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We previously showed that the silkworm holocentric spindles are square-shaped, compared to the canonical oval shape of human monocentric spindles (Vanpoperinghe et al. 2021). Further, while kinesin-5 depletion resulted in monopolar spindles in both cells, kinesin-14 depletion affected only the silkworm cells, resulting in mal-shaped spindles (Vanpoperinghe et al. 2021). We now extend our study to quantify the effect of kinesin-5 and kinesin-14 on spindle assembly dynamics and chromosome segregation in holocentric silkworm BmN4 cells. We find that mal-shaped spindle and prolonged mitosis duration are highly correlated with chromosome segregation error, leading to aneuploidy and cell death in BmN4 cells. Further, double RNAi-mediated depletion of kinesin-5 and kinesin-14 partially rescue the monopolar spindle and mal-shaped spindle phenotypes in kinesin-5 and kinesin 14-depleted cells, respectively.

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.

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 in a classical model system, the silkwormBombyx 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 chromosome spreads. We show that meiotic pairing is robust in both males and female meiosis. Additionally, we show that meiotic bivalent formation inB. morimales is highly similar to bivalent formation inC. elegans, with both of these pathways ultimately resulting in the pairing of chromosome ends with non-paired ends facing the spindle pole and microtubule recruitment independent of the centromere-specifying factor CENP-A.Author’s SummaryMeiosis 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. We illustrate that Oligopaints are highly specific inB. moriand demonstrate how Oligopaints can be used to study the dynamics of meiotic chromosomes in diverse species.

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.

International audience; 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 (also called CENP-A), which physically interacts with components of the inner kinetochore constitutive centromere-associated network (CCAN). Unexpectedly, regarding 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, that are required for accurate mitotic progression. Our study focuses on CENP-T, which we found to be sufficient to recruit the Mis12 and Ndc80 outer kinetochore complexes. In addition, CRISPR-mediated gene editing in Bombyx mori establishes an essential function of CENP-T in vivo. Finally, the retention of CENP-T and additional CCAN 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, contributing to the emerging picture of an unexpected plasticity to build a kinetochore.

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 fromSaccharomyces cerevisiae, RNAi is present in other budding-yeast species, includingNaumovozyma 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 ofXRN1impaired gene silencing inN. 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.

Over the past few years, interest in chromatin and its evolution has grown. To further advance these interests, we organized a workshop with the support of The Company of Biologists to debate the current state of knowledge regarding the origin and evolution of chromatin. This workshop led to prospective views on the development of a new field of research that we term ‘EvoChromo’. In this short Spotlight article, we define the breadth and expected impact of this new area of scientific inquiry on our understanding of both chromatin and evolution.

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.

Summary Despite the essentiality for faithful chromosome segregation, centromere architectures are diverse among eukaryotes 1 , 2 and embody two main configurations: mono- and holocentromeres, referring, respectively, to localized or unrestricted distribution of centromeric activity. Of the two, some holocentromeres offer the curious condition of having arisen independently in multiple insects, most of which have lost the otherwise essential centromere-specifying factor CenH3 3 (first described as CENP-A in humans). 4 , 5 , 6 , 7 The loss of CenH3 raises intuitive questions about how holocentromeres are organized and regulated in CenH3-lacking insects. Here, we report the first chromatin-level description of CenH3-deficient holocentromeres by leveraging recently identified centromere components 6 , 7 and genomics approaches to map and characterize the holocentromeres of the silk moth Bombyx mori, a representative lepidopteran insect lacking CenH3. This uncovered a robust correlation between the distribution of centromere sites and regions of low chromatin activity along B. mori chromosomes. Transcriptional perturbation experiments recapitulated the exclusion of B. mori centromeres from active chromatin. Based on reciprocal centromere occupancy patterns observed along differentially expressed orthologous genes of Lepidoptera, we further found that holocentromere formation in a manner that is recessive to chromatin dynamics is evolutionarily conserved. Our results help us discuss the plasticity of centromeres in the context of a role for the chromosome-wide chromatin landscape in conferring centromere identity rather than the presence of CenH3. Given the co-occurrence of CenH3 loss and holocentricity in insects, 7 we further propose that the evolutionary establishment of holocentromeres in insects was facilitated through the loss of a CenH3-specified centromere.

The kinetochore is a multi-protein complex that mediates the attachment of a eukaryotic chromosome to the mitotic spindle. The protein composition of kinetochores is similar across species as divergent as yeast and human. However, recent findings have revealed an unexpected degree of compositional diversity in kinetochores. For example, kinetochore proteins that are essential in some species have been lost in others, whereas new kinetochore proteins have emerged in other lineages. Even in lineages with similar kinetochore composition, individual kinetochore proteins have functionally diverged to acquire either essential or redundant roles. Thus, despite functional conservation, the repertoire of kinetochore proteins has undergone recurrent evolutionary turnover.

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.

Histones wrap DNA to form nucleosomes that package eukaryotic genomes. Histone variants have evolved for diverse functions including gene expression, DNA repair, epigenetic silencing, and chromosome segregation. With the rapid increase of newly sequenced genomes the repertoire of histone variants expands, demonstrating a great diversification of these proteins across eukaryotes. In this chapter, we are providing guidelines for the computational characterization and annotation of histone variants. We describe methods to predict the characteristic histone fold domain and list features specific to known histone variants that can be used to categorize newly identified histone fold proteins. We continue describing procedures to retrieve additional related histone variants for comparative sequence analyses and phylogenetic reconstructions to refine the annotation and to determine the evolutionary trajectories of the variant in question.

Histones wrap DNA to form nucleosomes that package eukaryotic genomes. Histone variants have evolved for diverse functions including gene expression, DNA repair, epigenetic silencing, and chromosome segregation. With the rapid increase of newly sequenced genomes the repertoire of histone variants expands, demonstrating a great diversification of these proteins across eukaryotes. In this chapter, we are providing guidelines for the computational characterization and annotation of histone variants. We describe methods to predict the characteristic histone fold domain and list features specific to known histone variants that can be used to categorize newly identified histone fold proteins. We continue describing procedures to retrieve additional related histone variants for comparative sequence analyses and phylogenetic reconstructions to refine the annotation and to determine the evolutionary trajectories of the variant in question.

The kinetochore is the multi-protein complex that drives chromosome segregation in eukaryotes. It assembles onto centromeric DNA and mediates attachment to spindle microtubules. Kinetochore research over the last several decades has been focused on a few animal and fungal model organisms, which revealed a detailed understanding of the composition and organization of their kinetochores. Yet, these traditional model organisms represent only a small fraction of all eukaryotes. To gain insights into the actual degree of kinetochore diversity, it is critical to extend these studies to nontraditional model organisms from evolutionarily distant lineages. In this chapter, we review the current knowledge of kinetochores across diverse eukaryotes with an emphasis on variations that arose in nontraditional model organisms. In addition, we also review the literature on species, in which the subcellular localization of kinetochores has changed from the nucleoplasm to the nuclear membrane. Finally, we speculate on the organization of the chromosome segregation machinery in an early eukaryotic ancestor to gain insights into fundamental principles of the chromosome segregation machinery, which are common to all eukaryotes.

SummaryUsing the yeast Cryptococcus neoformans, we describe a mechanism by which transposons are initially targeted for RNAi-mediated genome defense. We show that intron-containing mRNA precursors template siRNA synthesis. We identify a Spliceosome-Coupled And Nuclear RNAi (SCANR) complex required for siRNA synthesis and demonstrate that it physically associates with the spliceosome. We find that RNAi target transcripts are distinguished by suboptimal introns and abnormally high occupancy on spliceosomes. Functional investigations demonstrate that the stalling of mRNA precursors on spliceosomes is required for siRNA accumulation. Lariat debranching enzyme is also necessary for siRNA production, suggesting a requirement for processing of stalled splicing intermediates. We propose that recognition of mRNA precursors by the SCANR complex is in kinetic competition with splicing, thereby promoting siRNA production from transposon transcripts stalled on spliceosomes. Disparity in the strength of expression signals encoded by transposons versus host genes offers an avenue for the evolution of genome defense.

Yeast Joins RNAi Club RNA interference (RNAi) silences gene expression via small interfering (si) RNAs that bind to target sequences. RNAi has been found in almost all eukaryotes examined, with the notable exception of the budding yeast, Saccharomyces cerevisiae , one of the most prominent organisms used in the study of molecular biology. Indeed, RNAi has been thought to be missing from all budding yeast. Drinnenberg et al. (p. 544 , published online 10 September; see the Perspective by Moazed ) now show that several species of budding yeast do possess RNAi, which seems to act primarily on transposable elements and subtelomeric repeats. A noncanonical Dicer protein generates siRNAs that are loaded onto Ago1 protein in S. castellii . Introduction of these two genes into S. cerevisiae was sufficient to reconstitute RNAi, where it also acted to dampen the activity of transposons.

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 RNA interference (RNAi) pathway is found in most eukaryotic lineages but curiously is absent in others, including that of Saccharomyces cerevisiae. We show that reconstituting RNAi in S. cerevisiae causes loss of a beneficial double-stranded RNA virus known as killer virus. Incompatibility between RNAi and killer viruses extends to other fungal species in that RNAi is absent in all species known to possess double-stranded RNA killer viruses, whereas killer viruses are absent in closely related species that retained RNAi. Thus, the advantage imparted by acquiring and retaining killer viruses explains the persistence of RNAi-deficient species during fungal evolution.

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.