InteractiveFly: GeneBrief

chromosome alignment defect 1: Biological Overview | References

BIOLOGICAL OVERVIEW

Propagation of centromere identity during cell cycle progression in higher eukaryotes depends critically on the faithful incorporation of a centromere-specific histone H3 variant encoded by CENPA in humans and cid in Drosophila. Cenp-A/Cid is required for the recruitment of Cenp-C, another conserved centromere protein. With yeast three-hybrid experiments, this study demonstrates that the essential Drosophila centromere protein Cal1 can link Cenp-A/Cid and Cenp-C. Cenp-A/Cid and Cenp-C interact with the N- and C-terminal domains of Cal1, respectively. These Cal1 domains are sufficient for centromere localization and function, but only when linked together. Using quantitative in vivo imaging to determine protein copy numbers at centromeres and kinetochores, it was demonstrates that centromeric Cal1 levels are far lower than those of Cenp-A/Cid, Cenp-C and other conserved kinetochore components, which scale well with the number of kinetochore microtubules when comparing Drosophila with budding yeast. Rather than providing a stoichiometric link within the mitotic kinetochore, Cal1 limits centromeric deposition of Cenp-A/Cid and Cenp-C during exit from mitosis. The low amount of endogenous Cal1 prevents centromere expansion and mitotic kinetochore failure when Cenp-A/Cid and Cenp-C are present in excess (Schittenhelm, 2010).

The centromeric regions of chromosomes direct formation of kinetochores, which allow chromosome attachment to spindle microtubules. Centromeres and kinetochores are therefore of paramount importance for faithful propagation of genetic information. However, centromeric DNA sequences are not conserved. Most eukaryotes (including Drosophila melanogaster and humans) have regional centromeres with up to several megabases of repetitive DNA. Importantly, these repetitive sequences are neither necessary nor sufficient for centromere function, indicating that there is an epigenetic centromere specification (Schittenhelm, 2010).

A centromere-specific histone H3 variant (CenH3) is thought to be crucial for epigenetic centromere marking. CenH3 proteins are present in all eukaryotes (e.g. CENP-A in humans and Cid in Drosophila). They replace histone H3 in canonical nucleosomes or possibly variant complexes. Depletion of CenH3 results in a failure to localize most or all other centromere and mitosis-specific kinetochore proteins. Strong overexpression of Drosophila Cenp-A/Cid results in incorporation at ectopic chromosomal sites, which in part also assemble ectopic kinetochores during mitosis (Schittenhelm, 2010).

Ectopic kinetochores result in chromosome segregation errors and genetic instability. Ectopic CenH3 incorporation therefore must be prevented. Although still fragmentary, the understanding of the molecular mechanisms that regulate CenH3 incorporation is progressing rapidly. In proliferating cells, an additional complement of CenH3 needs to be incorporated during each cell cycle. In syncytial Drosophila embryos, this occurs during exit from mitosis. Similar findings were made in human cells, where Cenp-A deposition occurs during late telophase and early G1 phase. The number of factors shown to be required for normal CenH3 deposition is increasing rapidly, which suggests that there is an intricate control mechanism. Various and often dedicated chaperones (see Dunleavy, 2009; Foltz, 2009), chromatin modifying and remodelling factors (see Perpelescu, 2009), as well as other centromere components (see Pidoux, 2009; Williams, 2009) are involved (Schittenhelm, 2010).

In Drosophila, Cenp-C is incorporated into centromeres concomitantly with Cenp-A/Cid (Schuh, 2007). High-resolution mapping with native Drosophila chromosomes has indicated that these two proteins do not have an identical localization within the kinetochore. Although these localization studies cannot exclude an association between subfractions of Cenp-A and Cenp-C, direct molecular interactions between these centromere proteins have not yet been reported. Recently, however, Cal1 has been identified in Drosophila and shown to be required for normal centromeric localization of Cenp-A/Cid and Cenp-C (Goshima, 2007; Erhardt, 2008). Moreover, these three Drosophila centromere proteins can be co-immunoprecipitated from soluble chromatin preparations (Erhardt, 2008). Cal1 might therefore provide a physical link between Cenp-A/Cid and Cenp-C (Schittenhelm, 2010).

This study reports that Cal1 has distinct binding sites for Cenp-A/Cid and Cenp-C. It can link these proteins together according to yeast three-hybrid experiments. However, the level of centromeric Cal1 is far lower than that of Cenp-A/Cid and Cenp-C. Cal1 therefore cannot function as a stoichiometric linker connecting each monomer or dimer of Cenp-C to Cenp-A within the centromere. But the low levels of Cal1 effectively protect cells against mitotic defects resulting from increased centromeric incorporation of excess Cenp-A/Cid and Cenp-C (Schittenhelm, 2010).

cal1 is an essential gene that is expressed specifically in mitotically proliferating cells. To provide its function, the protein product needs its N-terminal domain, which interacts with Cenp-A/Cid, as well as its C-terminal domain, which interacts with Cenp-C. By contrast, the most rapidly diverging middle region of Cal1 seems to be of lesser importance because expression of the N-C version, which lacks the M domain, is sufficient to prevent the characteristic defects in cal1 mutant embryos. The obvious functionality of the N-C version also emphasizes the importance of the centromeric localization of Cal1. The complete Cal1 protein is observed not only at the centromere, but also in the nucleolus. The M region is both sufficient and required for nucleolar localization. However, because this M domain is not required for cal1 mutant rescue, the significance of the nucleolar Cal1 localization remains unclear (Schittenhelm, 2010).

Rescue of cal1 mutants is not observed when the N- and C-terminal domains of Cal1 are expressed without a covalent linkage. The ability to recruit Cenp-A/Cid and Cenp-C into a complex, as clearly evidenced by yeast three-hybrid experiments, is therefore likely to be crucial for Cal1 function. Co-immunoprecipitation of Cal1, Cenp-A/Cid and Cenp-C has previously indicated that these components can associate in vivo (Erhardt, 2008). However, quantification of protein levels, which is largely dependent on the accuracy of EGFP signal quantifications, demonstrates that Cenp-C is not exclusively anchored to centromeric chromatin via persistent and stoichiometric Cal1-mediated links to Cenp-A/Cid. Centromeric Cal1 levels are more than 40-times lower than those of Cenp-A/Cid and Cenp-C (Schittenhelm, 2010).

The centromeric amount of Cal1 is also far lower than that of the other kinetochore components that have been quantified (Spc105, Spc25, Nuf2). Interestingly, per kinetochore, the copy numbers of these components appear to be scaling well with the number of kinetochore microtubules (kMTs) when comparing the current results from Drosophila with those described for budding and fission yeast (Joglekar, 2006; Joglekar, 2008). Spc25 and Nuf2 are constituents of the heterotetrameric Ndc80 complex, which binds directly to kinetochore microtubules (kMTs) (Santaguida, 2009; see Models of kinetochore assembly). Eight copies of the Ndc80 complex are thought to bind a single kMT to the budding yeast kinetochore (Joglekar, 2006). In Drosophila, where the number of kMTs per kinetochore appears to be around 11 (Maiato, 2006), about seven copies appear to be present per kMT according to the quantification. This quantification of kinetochore proteins fits very well with the notion that the kinetochores of higher eukaryotes might be composed of several copies of a module that is present in one copy in budding yeast. By contrast, the centromere proteins Cenp-A and Cenp-C are scaling less well with the number of kMTs. The increased complexity of lateral co-ordination within animal kinetochores and of epigenetic specification of centromere identity might explain the higher relative amount of centromere proteins apparent in Drosophila. Despite this relative increase, centromeric Cenp-A/Cid allows packaging of only about 5% of the centromeric DNA in Drosophila under the assumption that Cenp-A/Cid nucleosomes wrap about 200 bp of a 200 kb centromere (Schittenhelm, 2010).

Although these quantifications exclude the notion that Cal1 functions as a stable stoichiometric linker of Cenp-A/Cid and Cenp-C in mitotic kinetochores, the overexpression experiments provide further support for a role as a centromere protein-loading factor (Erhardt, 2008). Moreover, these experiments reveal additional layers of regulation that prevent excess incorporation of centromere proteins within the centromeric region. They also indicate that such excess incorporation is highly detrimental to kinetochore function. Previous work in Drosophila has demonstrated that strong overexpression of Cenp-A/Cid (about 70-fold) can lead to ectopic kinetochore formation (Heun, 2006). However, almost all Cenp-A/Cid that is incorporated ectopically within the chromosome arm regions is degraded rapidly, which is also observed in yeast. This study shows that the limiting amounts of Cal1 provide additional, highly efficient protection against excessive chromosomal incorporation of Cenp-A/Cid. After bypassing this protection by Cal1 overexpression, even low levels of Cenp-A/Cid overexpression (about 2.5-fold) result in increased incorporation into centromeres (about 1.6-fold). When, in addition to Cal1 and Cenp-A/Cid, Cenp-C is also mildly co-overexpressed (about 3.5-fold), the levels of centromeric Cenp-A/Cid are further increased (about 2-fold) along with those of Cal1 and Cenp-C. Importantly, co-overexpression of these centromere proteins resulted not only in increased centromeric levels, but also in severe mitotic defects (Schittenhelm, 2010).

Although other interpretations are not excluded, these findings strongly suggest that the mitotic defects observed after overexpression of Cal1 and Cenp-A/Cid, and even more strongly when Cenp-C was also overexpressed, reflect the consequence of the increase in the centromeric levels of these proteins. The increase in centromeric levels of centromere proteins was accompanied by a significant increase in kinetochore proteins (Spc105 and the Mis12 and Ndc80 complex) but only to a very limited extent and only when all three centromere proteins were co-expressed. The increased amounts of centromeric Cenp-A/Cid observed after co-expression of Cal1 and Cenp-A/Cid, which were not accompanied by a statistically significant increase in kinetochore protein levels, might therefore be sufficient to disturb the spatial organization of the kinetochore, leading to inefficient chromosome congression, spindle checkpoint hyperactivation and chromosome segregation defects in anaphase (Schittenhelm, 2010).

Experiments in stg mutant embryos, demonstrate that co-overexpression of centromeric proteins during interphase is not sufficient to cause excess centromeric incorporation, consistent with the previously demonstrated dependence of centromeric deposition of Cenp-A/Cid and Cenp-C on exit from mitosis (Schuh, 2007). Indeed, forcing progression through mitosis (by hs-stg induction) was observed to be sufficient to cause centromeric deposition of the overexpressed proteins. Moreover, the fact that the excess centromere proteins that were not yet incorporated into the centromere did not disturb the hs-stg induced mitosis, further supports the suggestion that the mitotic defects observed after co-expression of centromeric proteins depend on excessive incorporation into the centromere (Schittenhelm, 2010).

The severe mitotic defects observed after co-overexpression of Cal1, Cenp-A/Cid and Cenp-C emphasize the importance of careful control of centromere protein deposition. Several levels of control are effective. The interdependence of Cal1, Cenp-A/Cid and Cenp-C functions in conjunction with cell cycle control to prevent detrimental excessive centromeric incorporation. The cell cycle regulators cyclin A, Rca1/Emi1 and Fzr/Cdh1 have recently been implicated in the control of deposition of Cenp-A/Cid and Cenp-C at the centromere (Erhardt, 2008). How these and possibly additional cell cycle regulators control centromere protein deposition has yet to be clarified (Schittenhelm, 2010).

A possible scenario for centromere protein deposition in Drosophila might include a release of nucleolar Cal1 at the onset of mitosis, followed by conversion into a form that associates with non-centromeric soluble Cenp-A/Cid during exit from mitosis. After binding of soluble Cenp-A/Cid to the N-terminal domain of Cal1, its C-terminal domain might become exposed so that it can bind to centromeric Cenp-C and promote Cenp-A/Cid transfer onto the neighboring centromeric chromatin and thereby indirectly also additional Cenp-C deposition (Schittenhelm, 2010).

The mechanisms and the extent of control of centromeric Cenp-A deposition appear to have evolved. In fission yeast, overexpression of Cenp-A/Cid alone is sufficient to obtain excess centromeric Cenp-A/Cnp1, and this excess does not result in increased kinetochore protein levels (Joglekar, 2008). Spreading of Cenp-A within centromeric chromatin has also been clearly demonstrated in human cells after mild overexpression of Cenp-A (Lam, 2006). Mitotic defects were not detected in this case, perhaps because of the very limited increase in centromeric Cenp-A. Cal1 homologs from non-Drosophilid genomes have not yet been identified so far. Conversely, with the exception of Cenp-C, homologs of the 15 components of the vertebrate centromere chromatin-associated network (CCAN), which is related to the yeast Ctf19 and Sim4 complexes, have not been revealed in Drosophilid genomes, neither by thorough bioinformatic analyses nor by genome-wide RNAi screens (Goshima, 2007; Erhardt, 2008). The CCAN seems also to be absent in C. elegans. It is conceivable therefore that Cal1 is a functional analog of the CCAN, which has also been implicated in Cenp-A loading (Okada, 2006). However, because the evolutionary sequence conservation of centromere and kinetochore components is generally very low, it remains a possibility that Cal1 homologs also exist and function in centromere loading of human Cenp-A and Cenp-C (Schittenhelm, 2010).

The checkpoint protein Zw10 connects CAL1-dependent CENP-A centromeric loading and mitosis duration in Drosophila cells

A defining feature of centromeres is the presence of the histone H3 variant CENP-A that replaces H3 in a subset of centromeric nucleosomes. In Drosophila cultured cells CENP-A deposition at centromeres takes place during the metaphase stage of the cell cycle and strictly depends on the presence of its specific chaperone CAL1. How CENP-A loading is restricted to mitosis is unknown. Overexpression of CAL1 is associated with increased CENP-A levels at centromeres and uncouples CENP-A loading from mitosis. Moreover, CENP-A levels inversely correlate with mitosis duration suggesting crosstalk of CENP-A loading with the regulatory machinery of mitosis. Mitosis length is influenced by the spindle assembly checkpoint (SAC), and this study found that CAL1 interacts with the SAC protein and RZZ complex component Zw10 and thus constitutes the anchor for the recruitment of RZZ. Therefore, CAL1 controls CENP-A incorporation at centromeres both quantitatively and temporally, connecting it to the SAC to ensure mitotic fidelity (Pauleau, 2019).

The formation of two genetically identical daughter cells with a correct and stable genome is of utmost importance during mitosis. Condensed chromosomes are attached and segregated to the opposing poles of the dividing cell at anaphase by the mitotic spindle. At the interface between the chromosomes and the spindle microtubules lies the kinetochore. This multi-protein complex is formed by the components of the KMN network (formed by the Knl1 complex, the Mis12 complex, and the Ndc80 complex) (Joglekar, 2017). The Ndc80 complex is mainly responsible for connecting microtubules with kinetochores while the Knl1 complex primarily coordinates the Spindle Assembly Checkpoint (SAC) (Musacchio, 2017). The SAC delays entry into anaphase until all chromosomes are properly attached and aligned at the metaphase plate. The metaphase to anaphase transition is controlled by the activation of Cdc20 of the APC/C, a multisubunit ubiquitin ligase that triggers the degradation of cell cycle regulators by the proteasome. The SAC proteins Mad2, BubR1, and Bub3 sequester Cdc20 by forming the Mitotic Checkpoint Complex (MCC) thereby preventing the activation of the APC/C. Besides, other proteins have been implicated in SAC activity including Bub1, Mad1, the Mps1 and Aurora B kinases, and the RZZ complex (formed by the three proteins Rough Deal (ROD), Zw10 and Zwilch (Musacchio, 2015). Finally, the Mis12 complex serves as a hub at the kinetochore interacting with all kinetochore complexes as well as with the centromere (Pauleau, 2019).

The kinetochore assembles on the centromere during mitosis, a highly specialized chromatin region that is defined by an enrichment of nucleosomes containing the histone H3 variant CENP-A, also called CID in Drosophila. In contrast to canonical histones, CENP-A deposition at centromeres is independent of DNA replication and is temporally restricted to a specific cell cycle stage, which varies between organisms: late telophase/early G1 in mammalian cultured cells, G2 in S. pombe and plants, and mitosis to G1 in Drosophila. The timing of CENP-A is particularly intriguing in Drosophila cultured cells as centromeric CENP-A is replenished during prometaphase-metaphase thus coinciding with kinetochore assembly. CENP-A loading requires the action of its dedicated chaperones: HJURP in humans, Scm3 in fungi and CAL1 in Drosophila (Chen, 2014, Erhardt, 2008, Schittenhelm, 2010). Deregulation of CENP-A and its loading machinery can result in the misincorporation of CENP-A into regions along the chromosome arms. Misincorporated CENP-A is usually rapidly degraded. If, however, CENP-A-containing nucleosomes remain at non-centromeric sites ectopic formation of functional kinetochores can occur that may lead to chromosome segregation defects and aneuploidy (Pauleau, 2019).

In Drosophila melanogaster, two other proteins are constitutively present at centromeres and essential for centromere function: the conserved protein CENP-C and the CENP-A chaperone CAL1. CENP-C has been shown to act as a linker between CENP-A nucleosomes and the Mis12 complex, therefore, providing a platform for kinetochore assembly (Przewloka, 2011). CENP-C is also implicated in CENP-A replenishment at centromeres during mitosis by recruiting CAL1 (Chen, 2009; Erhardt, 2008). CAL1 interacts with CENP-A in both pre-nucleosomal and nucleosomal complexes and is necessary for CENP-A protein stabilization via Roadkill-Cullin3-mediated mono-ubiquitination. Moreover, CAL1 has been previously shown to be the limiting factor for CENP-A centromeric incorporation in fly embryos. However, differences in centromere assembly have been reported between Drosophila cultured cells and embryos. Firstly, CENP-A loading has been shown to take place during mitotic exit in early embryos and prometaphase to early G1 in cultured cells. Second, CENP-C incorporates concomitantly to CENP-A in embryos while this time window seems to be larger in cultured cells. This study, therefore, set out to determine more precisely the function of CAL1 in CENP-A loading regulation in Drosophila cultured cells (Pauleau, 2019).

During the course of these studies, overexpression of CAL1 was found not only to increase endogenous and exogenous CENP-A abundance at centromeres, but also was found to uncouple CENP-A loading from mitosis. Strikingly, it was discovered a co-dependence of mitotic duration and accurate CENP-A loading that may be coordinated by an interaction of the CENP-A loading machinery with the SAC protein and RZZ subunit Zw10. These data suggest an intricate coordination of the spindle assembly checkpoint, CENP-A loading, and mitotic duration in order to safeguard accurate mitotic progression (Pauleau, 2019).

In Drosophila cells, CENP-A loading takes place primarily during prometaphase-metaphase. Additional turnover of CENP-A in G1 has been reported leading to the hypothesis that CENP-A could be further incorporated at this stage, which was not observe in FRAP experiments when centromeric CENP-A was bleached at the end of cytokinesis. However, the FRAP experiments and most importantly live staining of newly synthesized SNAP-CENP-A confirmed that the majority of CENP-A loading takes place during mitosis in Drosophila cultured cells (Pauleau, 2019).

In flies, CENP-A incorporation is controlled by its chaperone CAL1. It has been shown previously that co-overexpression of exogenous CENP-A and CAL1 leads to an increase of centromeric CENP-A in embryos. This study now shows that overexpression of CAL1 alone leads to increased endogenous CENP-A protein levels in Drosophila cultured cells. Ectopic incorporation of CENP-A, however, was never observed suggesting that CAL1 loads CENP-A exclusively to centromeres and that ectopic CENP-A incorporation in flies depends on alternative loading mechanisms similar to what has been suggested in human cells. Importantly, increased centromeric CENP-A levels following CAL1 overexpression correlated with faster mitosis. A similar acceleration of mitotic timing was observed when CENP-A was only mildly overexpressed, revealing a possible link between CENP-A loading and mitotic timing. Indeed, shortening mitosis duration by depleting Mad2 or BubR1 was associated with decreased CENP-A loading. However, just elongating the mitotic time window during which CENP-A can get loaded (Spindly or Cdc27 depletion, or by drug treatment) did not increase the amount of CENP-A incorporated at centromeres, showing that the length of mitosis alone is insufficient to control CENP-A amounts at centromeres. Rather, these experiments showed that only a defined amount of CENP-A can be incorporated at each mitosis probably correlating with CAL1's availability. Indeed, live analysis of CAL1-overexpressing cells allowed visualization of newly synthesized CENP-A incorporation to centromeres in all stages of the cell cycle. This strongly suggests that CAL1 controls CENP-A incorporation into centromeric chromatin both quantitatively and temporally. How exactly CENP-A levels at centromeres are sensed is unclear but this study identified the RZZ-component Zw10 as a new CAL1 interacting partner, which directly connects CENP-A loading to the SAC (Pauleau, 2019).

It has been proposed that SAC activation is a 2-steps process: at the end of G2-beginning of mitosis, before the kinetochores are assembled, cytosolic Mad1-Mad2 dimers initiate MCC formation inhibiting APC/CCdc20 and determining the timing of mitosis. After nuclear envelope disassembly, kinetochore-dependent MCC are generated and regulated by kinetochore-microtubules attachment. Therefore, the following model is suggested: efficient CENP-A loading by CAL1 during mitosis recruits Zw10 up to a threshold, which is sensed by the SAC. Low CENP-A levels at centromeres could lead to more cytosolic Mad2 thereby keeping the timer active longer. Higher CENP-A levels at centromeres during early mitosis would accelerate the recruitment of RZZ and consequently Mad2 to the kinetochores or capture microtubules more efficiently, therefore, releasing the timer and shortening mitosis duration in cells where kinetochores attach properly to the spindle microtubules. Interestingly, Nocodazole treatment did not affect CENP-A loading confirming previous observations that kinetochore attachment to the microtubule spindle does not play a role in CENP-A loading. These results are pointing further to an additional function of the SAC independent of the control of microtubule attachment. Interestingly, recent evidence shows that RZZ together with Spindly plays a central role in kinetochore expansion during early mitosis to form a fibrous corona that then compacts upon microtubule capture. Whether and -if so- how the kinetochore expansion by RZZ and spindly is involved in CENP-A loading needs to be investigated in the future (Pauleau, 2019).

Many essential components of the SAC require outer kinetochore components for their localization to centromeric regions. However, several outer components are missing from the Drosophila kinetochore and even though Mad1/2 recruitment to the kinetochore depends on the RZZ complex, the factors necessary for the localization of the RZZ to kinetochores are unknown. This study has shown that RZZ localization to the kinetochores does not require KNL1Spc105R but depends on the centromeric proteins CAL1 and CENP-A. Therefore, it is proposed that the Drosophila outer kinetochore and components of the SAC assemble through two independent pathways: the CENP-C-KMN-Bub1-Bub3/BubR1 branch or the CAL1-RZZ-Mad1/2 branch. How those two pathways communicate for the formation of MCC complexes remains to be determined. One link may be the KMN complex since Mad2 is diminished in the absence of KMN proteins. Interestingly, Spc105R mutation does not affect SAC function in fly embryos suggesting that flies rely more on the RZZ-Mad1/2 branch to engage the SAC (Pauleau, 2019).

CENP-A expression and its stability together with its dependence on the low abundant and highly specific loading factor CAL1 and the connection to mitotic events are likely interconnected cellular surveillance mechanisms to avoid misincorporation of CENP-A and, therefore, securing genome stability. How CAL1 itself is regulated to obtain such specificity is currently unknown. In conclusion, this study has shown that there is direct crosstalk between the SAC and the maintenance of centromeric chromatin, ensuring mitotic fidelity not only by controlling microtubule attachment but also by regulating the accurate composition of centromeres (Pauleau, 2019).

Asymmetric assembly of centromeres epigenetically regulates stem cell fate

Centromeres are epigenetically defined by CENP-A-containing chromatin and are essential for cell division. Previous studies suggest asymmetric inheritance of centromeric proteins upon stem cell division; however, the mechanism and implications of selective chromosome segregation remain unexplored. This study shows that Drosophila female germline stem cells (GSCs) and neuroblasts assemble centromeres after replication and before segregation. Specifically, CENP-A deposition is promoted by CYCLIN A, while excessive CENP-A deposition is prevented by CYCLIN B, through the HASPIN kinase. Furthermore, chromosomes inherited by GSCs incorporate more CENP-A, making stronger kinetochores that capture more spindle microtubules and bias segregation. Importantly, symmetric incorporation of CENP-A on sister chromatids via HASPIN knockdown or overexpression of CENP-A, either alone or together with its assembly factor CAL1, drives stem cell self-renewal. Finally, continued CENP-A assembly in differentiated cells is nonessential for egg development. This work shows that centromere assembly epigenetically drives GSC maintenance and occurs before oocyte meiosis (Dattoli, 2020).

Stem cells are fundamental for the generation of all tissues during embryogenesis and replace lost or damaged cells throughout the life of an organism. At division, stem cells generate two cells with distinct fates: (1) a cell that is an exact copy of its precursor, maintaining the 'stemness,' and (2) a daughter cell that will subsequently differentiate. Epigenetic mechanisms, heritable chemical modifications of the DNA/nucleosome that do not alter the primary genomic nucleotide sequence, regulate the process of self-renewal and differentiation of stem cells. In Drosophila male germline stem cells (GSCs), before division, phosphorylation at threonine 3 of histone H3 (H3T3P) preferentially associates with chromosomes that are inherited by the future stem cell (Xie, 2015). Furthermore, centromeric proteins seem to be asymmetrically distributed between stem and daughter cells in the Drosophila intestine and germline. These findings support the 'silent sister hypothesis', according to which epigenetic variations differentially mark sister chromatids driving selective chromosome segregation during stem cell mitosis. Centromeres, the primary constriction of chromosomes, are crucial for cell division, providing the chromatin surface where the kinetochore assembles. In turn, the kinetochore ensures the correct attachment of spindle microtubules and faithful chromosome partition into the two daughter cells upon division. Centromeric chromatin contains different kinds of DNA repeats (satellite and centromeric retrotransposons) wrapped around nucleosomes containing the histone H3 variant centromere protein A (CENP-A). Centromeres are not specified by a particular DNA sequence. Rather, they are specified epigenetically by CENP-A. Centromere assembly, classically measured as CENP-A deposition to generate centromeric nucleosomes, occurs at the end of mitosis (between telophase and G1) in humans. Additional cell cycle timings for centromere assembly have been reported in flies. Interestingly, Drosophila spermatocytes and starfish oocytes are the only cells known to date to assemble centromeres before chromosome segregation, during prophase of meiosis I. These examples show that centromere assembly dynamics can differ among metazoans and also among different cell types in the same organism (Dattoli, 2020).

A key player in centromere assembly in vertebrates is HJURP (holliday junction recognition protein), which localizes at centromeres during the cell cycle window of CENP-A deposition. Furthermore, centromere assembly is regulated by the cell cycle machinery. In flies, deposition of CID (the homologue of CENP-A) requires activation of the anaphase promoting complex/cyclosome (APC/C) and degradation of CYCLIN A (CYCA). In humans, centromere assembly is antagonized by Cdk1 activity, while the kinase Plk1 promotes assembly. Additionally, the CYCLIN B (CYCB)/Cdk1 complex inhibits the binding of CENP-A to HJURP, preventing CENP-A loading at centromeres. To date, little is known about centromere assembly dynamics and functions in stem cell asymmetric divisions. Drosophila melanogaster ovaries provide an excellent model to study stem cells in their native niche. In this tissue, germline stem cells (GSCs) are easily accessible and can be manipulated genetically. Moreover, centromere assembly mechanisms in GSCs and their differentiated cells, cystoblasts (CBs), could be used to epigenetically discriminate between these two cell types. In Drosophila, CID binds to CAL1 (fly functional homologue of HJURP) in a prenucleosomal complex, and its localization to centromeres requires CAL1 and CENP-C (Dattoli, 2020).

This study investigated the dynamics of CENP-A deposition in Drosophila GSCs. GSC centromeres are assembled after replication, but before chromosome segregation, with neural stem cells following the same trend. Centromere assembly in GSCs is tightly linked to the G2/M transition. Indeed, CYCA localizes at centromeres, and its knockdown is responsible for a marked reduction of centromeric CID and CENP-C, but not CAL1. Surprisingly, excessive CID deposition is prevented by CYCB, through the kinase HASPIN. Superresolution microscopy analysis of GSCs at prometaphase and metaphase shows that CID incorporation on sister chromatids occurs asymmetrically, and chromosomes that will be inherited by the stem cell are loaded with more CID. Moreover, GSC chromosomes make stronger kinetochores, which anchor more spindle fibers. This asymmetric distribution of CID between GSC and CB is maintained also at later stages of the cell cycle, while it is not observed in differentiated cells outside of the niche. This study also found that the depletion of CAL1 at centromeres blocks GSC proliferation and differentiation. Notably, overexpression of both CID and CAL1, as well as HASPIN knockdown, promotes stem cell self-renewal and disrupts the asymmetric inheritance of CID. Conversely, overexpression of CAL1 causes GSC-like tumors. Finally, CAL1 and CID knockdown at later stages of egg development have no obvious effect on cell division, suggesting that these cells inherit CID from GSCs. Taken together, these findings establish centromere assembly as a new epigenetic pathway that regulates stem cell fate (Dattoli, 2020).

In this study a detailed characterization of centromere dynamics was performed throughout the cell cycle in Drosophila female GSCs. This analysis reveals that GSCs initiate CID incorporation after replication and that its deposition continues until at least prophase. Drosophila neural stem cells follow the same trend. Notably, this timing is different from existing studies in other metazoans. It was also found that CYCA, CYCB, and HASPIN are critically involved in CID (and CENP-C) loading at centromeres. According to the model, CYCA promotes centromere assembly, while CYCB prevents excessive deposition of CID, through the HASPIN kinase. Moreover, chromosomes that will be inherited by GSCs are labeled with a higher amount of CID and capture more spindle microtubules. Importantly, this study shows that overexpression of CAL1 and CID together, as well as HASPIN knockdown, promotes stem cell self-renewal, disrupting the asymmetric inheritance of CID. Depletion of CAL1 in stem cells blocks cell division, while CAL1 overexpression causes GSC-like tumors, highlighting its crucial role in cell proliferation. Three main points of discussion are raised: (1) the biological significance of centromere assembly in G2-M phase; (2) CAL1 is a cell proliferation marker; and (3) CID incorporation into centromeric chromatin occurs before meiosis (Dattoli, 2020).

According to the data, CID deposition occupies a wide window of time from after replication and early G2 phase to prophase. The assembly of GSC centromeres during the G2/M transition could be due to the contraction of the G1 phase, a characteristic of stem cells. Yet, in fly embryonic divisions, G1 phase is missing, and instead CID loading occurs at anaphase. Therefore, G2/M assembly might be a unique property of stem cells. This timing is also similar to the one found for Drosophila spermatocytes, which assemble centromeres in prophase of meiosis I. These spermatocytes undergo an arrest in prophase I for days, indicating a gradual loading of CID over a long period of time. Intriguingly, a similar phenomenon has been recently observed in G0-arrested human tissue culture cells and starfish oocytes. Given that GSCs are mostly in G2 phase, Drosophila stem cells might show similar properties to quiescent cells. According to the most recent models, there is a dual mechanism for CENP-A deposition: (a) a rapid pulse during G1 in mitotically dividing cells; and (b) a slow but constant CENP-A deposition in nondividing cells to actively maintain centromeres. Indeed, while previous studies in Drosophila NBs show a rapid pulse of CENP-A incorporation at telophase/G1, the majority of the loading could occur between G2 and prophase. The new results also support this model (Dattoli, 2020).

Incorporation of CID before chromosome segregation might reflect a different CYCLIN-CDK activity in these cells. For instance, it has been already shown that in Drosophila GSCs CYCLIN E, a canonical G1/S cyclin, exists in its active form (in combination with Cdk2) throughout the cell cycle, indicating that some of the biological process commonly occurring in G1 phase might actually take place in G2 phase. This is in line with the current functional findings, where depletion of CYCA causes a decreased efficiency in CID and CENP-C assembly. This study also found that this loss might be independent from CAL1. Surprisingly, correct CID deposition in GSCs also requires CYCB and HASPIN. Indeed, an inhibitory mechanism for CID deposition through CYCB has already been proposed in mammals (Stankovic, 2017). Interestingly, in Drosophila male GSCs, centromeric CAL1 is reduced between G2 and prometaphase (Ranjan, 2019), further suggesting a role for additional regulators of CID assembly, such as CYCA/B or HASPIN, at this time (Dattoli, 2020).

According to the current results, asymmetric cell division of GSCs is epigenetically regulated by differential amounts of centromeric proteins deposited at sister chromatids, which in turn can influence the attachment of spindle microtubules and can ultimately bias chromosome segregation. It is interesting to speculate on the temporal sequence of these events. Two scenarios can be proposed: (a) the nucleation of microtubules from the GSC centrosome requires bigger kinetochores; or (b) bigger kinetochores require a higher amount of spindle fibers to attach. The current results together with recent studies support the latter scenario. In fact, in Drosophila male GSCs, asymmetric distribution of centromeric proteins is established before microtubule attachment. Furthermore, microtubule disruption leaves asymmetric loading of CID intact, while it disrupts the asymmetric segregation of sister chromatids. The current data confirm this model, symmetric segregation of CID was observed upon HASPIN knockdown. Indeed, in vertebrates HASPIN knockdown causes spindle defects. Specifically, it was observed that a 1.2-fold difference in CID and CENP-C levels between GSC and CB chromosomes can bias segregation. While this difference is small, it fits with the observation that small changes in CENP-A level (on the order of 2-10% per day) impact on centromere functionality in the long run. In Drosophila male GSCs, an asymmetric distribution of CID on sister chromatids >1.4-fold was reported. This higher value might reflect distinct systems in males and females or the quantitation methods used. Importantly, CID asymmetry in males is established in G2/prophase, in line with the time window this study defines for CID assembly. Further support for unexpected CID loading dynamics comes from the finding that GSCs in G2/prophase contain ~30% more CID on average compared with S phase, indicating that CID is not replenished to 100% each cell cycle. Interestingly, the time course of H3T3P appearance during the GSC cell cycle closely follows the timing of CID incorporation, suggesting that the asymmetric deposition of CID might drive the differential phosphorylation of the histone H3 on sister chromatids. Finally, the results are in line with findings that the long-term retention of CENP-A in mouse oocytes has a role in establishing asymmetric centromere inheritance in meiosis (Dattoli, 2020).

These functional studies support a role for CAL1 in cell proliferation, with no apparent role in asymmetric cell division. Indeed, centromeric proteins have been already proposed as biomarkers for cell proliferation. Specifically, functional analysis of centromeric proteins, as well as the HASPIN kinase, allowed discrimination between the classic role of centromeres in cell division and a role in asymmetric cell division. In a favorite scenario, CAL1 is needed to make functional centromeres crucial for cell division, while the asymmetric distribution of CID sister chromatids regulates asymmetric cell division and might depend on other factors, such as HASPIN. However, it cannot be rule out that the effects on cell fate observed with the functional analysis might reflect alternative CAL1 functions outside of the centromere, for example due to changes in chromosome structure or gene expression (Dattoli, 2020).

Centromeres are crucially assembled in GSCs and therefore before meiosis of the oocyte takes place. Thus, it is possible that the 16-cell cysts inherit centromeric proteins synthesized and deposited in the GSCs, and the rate of new CID loading is reduced. This would explain why CAL1 function at centromeres is dispensable at this developmental stage (Dattoli, 2020).

Ultimately, the results provide the first functional evidence that centromeres have a role in the epigenetic pathway that specifies stem cell identity. Furthermore, these data support the silent sister hypothesis (Lansdorp, 2007), according to which centromeres can drive asymmetric division in stem cells (Dattoli, 2020).

Assembly of Drosophila centromeric chromatin proteins during mitosis

Semi-conservative segregation of nucleosomes to sister chromatids during DNA replication creates gaps that must be filled by new nucleosome assembly. This study analyzed the cell-cycle timing of centromeric chromatin assembly in Drosophila, which contains the H3 variant CID (CENP-A in humans), as well as CENP-C and Chromosome alignment defect 1 (CAL1), which are required for CID localization. Pulse-chase experiments show that CID and CENP-C levels decrease by 50% at each cell division, as predicted for semi-conservative segregation and inheritance, whereas CAL1 displays higher turnover. Quench-chase-pulse experiments demonstrate that there is a significant lag between replication and replenishment of centromeric chromatin. Surprisingly, new CID is recruited to centromeres in metaphase, by a mechanism that does not require an intact mitotic spindle, but does require proteasome activity. Interestingly, new CAL1 is recruited to centromeres before CID in prophase. Furthermore, CAL1, but not CENP-C, is found in complex with pre-nucleosomal CID. Finally, CENP-C displays yet a different pattern of incorporation, during both interphase and mitosis. The unusual timing of CID recruitment and unique dynamics of CAL1 identify a distinct centromere assembly pathway in Drosophila and suggest that CAL1 is a key regulator of centromere propagation (Mellone, 2011).

CID, CAL1 and CENP-C display different turnover and assembly dynamics, despite the fact that these essential centromeric components interact physically, and are interdependent for centromere localization. Epitope tagged SNAP-CID, SNAP-CAL1 and SNAP-CENP-C were expressed from the identical Copia promoter; thus it is unlikely that these distinctions are due to different rates of new protein synthesis. Using a pulse-chase strategy, it was shown that CID levels are reduced by ~50% after one cell cycle, which could result from semi-conservative distribution of pre-existing CID nucleosomes, or random redistribution of parental CID-H4 tetramers, to replicated sister chromatids. While CID and CENP-C display stable association with centromeres and 50:50 distribution after each cell cycle, 66% of TMR-CAL1 is replaced by new protein. Thus, CAL1 is either less stably bound, or its replenishment involves partial removal of pre-existing protein. Alternatively, CAL1 could undergo an even higher turnover and the quantification could be an underestimation; CAL1 could be entirely recruited de novo and the measured centromeric TMR-CAL1 could reflect recruitment from an initial soluble pool at the time of labeling (Mellone, 2011).

An additional difference is that while SNAP-CID and CAL1 are detectable at centromeres 1 h after quenching the SNAP epitopes, 10 h of chase time are necessary for CENP-C to be visible by fluorescent substrate tetramethylrhodamine (TMR) labeling. This suggests that at each cell cycle the recruitment of CID and CAL1 relies for the most part on newly-synthesized protein, while CENP-C recruitment also involves a pre-existing non-centromeric or soluble pool. Indeed, the cellular fractionation analysis demonstrated the presence of low levels of CENP-C in chromatin-free extracts, supporting the possibility that there is a soluble pool of CENP-C available to replenish the centromere-associated CENP-C diluted during the cell cycle (Mellone, 2011).

CENP-C is targeted to centromeres during multiple cell cycle stages, consistent with previous findings in human cells. In contrast, newly-synthesized CAL1 and CID are recruited to centromeres during discrete stages of mitosis. Using quench-chase-pulse time-courses in both asynchronous and arrested cultures, it was demonstrated that the contribution of interphase to CID loading is minimal, since the percent of interphase cells displaying newly-synthesized SNAP-CID and the signal intensity of TMR-CID differ dramatically from those measured for mitotic cells. These observations distinguish Drosophila from human HeLa cells, where CENP-A is recruited during G1, from fission yeast, where CENP-A assembles at centromeres in both S and G2 phases, as well as from plants and Dictyostelium (G2/prophase) (Mellone, 2011).

Both new CID and CAL1 are assembled at centromeres in mitosis, but each protein is recruited during discrete stages: prophase for CAL1 and metaphase for CID. It is possible that CID and CAL1 loading are initiated simultaneously in prophase, but CAL1 levels accumulate faster than CID at centromeres. Regardless, the observed temporal distinction suggests that CAL1 acts upstream of CID recruitment (see Model for centromere assembly in Drosophila cells.). Incorporation of nascent CAL1 at centromeres during prophase could be mediated by binding to pre-existing centromeric CID and CENP-C. This could in turn promote new incorporation of nascent CID during metaphase, either by gap-filling or exchange of space-holder histone H3 (Mellone, 2011).

Interestingly, a similar temporal distinction has been described for the human centromere proteins hMis18α, β and M18BP, which localize to centromeres in anaphase, before new CENP-A assembly in late telophase/G1. The lack of any physical interaction between hMis18α, β, M18BP and CENP-A, and the observation that hMis18α can localize to centromeres even if CENP-A is depleted, has led to the proposal that this complex may 'prime' centromeres to receive new CENP-A (Fujita, 2007) from the HJURP chaperone, whose centromeric targeting coincides temporally with deposition of new CENP-A. Homologs for hMis18 complex components and HJURP (or the budding and fission yeast Scm3 homologs) have not been identified in the Drosophila genome (Mellone, 2011).

Collectively these data support a model in which CAL1 performs functions attributable to both HJURP and hMis18, despite the lack of sequence homology. hMis18 proteins are recruited to centromeres before CENP-A, and CAL1 loading precedes CID assembly. However, the hMis18 complex does not interact with CENP-A, whereas CAL1 and CID are associated in chromatin-free extracts, identifying the first Drosophila protein that binds CID in its pre-nucleosomal form. HJURP also interacts with pre-nucleosomal CENP-A (Foltz, 2009), and both HJURP and CAL1 strongly colocalize with nucleoli. Thus, CAL1 could 'prime' the centromere in prophase, and also mediate CID recruitment directly in metaphase (Mellone, 2011 and references therein).

It has been showm that gross-levels of centromeric GFP-CID and GFP-CENP-C did not visibly change through the cell cycle in time-lapse analysis, consistent with the 50:50 segregation observed in this study during one division. In contrast, GFP-CAL1 levels were significantly reduced in metaphase, increased again in telophase, and remained stable through interphase (Ehrlich, 2008). The transient reduction in GFP-CAL1 levels at metaphase is intriguing, given that it coincides with new CID assembly. The observation that newly assembled TMR-CAL1 intensities were constant from prophase to cytokinesis suggests that most of the GFP-CAL1 reduction at metaphase and increase at telophase involves pre-existing protein. One model to account for these observations is that free CAL1 (not bound to CID) is recruited to centromeres in prophase where it performs a yet undefined ‘priming’ function; then, the subset of CAL1 bound to pre-nucleosomal CID escorts it to centromeres in metaphase while 'old' CAL1 is displaced (Model for centromere assembly in Drosophila cells.). The interdependency of CAL1, CID and CENP-C in centromere localization (Ehrlich, 2008) could be explained by the requirement of pre-existing CID and CENP-C for CAL1 assembly in prophase (Mellone, 2011).

The loading of CID and CAL1 in specific, early stages of mitosis also raises questions about the nature of the signal(s) that initiate assembly of centromeric chromatin. Centromere replenishment signaling by kinetochore-microtubule interactions is inconsistent with the demonstration that CID loading in metaphase is not affected by colchicine treatment, and therefore does not require spindles (as also observed in human cells), SAC inactivation, chromosome segregation, or inter-kinetochore tension. However, it has been shown that premature activation of the Anaphase Promoting Complex, by Cyclin A or RCA1 depletion, interferes with CID localization to centromeres, demonstrating that centromeric chromatin assembly is linked to key regulators of mitotic progression. Interestingly, Cyclin A localizes to centromeres and is degraded in metaphase; this study has demonstrated that metaphase loading depends on proteasome activity, which could include degradation of key mitotic regulators. MG132 treatment prior to BTP block prevented CID loading while transfecting cells with a non-degradable form of CYCA abrogated new CID recruitment in a subset of cells, and TMR-CID levels were significantly reduced in most cells. One possibility to explain the stronger impact of proteasome inhibition is that proteasome targets in addition to CYCA need to be degraded for efficient CID deposition. Alternatively, the presence of centromeric endogenous CYCA, which is probably degraded normally in the presence of excess ND-CYCA, might trigger a sufficient signal to initiate CID incorporation in some cells. Interestingly, Cyclin A is degraded in the presence of microtubule drugs and escapes inhibition of the APC by the SAC, which would explain why new CID recruitment takes place efficiently in the presence of colchicine. Proteasome and ubiquitin-ligase activities have been implicated in controlling proper CENP-A centromeric incorporation by degradation of euchromatic CENP-A in budding yeast and Drosophila. Understanding the relationship between the CENP-A degradation pathway and the implication of proteasome activity in the recruitment of nascent CENP-A will require further investigation (Mellone, 2011).

It is unclear at this point how degradation of CYCA contributes to CID assembly. One possibility is that high CDK-CYCA activity at the centromere inhibits CID recruitment, and that local inhibition of CDK activity through degradation of CYCA or other substrates triggers CID assembly. Understanding the role of degradation of Cyclin A and other APC and proteasome substrates in CID recruitment will be crucial to elucidating how centromere assembly is coupled to the cell cycle (Mellone, 2011).

The dynamics of centromere replenishment in Drosophila cultured cells differs from those observed in S. pombe and human HeLa cells. Early syncytial fly embryos display slightly later recruitment of new CID in anaphase, but this difference could be due to the unusually short nuclear cycles that lack G1 and G2 phases. Although CENP-A loading in HeLa cells is first observed in telophase, it is possible that the primary signal to initiate CENP-A loading (e.g. inhibiting local CDK-CYCA activity at the centromere) is conserved, and occurs during prophase or metaphase in both Drosophila and human cells (Mellone, 2011).

It is also puzzling that key proteins required in trans for CENP-A assembly, such as HJURP and CAL1, are not always conserved, in contrast to the universality of centromeric chromatin components such as CENP-A and CENP-C. It is possible that highly diverged proteins, such as CAL1, perform the same function(s) as human regulators such as HJURP and hMis18. Thus, although this data challenges the universality of centromere propagation dynamics in metazoans, it will be important to determine whether some mechanisms and signals required for CENP-A replenishment are conserved, despite different times of assembly in the cell cycle, and the lack of conservation for key regulatory proteins (Mellone, 2011).

Centromere proteins CENP-C and CAL1 functionally interact in meiosis for centromere clustering, pairing, and chromosome segregation

Meiotic chromosome segregation involves pairing and segregation of homologous chromosomes in the first division and segregation of sister chromatids in the second division. Although it is known that the centromere and kinetochore are responsible for chromosome movement in meiosis as in mitosis, potential specialized meiotic functions are being uncovered. Centromere pairing early in meiosis I, even between nonhomologous chromosomes, and clustering of centromeres can promote proper homolog associations in meiosis I in yeast, plants, and Drosophila. It was not known, however, whether centromere proteins are required for this clustering. This study exploited Drosophila mutants for the centromere proteins centromere protein-C (CENP-C) and chromosome alignment 1 (CAL1) to demonstrate that a functional centromere is needed for centromere clustering and pairing. The cenp-C and cal1 mutations result in C-terminal truncations, removing the domains through which these two proteins interact. The mutants show striking genetic interactions, failing to complement as double heterozygotes, resulting in disrupted centromere clustering and meiotic nondisjunction. The cluster of meiotic centromeres localizes to the nucleolus, and this association requires centromere function. In Drosophila, synaptonemal complex (SC) formation can initiate from the centromere, and the SC is retained at the centromere after it disassembles from the chromosome arms. Although functional CENP-C and CAL1 are dispensable for assembly of the SC, they are required for subsequent retention of the SC at the centromere. These results show that integral centromere proteins are required for nuclear position and intercentromere associations in meiosis (Unhavaithaya, 2013).

Localization studies demonstrated centromere pairing in yeast, Drosophila, and plants, and it showed that the centromeres cluster together in Drosophila meiosis I. This study has establish that centromere function is required for both pairing and clustering. Thus, centromeres are integrally involved in these two processes and not brought together solely by external factors. Because these events occur before assembly of the kinetochore, it is likely that the chromatin and associated proteins at the centromere are critical. The mutations in cenp-C reveal that functional CENP-C is necessary at a minimum for maintenance of centromere pairing and clustering in Drosophila oocytes. The noncomplementation between truncated CENP-C and CAL1 protein forms implicates CAL1 as also being crucial for centromere pairing and clustering. Given the role of CENP-C in recruiting proteins to the centromere, the requirement for this protein could reflect a direct role in centromere pairing and clustering or the need for a protein whose localization is dependent on CENP-C and/or CAL1. In the cenp-C mutant and the cenp-C cal1 double-heterozygous mutant, CID is still localized to the centromere, as evidenced by its presence at brightly DAPI-stained heterochromatin at levels that, by immunofluorescence, are not significantly lower than WT. Thus, CID presence is insufficient for centromere clustering and pairing. The reduced level of CID staining in the double-heterozygous mutant is nearly significant, however; thus, the possibility that reduced CID levels contribute to the mutant defects is not excluded (Unhavaithaya, 2013).

The proteins at the centromere may interact with nuclear structures to promote centromere clustering. This study identifies the nucleolus as a likely candidate. The centromere clusters are associated with the nucleolus in WT oocytes, and this association requires cenp-C and cal1 function. In Drosophila female meiosis, the nucleolus may serve as an anchor site for centromeres throughout prophase I. The SC also may cluster centromeres. Clustering has been shown to be disrupted in mutants for the SC transverse and central elements. The observation that the SC protein C(3)G fails to be retained at the centromere in cenp-C and cal1 mutants raises the possibility that the failure of clustering in these centromere protein mutants is a consequence of the absence of the SC. The hypothesis of this causality is consistent with the timing of defects; as early as pachytene, both centromere SC and clustering are absent. It remains to be determined how the SC, a structure contained between pairs of homologs, could gather centromeres into a cluster. In c(3)g mutants, more than four CID foci can be observed, indicating that both centromere pairing and clustering can be affected. Thus, failure of centromere retention of the SC also could account for the pairing defects in the centromere protein mutants (Unhavaithaya, 2013).

The allele-specific noncomplementation (type I second-site noncomplementation) between the mutations causing C-terminal truncations of CENP-C and CAL1 is unusual and informative. Such mutations that alter protein structure rather than simply reducing protein levels provide the opportunity to investigate genetic interactions. This allele-specific noncomplementation affects all the processes analyzed: centromere pairing, centromere clustering and nucleolar association, SC retention at the centromere, and meiotic segregation. The antagonistic genetic interaction requires the truncated protein forms, because deficiencies for each of the genes complement the truncation allele of the other for meiotic segregation and cause only slight defects in centromere pairing and clustering. This is also true for the cenp-CZ3-4375 allele that reduces protein levels. Thus, simply decreasing the levels of the proteins does not perturb these processes. The C-terminal region of CAL1 binds to CENPC, whereas the N terminus binds to CID; thus, the truncated form could have a dominant negative effect by binding CID and blocking its link to CENP-C. The C terminus of CENP-C is required for its localization to the centromere as well as binding to CAL1, whereas it binds the KNL-1/Mis12 complex/Ndc80 complex (KMN) kinetochore network via its N terminus. Thus, C-terminal truncated CENP-C also could act as a dominant negative to uncouple the KMN complex from a functional centromere association, particularly given that the N terminus alone can bind to kinetochore proteins but not to the centromere. Expression of the N terminus alone also can disrupt the spindle assembly checkpoint. The truncation alleles of cenp-C and cal1 each alone have slight semidominant effects on centromere pairing, clustering, and meiotic segregation, consistent with dominant negative activities. The combination of the two dominant negative effects could account for perturbation of the meiotic processes. It cannot be excluded, however, that these truncation alleles act as recessive neomorphs, conferring novel properties on the proteins (Unhavaithaya, 2013).

A critical question is whether centromere clustering is required for proper meiotic segregation. It remains to be determined whether the meiotic nondisjunction that occurs in these centromere protein mutants is linked to the failure of centromere clustering and/or centromere pairing. The meiotic segregation errors in oocytes affect both the X chromosome, which undergoes recombination, and the 4th chromosome, which is achiasmate and lacks SC. One way that meiotic segregation of both types of chromosomes could be dependent on clustering would be if association with the nucleolus is necessary for proper assembly of the kinetochore later in prophase I. It is notable, however, that the meiotic segregation errors in oocytes assayed for the X chromosome occurred exclusively in meiosis I; thus, a defect in kinetochore function necessary for both meiosis I and II was not evident. There are known meiosis I-specific requirements of the kinetochore, such as the need for the two sister kinetochores to co-orient in meiosis I, and establishment of these may require centromere clustering and/or nucleolar association (Unhavaithaya, 2013).

This proposal is consistent with the demonstrated effects of cenp-C mutants in meiosis in Saccharomyces pombe. An alternative possibility is that the centromere mutations have independent effects on centromere clustering and subsequent segregation. For example, the centromere clustering defects could result from failure to retain the SC at the centromere and the meiotic nondisjunction could be an independent consequence of improperly assembled kinetochores later in meiosis I. The centromere mutations clearly can affect meiotic segregation independent of centromere pairing and clustering, given the meiotic nondisjunction in males double-heterozygous for the cenp-C and cal1 alleles. In Drosophila male meiosis, centromere clustering, SC formation, and recombination do not occur (Unhavaithaya, 2013).

Although observed in yeast, plants, and Drosophila, a role for intrinsic centromere function in the nuclear localization of centromeres and associations between centromeres in meiosis has not yet been defined. The demonstration that proper centromere architecture is necessary for these interactions opens a path to define the molecular basis of centromere pairing and clustering across these species in meiosis (Unhavaithaya, 2013).

Genome-wide analysis reveals a cell cycle-dependent mechanism controlling centromere propagation

Centromeres are the structural and functional foundation for kinetochore formation, spindle attachment, and chromosome segregation. In this study, factors required for centromere propagation were isolated using genome-wide RNA interference screening for defects in centromere protein A (CENP-A; centromere identifier [CID]) localization in Drosophila. The proteins CAL1 and CENP-C were identified as essential factors for CID assembly at the centromere. CID, CAL1, and CENP-C coimmunoprecipitate and are mutually dependent for centromere localization and function. The mitotic cyclin A (CYCA) and the anaphase-promoting complex (APC) inhibitor RCA1/Emi1 were identified as regulators of centromere propagation. CYCA was shown to be centromere localized, and CYCA and RCA1/Emi1 were shown to couple centromere assembly to the cell cycle through regulation of the fizzy-related/CDH1 subunit of the APC. These findings identify essential components of the epigenetic machinery that ensures proper specification and propagation of the centromere and suggest a mechanism for coordinating centromere inheritance with cell division (Erhardt, 2008).

This is the first example of a genome-wide RNAi screen for mislocalization of an endogenous chromosomal protein and provides the distinct advantage that the primary screen output is a direct readout of the phenotype of interest. This approach identified novel and known factors that control the assembly of centromeric chromatin and link centromere assembly and propagation to the cell cycle (Erhardt, 2008).

Although centromere assembly has been described as a hierarchical process directed by CENP-A, the data show that CID, CENP-C, and CAL1 are interdependent for centromere propagation, which is consistent with experiments in vertebrate cells showing interdependence between the CENP-H-CENP-I complex and CENP-A. However, studies in C. elegans and vertebrates have not detected a role for CENP-C in CENP-A chromatin assembly, suggesting that CENP-C plays a more prominent role in regulating centromere propagation in flies. Collectively, these results suggest that CENPs that depend on CENP-A for their localization may 'feed back' to control CENP-A assembly. Histone variants are assembled into chromatin both by histone chaperones (e.g., the histone H3.3-specific chaperone HIRA [histone regulatory A] that provides specificity to the CHD1 chromatin-remodeling ATPase) and by histone variant-specific ATPases (e.g., Swr1 that can use the general chaperone Nap1 or the specific chaperone Chz1 to assemble H2A.Z). CENP-C or CAL1 might facilitate centromere-specific CID localization by providing centromere specificity to a chromatin-remodeling ATPase in a manner analogous to HIRA or might direct the localization of chromatin assembly factors to the centromere. It will be interesting to determine what factors associate with CAL1 and CENP-C as a route to elucidating the mechanisms of centromere assembly and propagation (Erhardt, 2008).

The loading of CENP-A in human somatic cells and in Drosophila embryos occurs after anaphase initiation when APC^FZR/CDH1 activity is high. Ubiquitin-mediated proteolysis facilitates formation of a single centromere by degrading noncentromeric CENP-A, and subunits of the APC are localized to kinetochores. The results demonstrate that normal regulation of APC^FZR/CDH1 activity is required for centromere propagation, providing a link between centromere assembly and cell cycle regulation (Erhardt, 2008).

Two alternative models are proposed for the role of APC^FZR/CDH1 in centromere function. The first model is that CYCA is the relevant substrate of APC^FZR/CDH1 and that the kinase activity of the CYCA-Cdk1 complex is required for the localization of CID, CENP-C, and CAL1 to the centromere. CYCA is normally degraded as cells proceed through mitosis, suggesting that CYCA-Cdk1 would likely act during G2 or early M to phosphorylate a substrate involved in centromere assembly. The CID and CENP-C localization defect caused by CYCA depletion was rescued by the simultaneous depletion of FZR/CDH1 even though the levels of CYCA protein remained low in the double depletion. The rescue of the CID and CENP-C localization defect in cells with low CYCA protein suggests that maintaining high levels of CYCA-Cdk1 activity is not required for centromere propagation, but it cannot be ruled out that the residual CYCA protein in these cells is sufficient to rescue the centromeric phenotype when APC activity is compromised by FZR/CDH1 depletion (Erhardt, 2008).

The second model that is consistent with these observations is that one or more APC^FZR/CDH1 substrates (“X”) regulate the interdependent localization of CID, CENP-C, and CAL1 to the centromere. RCA1 and CYCA inhibit the APC in G2 to allow mitotic cyclin accumulation. An APC^FZR/CDH1 substrate could repress centromere assembly until anaphase/G1, when proteolysis would remove the repression in a manner analogous to replication licensing. If an APC^FZR/CDH1 substrate acted solely as a negative regulator of centromere assembly, FZR/CDH1 depletion should prevent CID assembly at centromeres, and premature APC^FZR/CDH1 activation by CYCA or RCA1 depletion might cause an increase of CID at centromeres as a result of premature assembly. It was observed that neither CDH1 nor CDC20 depletion alone impacted CID, CAL1, or CENP-C assembly at centromeres or the overall levels of these proteins but that premature APC activation resulted in failed centromere assembly (Erhardt, 2008).

A simple interpretation of the results is that CYCA-Cdk1 or another APC^FZR/CDH1 substrate acts during G2/metaphase before APC^FZR/CDH1 activation to make centromeres competent for assembly during anaphase and/or G1. Premature removal of the APC^FZR/CDH1 substrate would cause failure to relicense the centromeres for assembly in the next G1 phase. When compared with the process of replication licensing, in which the positive regulator CDC6 and the negative regulators geminin and CYCA are all substrates of APC^FZR/CDH1, the model of a single APC^FZR/CDH1 substrate that controls centromere licensing or propagation may be oversimplified. This study observed that defective centromere localization of CID and CENP-C after CYCA or RCA1 depletion was not rescued by CDC20 depletion, but a role for APC^FZY/CDC20 in centromere propagation cannot be ruled out because premature APC^FZR/CDH1 activation could mask a subsequent role for FZY/CDC20, which is activated at the metaphase/anaphase transition (Erhardt, 2008).

It is not yet known whether the localization of CYCA at centromeres is important for the regulation of centromere assembly. In Drosophila, it has been demonstrated that the subcellular localization of CYCA is not important for proper progression through the cell cycle; however, these experiments did not directly address whether mislocalization of CYCA prevented the association of CYCA with centromeres. It will be interesting to determine whether CID, CENP-C, and CAL1 localization require centromere-localized CYCA-Cdk1 activity or whether any of these proteins are a direct target of CYCA-Cdk1 (Erhardt, 2008).

The results suggest that CID or CAL1 levels are indirectly controlled by APC activity. Interestingly, the human M18BP1 has recently been proposed to act as a 'licensing factor' for centromere assembly. Although no clear homologues of M18BP1/KNL2 have been identified in Drosophila, both CAL1 in flies and M18BP1/KNL2 in other species are interdependent with CENP-A for centromere localization. Strikingly, levels of CAL1 and M18BP1/KNL2 are reduced on metaphase centromeres and increase coincident with CENP-A loading in late anaphase/telophase. Further analysis is required to determine whether CAL1 and M18BP1/KNL2 function analogously in centromere assembly. It will be important to determine whether fly homologues of other Mis18 complex components are associated with CAL1 and important for centromere assembly. Identifying the APC substrates involved in centromere assembly will be necessary to distinguish between these models and to determine how these proteins epigenetically regulate centromere assembly and couple this essential process to the cell cycle (Erhardt, 2008).

Search PubMed for articles about Drosophila Cal1

Chen, C. C., Dechassa, M. L., Bettini, E., Ledoux, M. B., Belisario, C., Heun, P., Luger, K. and Mellone, B. G. (2014). CAL1 is the Drosophila CENP-A assembly factor. J Cell Biol 204(3): 313-329. PubMed ID: 24469636

Dattoli, A. A., Carty, B. L., Kochendoerfer, A. M., Morgan, C., Walshe, A. E. and Dunleavy, E. M. (2020). Asymmetric assembly of centromeres epigenetically regulates stem cell fate. J Cell Biol 219(4). PubMed ID: 32328637

Dunleavy, E. M., et al. (2009). HJURP is a cell-cycle-dependent maintenance and deposition factor of CENP-A at centromeres. Cell 137: 485-497. PubMed ID: 19410545

Erhardt, S., et al. (2008). Genome-wide analysis reveals a cell cycle-dependent mechanism controlling centromere propagation. J. Cell Biol. 183: 805-818. PubMed ID: 19047461

Foltz, D. R., et al. (2009). Centromere-specific assembly of CENP-a nucleosomes is mediated by HJURP. Cell 137: 472-484. PubMed ID: 19410544

Fujita, Y., et al. (2007). Priming of centromere for CENP-A recruitment by human hMis18alpha, hMis18beta, and M18BP1. Dev. Cell 12: 17-30. PubMed ID: 17199038

Goshima, G., et al. (2007). Genes required for mitotic spindle assembly in Drosophila S2 cells. Science 316: 417-421. PubMed ID: 17412918

Heun, P., et al. (2006). Mislocalization of the Drosophila centromere-specific histone CID promotes formation of functional ectopic kinetochores. Dev. Cell 10: 303-315. PubMed ID: 16516834

Joglekar, A. P., et al. (2006). Molecular architecture of a kinetochore-microtubule attachment site. Nat. Cell Biol. 8: 581-585. PubMed ID: 16715078

Joglekar, A. P., et al. (2008). Molecular architecture of the kinetochore-microtubule attachment site is conserved between point and regional centromeres. J. Cell Biol. 181: 587-594. PubMed ID: 18474626

Joglekar, A. P. and Kukreja, A. A. (2017). How kinetochore architecture shapes the mechanisms of its function. Curr Biol 27(16): R816-R824. PubMed ID: 28829971 .

Lam, A. L., et al. (2006). Human centromeric chromatin is a dynamic chromosomal domain that can spread over noncentromeric DNA. Proc. Natl. Acad. Sci. 103: 4186-4191. PubMed ID: 16537506

Maiato, H., et al. (2006). The ultrastructure of the kinetochore and kinetochore fiber in Drosophila somatic cells. Chromosoma 115: 469-480. PubMed ID: 16909258

Mellone, B. G., et al. (2011). Assembly of Drosophila centromeric chromatin proteins during mitosis. PLoS Genet. 7(5): e1002068. PubMed ID: 21589899

Musacchio, A. (2015). The molecular biology of spindle assembly checkpoint signaling dynamics. Curr Biol 25(20): R1002-1018. PubMed ID: 26485365

Musacchio, A. and Desai, A. (2017). A molecular view of kinetochore assembly and function. Biology (Basel) 6(1). PubMed ID: 28125021

Okada, M., et al. (2006). The CENP-H-I complex is required for the efficient incorporation of newly synthesized CENP-A into centromeres. Nat. Cell Biol. 8: 446-457. PubMed ID: 16622420

Pauleau, A. L., Bergner, A., Kajtez, J. and Erhardt, S. (2019). The checkpoint protein Zw10 connects CAL1-dependent CENP-A centromeric loading and mitosis duration in Drosophila cells. PLoS Genet 15(9): e1008380. PubMed ID: 31553715

Perpelescu, M., et al. (2009). Active establishment of centromeric CENP-A chromatin by RSF complex. J. Cell Biol. 185: 397-407. PubMed ID: 19398759

Pidoux, A. L., et al. (2009). Fission yeast Scm3: A CENP-A receptor required for integrity of subkinetochore chromatin. Mol. Cell 33: 299-311. PubMed ID: 19217404

Santaguida, S. and Musacchio, A. (2009). The life and miracles of kinetochores. EMBO J. 28: 2511-2531. PubMed ID: 19629042

Schittenhelm, R. B., Althoff, F., Heidmann, S. and Lehner, C. F. (2010). Detrimental incorporation of excess Cenp-A/Cid and Cenp-C into Drosophila centromeres is prevented by limiting amounts of the bridging factor Cal1. J. Cell Sci. 123(Pt 21): 3768-79. PubMed ID: 20940262

Schuh, M., Lehner, C. F. and Heidmann, S. (2007). Incorporation of Drosophila CID/CENP-A and CENP-C into centromeres during early embryonic anaphase. Curr. Biol. 17: 237-243. PubMed ID: 17222555

Unhavaithaya, Y. and Orr-Weaver, T. L. (2013). Centromere proteins CENP-C and CAL1 functionally interact in meiosis for centromere clustering, pairing, and chromosome segregation. Proc Natl Acad Sci U S A 110: 19878-19883. PubMed ID: 24248385

Williams, J. S., Hayashi, T., Yanagida, M. and Russell, P. (2009). Fission yeast Scm3 mediates stable assembly of Cnp1/CENP-A into centromeric chromatin. Mol. Cell 33: 287-298. PubMed ID: 19217403

date revised: 25 September 2023

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