomyces castellii , restoring RNAi-mediated gene repression. Unexpectedly, deletion of the C. albicans CaDCR1 results in a severe slow-growth phenotype, whereas deletion of another core component of the RNAi pathway ( CaAGO1 ) has little effect on growth, suggesting that CaDCR1 may have an essential function in addition to producing siRNAs. Indeed CaDcr1, the sole functional RNase III enzyme in C. albicans , has additional functions: it is required for cleavage of the 3′ external transcribed spacer from unprocessed pre-rRNA and for processing the 3′ tail of snRNA U4. Our results suggest two models whereby the RNase III enzymes of a fungal ancestor, containing both a canonical Dicer and Rnt1, evolved through a series of gene-duplication and gene-loss events to generate the variety of RNase III enzymes found in modern-day budding yeasts.

The kinetochore is a multiprotein complex that assembles on centromeric DNA and enables chromosome segregation during cell division. Essential for kinetochore assembly in most species is the histone variant CenH3 that initiates complex formation. Unexpected to this essential role, it was previously shown that CenH3 was lost in multiple insect orders including the Lepidoptera (butterflies, moths). Despite this loss, recent studies from my host lab revealed that other kinetochore homologs are present in Lepidoptera and must therefore be recruited in a different way. For my PhD I aimed to characterize the assembly of these CenH3-deficientt kinetochores in Bombyx mori cells. In order to address my aim, during the 1st and 2nd year of my PhD, I established tools and protocols in these cells with sparse experimental history. Those include the optimization of RNAI-mediated depletion, characterization of mitotic phenotypes, verification of antibodies and the generation of cell lines containing lacO arrays to able to study kinetochore assembly at ectopic sites. My studies focused on one particular kinetochore component, CENP-T that in other organisms makes important contributions to connect the kinetochore to DNA and to the spindle microtubules. Using the tools that I have generated, I could show that the B. mori CENP-T is essential for mitotic progression and both necessary and sufficient to recruit components to interact with the spindle.; Le kinétochore est un complexe multiprotéique qui s'assemble sur l'ADN centromérique et permet la ségrégation des chromosomes lors de la division cellulaire. Le variant d'histone CenH3 est essentiel pour l'assemblage des kinétochores dans la plupart des espèces où il initie leurs formation. De façon inattendue, compte tenu de leur rôle essentiel, il a été précédemment montré que CenH3 avait été perdu dans plusieurs ordres d'insectes dont les lépidoptères. Malgré cette perte, des études récentes au laboratoire ont révélé que d'autres composants du kinétochore ont des homologues chez les lépidoptères et doivent donc être recrutés de manière différente. Ma thèse a eu pour objectif de comprendre l'assemblage des kinétochores en absence de CenH3 dans des cellules de Bombyx mori. Pour cela, j'ai mis en place des outils dans ces cellules à l'histoire expérimentale sporadique. Par exemple: la déplétion d'un gène par ARNi et la validation des anticorps et la génération de lignées cellulaires contenant des répétitions lacO pour pouvoir étudier l'assemblage des kinétochores sur des sites ectopiques. Mes études se sont concentrées sur un composant particulier du kinétochore, CENP-T, qui dans d'autres organismes, contribue à la liaison du kinétochore à I'ADN et aux microtubules du fuseau. En utilisant les outils que j'ai générés, j'ai pu montrer que B. mori CENP-T est essentiel à la progression mitotique et à la fois nécessaire et suffisant pour recruter les composants interagissants avec le fuseau. Pris ensemble, les résultats de mon projet de doctorat généreront un nouveau système modèle pour l'étude de l'assemblage des kinétochores indépendants de CenH3.

The kinetochore is a multiprotein complex that assembles on centromeric DNA and enables chromosome segregation during cell division. Essential for kinetochore assembly in most species is the histone variant CenH3 that initiates complex formation. Unexpected to this essential role, it was previously shown that CenH3 was lost in multiple insect orders including the Lepidoptera (butterflies, moths). Despite this loss, recent studies from my host lab revealed that other kinetochore homologs are present in Lepidoptera and must therefore be recruited in a different way. For my PhD I aimed to characterize the assembly of these CenH3-deficientt kinetochores in Bombyx mori cells. In order to address my aim, during the 1st and 2nd year of my PhD, I established tools and protocols in these cells with sparse experimental history. Those include the optimization of RNAI-mediated depletion, characterization of mitotic phenotypes, verification of antibodies and the generation of cell lines containing lacO arrays to able to study kinetochore assembly at ectopic sites. My studies focused on one particular kinetochore component, CENP-T that in other organisms makes important contributions to connect the kinetochore to DNA and to the spindle microtubules. Using the tools that I have generated, I could show that the B. mori CENP-T is essential for mitotic progression and both necessary and sufficient to recruit components to interact with the spindle., Le kinétochore est un complexe multiprotéique qui s'assemble sur l'ADN centromérique et permet la ségrégation des chromosomes lors de la division cellulaire. Le variant d'histone CenH3 est essentiel pour l'assemblage des kinétochores dans la plupart des espèces où il initie leurs formation. De façon inattendue, compte tenu de leur rôle essentiel, il a été précédemment montré que CenH3 avait été perdu dans plusieurs ordres d'insectes dont les lépidoptères. Malgré cette perte, des études récentes au laboratoire ont révélé que d'autres composants du kinétochore ont des homologues chez les lépidoptères et doivent donc être recrutés de manière différente. Ma thèse a eu pour objectif de comprendre l'assemblage des kinétochores en absence de CenH3 dans des cellules de Bombyx mori. Pour cela, j'ai mis en place des outils dans ces cellules à l'histoire expérimentale sporadique. Par exemple: la déplétion d'un gène par ARNi et la validation des anticorps et la génération de lignées cellulaires contenant des répétitions lacO pour pouvoir étudier l'assemblage des kinétochores sur des sites ectopiques. Mes études se sont concentrées sur un composant particulier du kinétochore, CENP-T, qui dans d'autres organismes, contribue à la liaison du kinétochore à I'ADN et aux microtubules du fuseau. En utilisant les outils que j'ai générés, j'ai pu montrer que B. mori CENP-T est essentiel à la progression mitotique et à la fois nécessaire et suffisant pour recruter les composants interagissants avec le fuseau. Pris ensemble, les résultats de mon projet de doctorat généreront un nouveau système modèle pour l'étude de l'assemblage des kinétochores indépendants de CenH3.

Despite their essentiality for faithful chromosome segregation, centromere architectures are diverse among eukaryotes and embody two main configurations: mono- and holocentromeres, referring respectively to a localized or unrestricted distribution of centromeric activity. Previous studies revealed that holocentricity in many insects strongly coincides with the loss of the otherwise essential centromeric marker CenH3 (CENP-A), suggesting a molecular link between the two events. In this study, we leveraged recently- identified centromere components and genomics approaches to map and characterize the centromeres of the silk moth Bombyx mori, a holocentric insect representative lacking CenH3. This uncovered a robust correlation between centromere profiles and regions of low chromatin dynamics found anywhere along the chromosome. Transcriptional perturbation experiments showed that centromeres become excluded from active chromatin regions but can form de novo in regions where chromatin activity is low. The identified link to chromatin dynamics helps to discuss the plasticity of centromere identity. In this context, our study points to a novel mechanism of centromere formation that occurs in a manner recessive to the chromosome-wide chromatin landscape rather than being defined by the presence of CenH3. Based on similar profiles observed in additional Lepidoptera, we propose an evolutionarily conserved mechanism that underlies the establishment of holocentromeres through loss of a specified centromere., Malgré leur caractère essentiel pour une ségrégation fidèle des chromosomes, il existe diverses architectures de centromères parmi les eucaryotes représentés en deux principales configurations : mono- et holocentromères pour, respectivement une distribution localisée ou non localisée de l’activité centromérique. De précédentes études ont révélé que l’holocentrisme chez de nombreux insectes coïncide fortement avec la perte du marqueur centromérique – par ailleurs essentiel – CenH3 (CENP-A), suggérant un lien moléculaire entre les deux évènements. Nous avons exploité des éléments de centromères récemment identifiés ainsi que des approches génomiques pour cartographier et caractériser les centromères du vers à soie Bombyx mori, un insecte représentatif holocentrique dépourvu de CenH3. Cela a révélé une corrélation robuste entre les profils de centromères et les régions de faible dynamique de la chromatine trouvée n’importe où le long du chromosome. Des expériences de perturbation transcriptionnelle ont montré que les centromères sont exclus des régions actives de la chromatine, mais peuvent se former de novo dans les régions où l’activité de la chromatine est faible. Le lien identifié avec la chromatine aide à discuter de la plasticité de l’identité du centromère. Dans ce contexte, notre étude pointe vers un nouveau mécanisme de formation des centromères qui se produit de manière récessive sur le chromosome – large paysage chromatinien plutôt que d’être défini par la présence de CenH3. Sur la base de profils similaires observés chez d’autres lépidoptères, nous proposons un mécanisme conservé qui sous-tend l’établissement d’holocentromères par la perte d’un centromère précis.