InteractiveFly: GeneBrief

chromosome alignment defect 1: Biological Overview | References

Gene name - chromosome alignment defect 1

Synonyms -

Cytological map position - 89E12-89E12

Function - chromatin protein

Keywords - regulator of centromeric deposition of Cenp-A/Cid and Cenp-C during exit from mitosis, substochiometric centromeric linker protein involved in centromere propagation

Symbol - cal1

FlyBase ID: FBgn0038478

Genetic map position - chr3R:12,914,013-12,917,663

Classification - N- and C-terminal interaction domains

Cellular location - nuclear

NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Rosin, L. and Mellone, B.G. (2016). Co-evolving CENP-A and CAL1 domains mediate centromeric CENP-A deposition across Drosophila species. Dev Cell 37: 136-147. PubMed ID: 27093083
Centromeres mediate the conserved process of chromosome segregation, yet centromeric DNA and the centromeric histone, CENP-A, are rapidly evolving. The rapid evolution of Drosophila CENP-A loop 1 (L1) is thought to modulate the DNA-binding preferences of CENP-A to counteract centromere drive, the preferential transmission of chromosomes with expanded centromeric satellites. Consistent with this model, CENP-A from Drosophila bipectinata (bip) cannot localize to Drosophila melanogaster (mel) centromeres. It was shown that this result is due to the inability of the mel CENP-A chaperone, CAL1, to deposit bip CENP-A into chromatin. Co-expression of bip CENP-A and bip CAL1 in mel cells restores centromeric localization, and similar findings apply to other Drosophila species. Two co-evolving regions, CENP-A L1 and the CAL1 N terminus, were identified as critical for lineage-specific CENP-A incorporation. Collectively, these data show that the rapid evolution of L1 modulates CAL1-mediated CENP-A assembly, suggesting an alternative mechanism for the suppression of centromere drive.


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).

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 APCFZR/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 APCFZR/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 APCFZR/CDH1 in centromere function. The first model is that CYCA is the relevant substrate of APCFZR/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 APCFZR/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 APCFZR/CDH1 substrate could repress centromere assembly until anaphase/G1, when proteolysis would remove the repression in a manner analogous to replication licensing. If an APCFZR/CDH1 substrate acted solely as a negative regulator of centromere assembly, FZR/CDH1 depletion should prevent CID assembly at centromeres, and premature APCFZR/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 APCFZR/CDH1 substrate acts during G2/metaphase before APCFZR/CDH1 activation to make centromeres competent for assembly during anaphase and/or G1. Premature removal of the APCFZR/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 APCFZR/CDH1, the model of a single APCFZR/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 APCFZY/CDC20 in centromere propagation cannot be ruled out because premature APCFZR/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

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

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

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

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

Biological Overview

date revised: 18 September 2011

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