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GFP::CPAR-1 signal is lost from chromosomes at the metaphase-anaphase transition of oocyte meiosis I. Image frames from time-lapse sequences of GFP::CPAR-1 (OD416) and GFP::HCP-3 (OD421) meiosis I embryos (single copy insertions as in Fig 1). Chromosomes were labeled with mCherry::H2b. In the GFP::HCP-3 strain, the GFP fusion is the sole source of HCP-3. The images shown are from prometaphase through late anaphase. Metaphase is defined as the first frame in which chromosomes are aligned and spindle is rotated to be perpendicular to cortex. Schematics on the left indicate the stages shown. Similar results were obtained for n = 10 time-lapse sequences. Scale bar, 5 μm.
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Centromeres are defined epigenetically in the majority of eukaryotes by the presence of chromatin containing the centromeric histone H3 variant CENP-A. Most species have a single gene encoding a centromeric histone variant whereas C. elegans has two: HCP-3 (also known as CeCENP-A) and CPAR-1. Prior RNAi replacement experiments showed that HCP-3 is...
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Context 1
... signal continued to persist on chromosomes during anaphase segre- gation (S1 Movie). By contrast, GFP::CPAR-1 signal was abruptly lost at the meiosis I meta- phase-anaphase transition (Fig 2 and S2 Movie). Consistent with the observed timing of signal loss, no GFP::CPAR-1 signal was observed on chromosomes during meiosis II or embryonic mitotic divisions ( Fig 1C). ...
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... distinguish be- tween these two possibilities, we generated pie-1 promoter-driven transgenes expressing GFP:: CPAR-1 (OD82) or CPAR-1::GFP (OD145)-where GFP was fused to the C-terminus of CPAR-1, distal to the histone fold rather than to the N-terminal tail-and integrated them into the genome using biolistic transformation [28]. Similar to the endogenous promoter-driven GFP::CPAR-1 (Fig 2), the pie-1 promoter-driven GFP::CPAR-1 signal was lost at the meta- phase-anaphase transition of meiosis I. CPAR-1::GFP localized similarly to GFP::CPAR-1 on bivalent chromosomes in oocytes and in prometaphase and metaphase of meiosis I (Fig 3A and S3 Movie). However, no loss of GFP signal from chromosomes was observed for CPAR-1:: GFP ( Fig 3A and S3 Movie). ...
Context 3
... WT GFP::CPAR-1, mCherry::H2b was crossed in and imaged at the same time to highlight the abrupt loss of GFP fluorescence. No loss of GFP fluorescence was observed at that the EQAR sequence is conserved in HCP-3, which does not appear to be cleaved by separ- ase to the same degree as CPAR-1 (Fig 2). We speculate that this difference could be due to two reasons. ...
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Citations
... Separase also cleaves itself (31), as well as some non-cohesin proteins (32)(33)(34), and also has distinct non-enzymatic regulatory functions (9,(35)(36)(37)(38). ...
Separase is a protease that performs critical functions in the maintenance of genetic homeostasis. Among them, the cleavage of the meiotic cohesin during meiosis is a key step in producing gametes in eukaryotes. However, the exact chromosomal localization of this proteolytic cleavage was not addressed due to the lack of experimental tools. To this end, we developed a method based on monoclonal antibodies capable of recognizing the predicted neo-epitopes produced by separase-mediated proteolysis in the RAD21 and REC8 cohesin subunits. To validate the epigenomic strategy of mapping cohesin proteolysis, anti-RAD21 neo-epitopes antibodies were used in ChIP-On-ChEPseq analysis of human cells undergoing mitotic anaphase. Second, a similar analysis applied for mapping of REC8 cleavage in germline cells in Macaque showed a correlation with a subset of alpha-satellites and other repeats, directly demonstrating that the site-specific mei-cohesin proteolysis hotspots are coincident but not identical with centromeres. The sequences for the corresponding immunoglobulin genes show a convergence of antibodies with close specificity. This approach could be potentially used to investigate cohesin ring opening events in other chromosomal locations, if applied to single cells.
... However, HCP-3 appears to be dispensable for oocyte meiotic segregation (Monen et al. 2005). A second CenH3 paralog in C. elegans, CPAR-1, shares high sequence similarity to HCP-3 in the histone fold domain (HFD) but is diverged in the N-terminal domain (Monen et al. 2015). Although CPAR-1 is enriched in meiotic chromosomes, it does not appear to localize to centromeres at all, and its precise function is not well understood (Gassmann et al. 2012;Monen et al. 2015). ...
... A second CenH3 paralog in C. elegans, CPAR-1, shares high sequence similarity to HCP-3 in the histone fold domain (HFD) but is diverged in the N-terminal domain (Monen et al. 2015). Although CPAR-1 is enriched in meiotic chromosomes, it does not appear to localize to centromeres at all, and its precise function is not well understood (Gassmann et al. 2012;Monen et al. 2015). An independent hcp-3 duplication occurred in a related species, C. remanei (Monen et al. 2015), but its function is also unknown. ...
... Although CPAR-1 is enriched in meiotic chromosomes, it does not appear to localize to centromeres at all, and its precise function is not well understood (Gassmann et al. 2012;Monen et al. 2015). An independent hcp-3 duplication occurred in a related species, C. remanei (Monen et al. 2015), but its function is also unknown. These previous studies left unclear whether CenH3 duplications in C. elegans and C. remanei were unusually rare or typical of Caenorhabditis nematodes. ...
Centromeric histones (CenH3s) are essential for chromosome inheritance during cell division in most eukaryotes. CenH3 genes have rapidly evolved and undergone repeated gene duplications and diversification in many plant and animal species. In Caenorhabditis species, two independent duplications of CenH3 (named hcp-3 for HoloCentric chromosome-binding Protein 3) were previously identified in C. elegans and C. remanei. Using phylogenomic analyses in thirty-two Caenorhabditis species, we find strict retention of the ancestral hcp-3 gene and ten independent duplications. Most hcp-3L (hcp-3-like) paralogs are only found in 1-2 species, are expressed in both males and females/hermaphrodites, and encode histone fold domains with 69-100% identity to ancestral hcp-3. We identified novel N-terminal protein motifs, including putative kinetochore protein-interacting motifs and a potential separase cleavage site, which are well conserved across Caenorhabditis HCP-3 proteins. Other N-terminal motifs vary in their retention across paralogs or species, revealing potential sub-functionalization or functional loss following duplication. An N-terminal extension in the hcp-3L gene of C. afra revealed an unprecedented protein fusion, where hcp-3L fused to duplicated segments from hcp-4 (nematode CENP-C). By extending our analyses beyond CenH3, we found gene duplications of six inner and outer kinetochore genes in Caenorhabditis, which appear to have been retained independent of hcp-3 duplications. Our findings suggest that centromeric protein duplications occur frequently in Caenorhabditis nematodes, are selectively retained for short evolutionary periods, then degenerate or are lost entirely. We hypothesize that unique challenges associated with holocentricity in Caenorhabditis may lead to this rapid ‘revolving door’ of kinetochore protein paralogs.
... In contrast to plants, centromeric histone specialization has not been previously observed in animal species. Although an estimated 10% of plant genomes harbor multiple CenH3 paralogs (Kawabe et al, 2006;Finseth et al, 2015;Maheshwari et al, 2015), CenH3 duplications were previously thought to be rare in animals (Li & Huang, 2008;Monen et al, 2015). Contrary to this view, the CenH3 gene (known as Cid) has duplicated at least four times in Drosophila (Kursel & Malik, 2017) and at least three times in mosquitoes (Kursel et al, 2020). ...
In most eukaryotes, centromeric histone (CenH3) proteins mediate mitosis and meiosis and ensure epigenetic inheritance of centromere identity. We hypothesized that disparate chromatin environments in soma versus germline might impose divergent functional requirements on single CenH3 genes, which could be ameliorated by gene duplications and subsequent specialization. Here, we analyzed the cytological localization of two recently identified CenH3 paralogs, Cid1 and Cid5, in Drosophila virilis using specific antibodies and epitope-tagged transgenic strains. We find that only ancestral Cid1 is present in somatic cells, whereas both Cid1 and Cid5 are expressed in testes and ovaries. However, Cid1 is lost in male meiosis but retained throughout oogenesis, whereas Cid5 is lost during female meiosis but retained in mature sperm. Following fertilization, only Cid1 is detectable in the early embryo, suggesting that maternally deposited Cid1 is rapidly loaded onto paternal centromeres during the protamine-to-histone transition. Our studies reveal mutually exclusive gametic specialization of divergent CenH3 paralogs. Duplication and divergence might allow essential centromeric genes to resolve an intralocus conflict between maternal and paternal centromeric requirements in many animal species.
... HCP-3 has been proven to be essential for mitosis but is not required for meiotic kinetochore formation or chromosome segregation (Monen et al. 2005). Although the functional importance of Cpar-1 is not completely understood, its enrichment on meiotic chromosomes was documented (Monen et al. 2015). In addition, recent data on Arabidopsis suggest that in species with a single-copy CenH3 gene, one protein probably must be customized for different centromere functions (Ravi et al. 2011). ...
Although centromeres have conserved function, centromere-specific histone H3 (CenH3) and centromeric DNA evolve rapidly. The centromere drive model explains this phenomenon as a consequence of the conflict between fast evolving DNA and CenH3, suggesting asymmetry in female meiosis as a crucial factor. We characterized evolution of the CenH3 protein in three closely related, polyploid mitotic parthenogenetic species of the Meloidogyne incognita group (MIG), and in the distantly related meiotic parthenogen Meloidogyne hapla. We identified duplication of the CenH3 gene in a putative sexual ancestral Meloidogyne. We found that one CenH3 (αCenH3) copy remained conserved in all extant species, including in distant Meloidogyne hapla, while the other copy evolved rapidly and under positive selection into four different CenH3 variants. This pattern of CenH3 evolution in Meloidogyne species suggests the sub-specialization of CenH3s in ancestral sexual species. Immunofluorescence performed on mitotic Meloidogyne incognita revealed a dominant role of αCenH3 on its centromere, while the other CenH3s have lost their function in mitosis. The observed αCenH3 chromosome distribution disclosed cluster-like centromeric organization. The ChIP-Seq analysis revealed that in M. incognita αCenH3-associated DNA dominantly comprises tandem repeats, composed of divergent monomers which share a completely conserved 19 bp-long box. Conserved αCenH3-associated DNA are also confirmed in the related mitotic MIG species suggesting preservation of both centromere protein and DNA constituents. We hypothesize that the absence of centromere drive in mitosis might allow for CenH3 and its associated DNA to achieve an equilibrium in which they can persist for long periods of time.
... In addition to α kleisins and separases, only three additional separase substrates have been identified (Slk19, kendrin/pericentrin, and CPAR-1) [59,[64][65][66]. These additional substrates are not cohesin subunits. ...
Regular chromosome segregation during the first meiotic division requires prior pairing of homologous chromosomes into bivalents. During canonical meiosis, linkage between homologous chromosomes is maintained until late metaphase I by chiasmata resulting from meiotic recombination in combination with distal sister chromatid cohesion. Separase-mediated elimination of cohesin from chromosome arms at the end of metaphase I permits terminalization of chiasmata and homolog segregation to opposite spindle poles during anaphase I. Interestingly, separase is also required for bivalent splitting during meiosis I in Drosophila males, where homologs are conjoined by an alternative mechanism independent of meiotic recombination and cohesin. Here we report the identification of a novel alternative homolog conjunction protein encoded by the previously uncharacterized gene univalents only (uno). The univalents that are present in uno null mutants at the start of meiosis I, instead of normal bivalents, are segregated randomly. In wild type, UNO protein is detected in dots associated with bivalent chromosomes and most abundantly at the localized pairing site of the sex chromosomes. UNO is cleaved by separase. Expression of a mutant UNO version with a non-functional separase cleavage site restores homolog conjunction in a uno null background. However, separation of bivalents during meiosis I is completely abrogated by this non-cleavable UNO version. Therefore, we propose that homolog separation during Drosophila male meiosis I is triggered by separase-mediated cleavage of UNO.
... After the duplication, one of the genes can assume more specialized functions, whereas the other one still performs its initial role. Interestingly, CENP-A duplicates differ mostly in the N-terminal tail sequence [51][52][53][54][55][56]. ...
... They have different expression patterns, with CPAR-1 present mostly in the germline, and HCP-3 in all dividing cells in both the germline and embryos (Figure 2A). The sequences of both proteins differ mainly in the N-terminal part, where CPAR-1 is recognized by separase, resulting in the removal of most of the N-terminal tail at the metaphase-anaphase transition of meiosis I [53]. Interestingly, only HCP-3 is an essential protein and seems to be the bona fide CENP-A. ...
The aim of mitosis is to segregate duplicated chromosomes equally into daughter cells during cell division. Meiosis serves a similar purpose, but additionally separates homologous chromosomes to produce haploid gametes for sexual reproduction. Both mitosis and meiosis rely on centromeres for the segregation of chromosomes. Centromeres are the specialized regions of the chromosomes that are attached to microtubules during their segregation. In this review, we describe the adaptations and layers of regulation that are required for centromere function during meiosis, and their role in meiosis-specific processes such as homolog-pairing and recombination. Since female meiotic divisions are asymmetric, meiotic centromeres are hypothesized to evolve quickly in order to favor their own transmission to the offspring, resulting in the rapid evolution of many centromeric proteins. We discuss this observation using the example of the histone variant CENP-A, which marks the centromere and is essential for centromere function. Changes in both the size and the sequence of the CENP-A N-terminal tail have led to additional functions of the protein, which are likely related to its roles during meiosis. We highlight the importance of CENP-A in the inheritance of centromere identity, which is dependent on the stabilization, recycling, or re-establishment of CENP-A-containing chromatin during meiosis.
... However, our previous investigation of CenH3 duplication events in Drosophila is the only study so far that has investigated CenH3 evolution and function in this light (Kursel and Malik 2017). Prior to this, the only known instances of CenH3 duplications in animals were two recent duplications in nematode species (Monen et al. 2005(Monen et al. , 2015, and several CenH3 duplications in Bovidae (cows and sheep), most of which have become pseudogenized (Li and Huang 2008). Our analysis of CenH3 (Cid) in Drosophila identified five independent Cid gene duplication events and revealed that the majority of Drosophila species encode two or three Cid paralogs, including some that have been coretained for over 40 My (Kursel and Malik 2017;Teixeira et al. 2018). ...
Despite their essential role in chromosome segregation in most eukaryotes, centromeric histones (CenH3s) evolve rapidly and are subject to gene turnover. We previously identified four instances of gene duplication and specialization of Cid, which encodes for the centromeric histone in Drosophila. We hypothesized that retention of specialized Cid paralogs could be selectively advantageous to resolve the intralocus conflict that occurs on essential genes like Cid, which are subject to divergent selective pressures to perform multiple functions. We proposed that intralocus conflict could be a widespread phenomenon that drives evolutionary innovation in centromeric proteins. If this were the case, we might expect to find other instances of co-retention and specialization of centromeric proteins during animal evolution. Consistent with this hypothesis, we find that most mosquito species encode two CenH3 (mosqCid) genes, mosqCid1 and mosqCid2, which have been co-retained for over 150 million years. In addition, Aedes species encode a third mosqCid3 gene, which arose from an independent gene duplication of mosqCid1. Like Drosophila Cid paralogs, mosqCid paralogs evolve under different selective constraints and show tissue-specific expression patterns. Analysis of mosqCid N-terminal protein motifs further supports the model that mosqCid paralogs have functionally diverged. Extending our survey to other centromeric proteins, we find that all Anopheles mosquitos encode two CAL1 paralogs, which are the chaperones that deposit CenH3 proteins at centromeres in Diptera, but a single CENP-C paralog. The ancient co-retention of paralogs of centromeric proteins adds further support to the hypothesis that intralocus conflict can drive their co-retention and functional specialization.
... Similar to R. pubera, the meiotic kinetochore does not form a line, but rather surrounds the chromosomes [173,174]. In oocyte meiosis, CENP-A, its meiosis-specific paralog CPAR-1, and CENP-C are coincident with chromatin, suggesting a broader distribution on DNA than in mitosis, but oddly they are reportedly dispensable for meiosis [173][174][175]. In spermatocyte meiosis CENP-A is barely detectable and is not present in sperm, while CENP-C appears around the chromatin and persists into the sperm [171,174]. ...
Centromeres are the eukaryotic chromosomal sites at which the kinetochore forms and attaches to spindle microtubules to orchestrate chromosomal segregation in mitosis and meiosis. Although centromeres are essential for cell division, their sequences are not conserved and evolve rapidly. Centromeres vary dramatically in size and organization. Here we categorizetheir diversity and explore the evolutionary forces shaping them. Nearly all centromeres favor AT-rich DNA that is gene-free and transcribed at a very low level. Repair of frequent centromere-proximal breaks probably contributes to their rapid sequence evolution. Point centromeres are only ∼125 bp and are specified by common protein-binding motifs, whereas short regional centromeres are 1-5 kb, typically have unique sequences, and may have pericentromeric repeats adapted to facilitate centromere clustering. Transposon-rich centromeres are often ∼100-300 kb and are favored by RNAi machinery that silences transposons, by suppression of meiotic crossovers at centromeres, and by the ability of some transposons to target centromeres. Megabase-length satellite centromeres arise in plants and animals with asymmetric female meiosis that creates centromere competition, and favors satellite monomers one or two nucleosomes in length that position and stabilize centromeric nucleosomes. Holocentromeres encompass the length of a chromosome and may differ dramatically between mitosis and meiosis. We propose a model in which low level transcription of centromeres facilitates the formation of non-B DNA that specifies centromeres and promotes loading of centromeric nucleosomes.
... 31 During anaphase I, separase cleaves the CENP-A related protein, CPAR-1, which may regu- late the metaphase-anaphase transition in C. elegans. 32 Separase is involved in centriole disengagement during male spermato- cyte meiosis 33 and regulates the separation and duplication of sperm-derived centrioles in embryos at the meiosis-mitosis transition. 34 During mitosis, separase cleaves the mitotic cohe- sin kleisin subunit SCC-1 to promote chromosome segrega- tion. ...
Separase cleaves cohesin to allow chromosome segregation. Separase also regulates cortical granule exocytosis and vesicle trafficking during cytokinesis, both of which involve RAB-11. We investigated whether separase regulates exocytosis through a proteolytic or non-proteolytic mechanism. In C. elegans, protease-dead separase (SEP-1(PD)::GFP) is dominant negative. Consistent with its role in cohesin cleavage, SEP-1(PD)::GFP causes chromosome segregation defects. As expected, partial depletion of cohesin rescues this defect, confirming that SEP-1(PD)::GFP acts through a substrate trapping mechanism. SEP-1(PD)::GFP causes cytokinetic defects that is synergistically exacerbated by depletion of the t-SNARE SYX-4. Furthermore, SEP-1(PD)::GFP delays furrow ingression, causes an accumulation of RAB-11 vesicles at the cleavage furrow site and delays the exocytosis of cortical granules during anaphase I. Depletion of syx-4 further enhanced RAB-11::mCherry and SEP-1(PD)::GFP plasma membrane accumulation during cytokinesis, while depletion of cohesin had no effect. In contrast, centriole disengagement appears normal in SEP-1(PD)::GFP embryos, indicating that chromosome segregation and vesicle trafficking are more sensitive to inhibition by the inactive protease. These findings suggest that separase cleaves an unknown substrate to promote the exocytosis of RAB-11 vesicles and paves the way for biochemical identification of substrates.
... Among our hits, two proteins previously reported as required for chromosome condensation and segregation in C. elegans stood out: CENP-A and topo-II ( Fig. 1, a and b; Maddox et al., 2006;Bembenek et al., 2013). C. elegans uniquely expresses two orthologues of CENP-A; HCP-3 is the dominant form required for mitosis, whereas CPAR-1 has no known function in embryos (Monen et al., 2015). Because these orthologues share extensive DNA sequence identity, it is not possible to independently target them by RNAi; therefore, we will refer to their depletion as CENP-A RNAi (Monen et al., 2005(Monen et al., , 2015. ...
... C. elegans uniquely expresses two orthologues of CENP-A; HCP-3 is the dominant form required for mitosis, whereas CPAR-1 has no known function in embryos (Monen et al., 2015). Because these orthologues share extensive DNA sequence identity, it is not possible to independently target them by RNAi; therefore, we will refer to their depletion as CENP-A RNAi (Monen et al., 2005(Monen et al., , 2015. Topo-II also has two C. elegans orthologues (CIN-4 and TOP-2, both hits in our screen). ...
The size of mitotic chromosomes is coordinated with cell size in a manner dependent on nuclear trafficking. In this study, we conducted an RNA interference screen of the Caenorhabditis elegans nucleome in a strain carrying an exceptionally long chromosome and identified the centromere-specific histone H3 variant CENP-A and the DNA decatenizing enzyme topoisomerase-II (topo-II) as candidate modulators of chromosome size. In the holocentric organism C. elegans , CENP-A is positioned periodically along the entire length of chromosomes, and in mitosis, these genomic regions come together linearly to form the base of kinetochores. We show that CENP-A protein levels decreased through development coinciding with chromosome-size scaling. Partial loss of CENP-A protein resulted in shorter mitotic chromosomes, consistent with a role in setting chromosome length. Conversely, topo-II levels were unchanged through early development, and partial topo-II depletion led to longer chromosomes. Topo-II localized to the perimeter of mitotic chromosomes, excluded from the centromere regions, and depletion of topo-II did not change CENP-A levels. We propose that self-assembly of centromeric chromatin into an extended linear array promotes elongation of the chromosome, whereas topo-II promotes chromosome-length shortening.
