The cid gene and the short upstream region constituting the promoter were cloned into a GFP fusion construct and the plasmid was introduced into D. melanogaster Kc cells by transient transfection. Cytological spread preparations were examined by fluorescence microscopy. As with anti-Cid antibody, Cid-GFP localizes to intense, point-like signals in interphase nuclei. In metaphase chromosomes, centromeres were found to be specifically labeled, based on precise colocalization with primary constrictions and colocalization with anti-Polo kinase antibody, which decorates Drosophila kinetochores of metaphase chromosomes. The slight offset of Cid-GFP and Polo kinase may reflect the offset of the centromere from the kinetochore (Henikoff, 2000).
Cid-GFP localizes to centromeres even when it is driven by a heterologous promoter. When the Drosophila hsp70 promoter was substituted for the cid promoter, sharp interphase spots were found indicative of centromeric localization in uninduced cells. Expression is probably due to low-level constitutive activity of the hsp70. Upon heat shock induction, spots increased 10-fold in intensity (Henikoff, 2000).
A report of cell cycle-limited expression of mRNA encoding CenpA led the authors to test for comparable behavior of cid mRNA. However, cid mRNA appears to be rare, since its cDNA is not represented among the 80,000 D. melanogaster expressed sequence tags found in GenBank, and cid mRNA was not detected in situ (Henikoff, 2000).
A serendipitous observation, that the C. elegans HCP-3 histone H3-like protein expressed from an uncharacterized genomic fragment displayed subnuclear localization in Kc cells, led the authors to consider that any H3-like heterolog might localize in an interesting manner. As was done for Cid, H3 and H2B, a full-length ORF from each of the other branches of the H3 phylogenetic tree was fused to GFP and driven by the cid promoter. These ORFs are Cse4p, human CENP-A, C. elegans HCP-3, and C. elegans D6H3. In striking contrast to the euchromatic pattern seen for histone-GFP constructs driven by the cid promoter, all four H3-like heterologs expressed from the same promoter localized preferentially to pericentric heterochromatin. To identify heterochromatin in metaphase chromosomes, its appearance in standard cytological preparations was utilized: heterochromatin remains attached between sister chromatids and so can be readily distinguished from euchromatic arms, which display no sister chromatid cohesion. On the acrocentric X chromosome, which is heterochromatic for the entire proximal one-half, the peak of localization was observed consistently over a subset of heterochromatin near the centromere. Localization of heterologs was seen to extend into pericentric regions of all chromosomes, sometimes with ubiquitous low-intensity labeling throughout the chromosomes. Preferential pericentric localization was seen over a 10x range of intensities; this range is presumably due to cell-to-cell differences in plasmid copy number (Henikoff, 2000).
Pericentric localization of H3-like proteins from yeast, worms, and humans is unlikely to result from sequence recognition, because any presumptive sequence target is not thought to be in common between the centromeres of these organisms and the pericentric regions of flies. Preferential localization is also unlikely to be due to recognition of a preexisting centromeric determinant, because the labeling encompasses a region that is much broader than the centromere itself (Henikoff, 2000).
To test the generality of preferential pericentric localization in Drosophila, H3-like heterologs were introduced into human cells. GFP fusion constructs of Cse4p and one of the worm H3-like proteins, HCP-3, were driven by the constitutive cytomegalovirus promoter after transient transfection. As a control, an H3-GFP construct was transfected; this gave a uniform nuclear pattern of fluorescence as expected for general chromatin localization. However, both yeast Cse4p-GFP and worm HCP-3-GFP display many small and six to nine large spots of localization over a weaker chromatin background. These spots were observed consistently over a 10x range of intensities corresponding to a range of plasmid copy numbers that typically are obtained in transient transfection experiments. Using an anticentromere antibody (ACA), it was confirmed that most centromeres were associated with GFP spots. Many of the small GFP spots were coincident with the ACA spots, and each large GFP spot typically encompassed at least one ACA spot. Therefore, heterologous H3-like proteins from yeast and worms localize on human chromatin to regions that include centromeres (Henikoff, 2000).
Localization of heterologous H3-like proteins to human centromeres was confirmed by examination of mitotic figures. Overall, GFP signals were less intense at metaphase than at interphase, perhaps because of the more condensed state of mitotic chromosomes. Mitotic spreads revealed that the large, intense spots correspond to pericentric regions of a few specific chromosomes. ACA labeling is confined to the primary constriction, whereas large-spot GFP fluorescence is seen to extend into adjacent regions. This consistent pattern of pericentric localization suggests that the large spots are sites of human classical satellites, which show similar pericentric localization. Certain additional regions occasionally were seen to be labeled, including telomeres. It is concluded that, in contrast to H3 itself, yeast and worm H3-like heterologs show preferential pericentric localization in both human and Drosophila cells (Henikoff, 2000).
Thus, deposition in heterochromatin appears to be a general feature of centromeric H3-like proteins in heterologous systems, because H3-like centromeric proteins from yeast and worms also localize to heterochromatic regions when they are constitutively expressed in human cells. This unexpected behavior of heterologs contradicts expectations based on specific recognition of centromere-specific or sequence-specific determinants. It is concluded that localization to heterochromatin must be a general property of centromeric H3-like proteins (Henikoff, 2000).
Pericentric localization behavior is especially surprising given the divergence of these proteins from one another in the core region. Yeast and worm H3-like proteins are no more similar (in fact, marginally less similar) to native fly and mammalian centromeric H3-like proteins than they are to H3 itself. Yet, H3 displays contrasting localization behavior when expressed identically to H3-like proteins in both fly and human cells. Therefore, preferential heterochromatic localization behavior in heterologous systems is not attributable to similarities that are shared with native centromere proteins and distinguish them from H3 itself (Henikoff, 2000).
Heterochromatin comprises ~10%-50% of the genomes of complex eukaryotes, and, yet, its function remains enigmatic, although the consistent presence of centromeres in heterochromatin suggests a mitotic role. However, deletion studies have not distinguished between a mitotic requirement for specific DNA or protein determinants vs. a requirement for heterochromatin per se. The demonstration that in both Drosophila and human cells heterologous centromeric histones localize to heterochromatic regions suggests that the heterochromatic state directly facilitates the localization of centromere proteins (Henikoff, 2000).
In light of the identical heterochromatic localization of diverse heterologous H3-like proteins, the precise localization of the native proteins presents a paradox: what prevents Cid and CenpA from also localizing broadly to heterochromatin? Precise localization to centromeres occurs even when Cid-GFP is induced at high levels, so it seems unlikely that the failure to localize more broadly is due to limiting amounts of protein. It is conceivable that native H3-like proteins are actively prevented from depositing in heterochromatin; however, this hypothesis leaves the preferential deposition of heterologs unexplained (Henikoff, 2000).
This paradox is resolved if it is supposed that both native and heterologous H3-like proteins deposit broadly to heterochromatin, but only native proteins come together to form a single, coherent structure that organizes a kinetochore. Such coming together of dispersed subunits has been proposed for mammalian centromeres and has been thought to involve large-scale looping within pericentric heterochromatin not detectable in standard cytological preparations. Support for this model comes from the observation that in C. elegans, dispersed HCP-3 comes together at prophase to form a ribbon-like centromere. The subunit model is consistent with evidence that centromere competence may be a general feature of satellite sequences and asserts that the mapping of centromeres to regions of a few hundred kilobases of satellite sequences within larger expanses of heterochromatin simply maps the location of the highest concentration of centromere subunits. By this model, the machinery responsible for the coming together of subunits would discriminate between native and heterologous H3-like proteins, and the in situ assay described in this study may be used to probe the biochemical nature of this process (Henikoff, 2000).
The centromere-specific histone variant CENP-A (CID in Drosophila) is a structural and functional foundation for kinetochore formation and chromosome segregation. Overexpressed CID is mislocalized into normally noncentromeric regions in Drosophila tissue culture cells and animals. Analysis of mitoses in living and fixed cells reveals that mitotic delays, anaphase bridges, chromosome fragmentation, and cell and organismal lethality are all direct consequences of CID mislocalization. In addition, proteins that are normally restricted to endogenous kinetochores assemble at a subset of ectopic CID incorporation regions. The presence of microtubule motors and binding proteins, spindle attachments, and aberrant chromosome morphologies demonstrate that these ectopic kinetochores are functional. It is concluded that CID mislocalization promotes formation of ectopic centromeres and multicentric chromosomes, which causes chromosome missegregation, aneuploidy, and growth defects. Thus, CENP-A mislocalization is one possible mechanism for genome instability during cancer progression, as well as centromere plasticity during evolution (Heun, 2006).
CENP-A has been demonstrated to provide a structural and functional foundation for the kinetochore in a variety of organisms. This study shows that ectopic incorporation of Drosophila CID into normally noncentromeric chromatin occurs in response to overexpression in S2 and animal cells, as observed previously in tissue culture cells and yeast. This study shows that CID mislocalization results in defective cell growth, cell and organismal death, and abnormal development (Heun, 2006).
The results strongly support the conclusion that these mitotic abnormalities and growth defects are caused by formation of ectopic kinetochores and multiple spindle attachments on individual chromatids. (1) Studies of fixed and live cells demonstrated that CID overexpression causes significant mitotic defects, including increased mitotic index and stretched, fragmented, and lagging chromosomes during anaphase. Time-lapse analysis in S2 cells revealed that CID overexpression also causes mitotic delays, as well as cut phenotypes, chromosome loss, and abnormal chromosome morphology during anaphase segregation (Heun, 2006).
(2) Proteins that are normally associated with endogenous centromeres are present at ectopic sites in response to CID mislocalization. The distribution and colocalization were examined of proteins involved in different centromere/kinetochore structures and functions, extending from the centromeric chromatin to the outer kinetochore. Proteins associated with centromeric chromatin (CENP-C), centromeric cohesion (MEI-S332, BUBR1), outer kinetochore formation and motor protein recruitment (ROD, POLO), and the SAC (BUBR1) are mislocalized and colocalize to normally noncentromeric regions in S2 and animal cells with mislocalized CID. Thus, proteins involved in a wide spectrum of centromere and kinetochore functions are recruited together to ectopic sites after CID mislocalization (Heun, 2006).
(3) CID mislocalization results in significantly elevated numbers of sites containing the kinetochore-associated KLP59C and Dynein motor proteins, and the microtubule plus-end binding protein MAST. KLP59C and Dynein are frequently colocalized at normally noncentromeric regions of chromosomes that display aberrant anaphase chromosome morphology. This data strongly suggests that ectopic CID incorporation can seed kinetochores that are able to form stable microtubule attachments, and that these attachments are able to transmit forces to chromosomes during mitosis. MAST localization in metaphase is likely to provide the best estimate for the number of ectopic functional kinetochores formed after CID induction, approximately twice the number observed in controls (Heun, 2006).
(4) CID mislocalization resulted in the appearance of cold-stable microtubule attachments at normally noncentromeric regions, in addition to endogenous centromeres. The presence of ectopic spindle forces was confirmed in fixed preparations and time-lapse analysis by observing chromosomes with bent or stretched chromosome arms, which can only result from forces directing different sites on a single chromatid to the same pole. Likewise, in fixed cells and time-lapse analysis, chromosomes were observed stretched along their longitudinal axes with endogenous centromeres in the middle, indicating that arms are under tension from opposite poles (Heun, 2006).
It is possible that other chromosome defects are caused by mislocalization of CID, in addition to ectopic kinetochore formation and multicentric chromosomes. However, inhibition of sister chromatid separation with the topoisomerase II inhibitor etoposide, and CID depletion by RNAi, produced mitotic defects that were qualitatively and quantitatively distinct from those observed after CID mislocalization. Thus, loss of endogenous centromere function or sister separation defects alone cannot account for the predominant chromosome phenotypes observed after CID mislocalization. Determining if other chromosome segregation defects in addition to ectopic kinetochores occur in response to CID mislocalization warrants further study (Heun, 2006).
It is concluded that CID induction results in broad incorporation into normally noncentromeric, predominantly euchromatic regions, a subset of which recruit key kinetochore proteins and exhibit kinetochore function. It is proposed that the mitotic, cellular, and organismal phenotypes are caused by the presence of more than one functional kinetochore and spindle attachment per chromatid. These results also provide further evidence that CENP-A is a key epigenetic mark for centromere identity (Heun, 2006).
Although CENP-A is currently the highest protein in the kinetochore assembly pathway, previous studies have not addressed whether CENP-A is also sufficient for kinetochore formation. The fact that most ectopic sites of CID incorporation are not associated with kinetochore proteins and spindle attachments indicates that CENP-A is not absolutely sufficient for kinetochore formation. However, there are several reasons why it is unlikely that a strict correlation between CENP-A incorporation and kinetochore formation would be observed in this system: (1) it seems unlikely that all kinetochore proteins are present in the vast excess required for kinetochore formation at all ectopic CID sites. Interestingly, the inner kinetochore protein CENP-C is recruited more efficiently to ectopic sites in comparison to all of the outer kinetochore proteins, suggesting that kinetochore formation may be limited by processes downstream from centromeric chromatin formation. (2) It is possible that only regions with CID incorporation above a threshold level, perhaps equivalent to the density at the endogenous centromere, are capable of establishing a functional kinetochore. This hypothesis is supported by the observation that the severity of chromosome segregation defects is closely correlated with CID expression levels. Lower levels of CENP-A induction are the most likely explanation for why ectopic kinetochores were not detected in the human study. Alternatively, human cells may possess a more efficient clearing mechanism for eliminating CENP-A from noncentromeric regions, as has been reported for S. cerevisiae. (3) Other broadly distributed chromatin factors may contribute to functional kinetochore formation in combination with CID. Centromeric chromatin in flies and humans contains histone modification patterns that are distinct from euchromatin and the flanking heterochromatin, which may also be required for the formation of ectopic, functional kinetochores (Heun, 2006).
Although centromere function and kinetochore assembly may require flanking heterochromatin, the presence of heterochromatin can also inhibit CID incorporation and kinetochore formation. In Drosophila, neocentromeres are produced when noncentromeric DNA and an endogenous centromere are juxtaposed, but not when heterochromatin separates these regions. The lack of CID incorporation into heterochromatin after induction is consistent with the hypothesis that heterochromatin antagonizes the spread of centromeric chromatin and normally acts to limit the size and distribution of centromeric chromatin. Thus, differences in the distribution of heterochromatin may also limit CID incorporation into ectopic sites, or the ability of ectopic sites to form functional kinetochores. Further studies are needed to determine exactly what factors limit kinetochore formation at ectopic sites, and to examine the sufficiency of CENP-A in more detail (Heun, 2006).
The centromere/kinetochore complex is indispensable for accurate segregation of chromosomes during cell divisions when it serves as the attachment site for spindle microtubules. Centromere identity in metazoans is believed to be governed by epigenetic mechanisms, because the highly repetitive centromeric DNA is neither sufficient nor required for specifying the assembly site of the kinetochore. A candidate for an epigenetic mark is the centromere-specific histone H3 variant CENP-A that replaces H3 in alternating blocks of chromatin exclusively in active centromeres. CENP-A acts as an initiator of kinetochore assembly, but the detailed dynamics of the deposition of metazoan CENP-A and of other constitutive kinetochore components are largely unknown. This study shows by quantitative fluorescence measurements in living early embryos that functional fluorescent fusion proteins of the Drosophila CENP-A and CENP-C homologs are rapidly incorporated into centromeres during anaphase. This incorporation is independent of ongoing DNA synthesis and pulling forces generated by the mitotic spindle, but strictly coupled to mitotic progression. Thus, these findings uncover a strikingly dynamic behavior of centromere components in anaphase (Schuh, 2007).
These results show that new CID and CENP-C incorporation takes place during anaphase of the syncytial divisions of Drosophila embryos. This incorporation is independent of DNA replication and of normal pulling forces generated by the mitotic spindle. While it is counterintuitive that CID and CENP-C incorporation occurs while the centromeres are under strain by the pulling forces generated by the mitotic spindle, mitosis is the only time point in syncytial embryos without ongoing DNA synthesis. Thus, it appears that CID and CENP-C incorporation concomitant with DNA replication needs to be prevented. The finding that CENP-C and CID incorporation during anaphase is independent of spindle pulling forces argues against the importance of tension in the epigenetic specification of the site of functional centromeres, at least for the syncytial divisions in Drosophila embryos. Nevertheless, as the mitotic spindle checkpoint enforces the dependence of anaphase on functional kinetochores, incorporation of centromere/kinetochore complex components only into functional kinetochores during anaphase may represent a safeguard mechanism to propagate centromeres (Schuh, 2007).
The centromere ensures correct segregation of chromosomes during mitosis by providing the site for kinetochore assembly and microtubule attachment. Most eukaryotic organisms contain only one centromere per chromosome, which is specifically positioned and faithfully propagated through each cell cycle. With the exception of Saccharomyces cerevisiae, DNA sequence is considered neither necessary nor sufficient to mark centromeres in most eukaryotes, suggesting that centromere identity in many organisms is determined epigenetically. CENH3 (CENP-A in mammals, CID in Drosophila) is a centromere-specific histone H3 variant that replaces canonical histone H3 in centromeric nucleosomes. It is essential for centromere function and kinetochore assembly and thus a prime candidate epigenetic mark for determining centromere identity (Mendiburo, 2011).
Global misincorporation of CID into chromosome arms leads to the formation of functional ectopic kinetochores only in a small subset of sites, hindering a direct correlation between CID presence and kinetochore formation. To determine whether CID is sufficient for directing kinetochore assembly and nucleate centromere identity, a CID-GFP-Lac Repressor (LacI) fusion protein was targeted to an array of lac operator (lacO) sequences stably integrated in Drosophila Schneider S2 cells. Inducible CID-GFP-LacI or GFP-LacI fusion proteins are efficiently targeted to lacO sequences only upon pulse induction, whereas low leaky expression of CID-GFP-LacI in uninduced cells correlates with an exclusive centromere localization (Mendiburo, 2011).
CENH3 has been shown to adopt a specialized nucleosomal structure, which is proposed to mark centromeric chromatin. To determine whether CID-GFP-LacI is incorporated in nucleosomes at the lacO, mononucleosomes from GFP-LacI and CID-GFP-LacI cells were separated on a sucrose gradient. Analysis of the fractions from both cell lines revealed that lacO DNA comigrates with fractions containing H3-nucleosomes, suggesting that they are chromatinized. GFP-LacI did not comigrate with nucleosomes, likely due to its release from lacO binding after micrococcal nuclease treatment. In contrast, CID-GFP-LacI was found in nucleosome-containing fractions, indicating that it can interact with chromatin independently of the LacI binding. Nucleosome incorporation was confirmed by coimmunoprecipitation of CID-GFP-LacI with histone H2A in the nucleosome fraction. To exclude that CID-GFP-LacI migration pattern is only due to the contribution of protein localized to endogenous centromeres, a mutant CID fused to GFP-LacI was created that does not target to centromeres and is therefore present only at the lacO array. In this mutated protein, three amino acids [G175S, L177V, and L178M (CIDsvm)] in the CENP-A targeting domain (CATD) of CID are replaced by the corresponding residues of histone H3.1, as shown for human CENP-A. The migration pattern of CIDsvm-GFP-LacI in sucrose gradients resembles that of CID-GFP-LacI, supporting the notion that CID-GFP-LacI is in nucleosomes also at lacO regions (Mendiburo, 2011).
Targeting of CID-GFP-LacI, but not GFP-LacI, to the lacO served to recruit kinetochore proteins, such as CENP-C and the microtubule-binding protein NDC80/HEC1, 1 day after pulse induction. Probing for the spindle assembly checkpoint proteins POLO kinase and MAD2 revealed the same pattern, suggesting that ectopically targeted CID is sufficient to direct kinetochore assembly (Mendiburo, 2011).
The formation of a functional ectopic kinetochore at the lacO is expected to generate a dicentric chromosome causing chromosome breakage and anaphase defects. Indeed, a higher number of aberrant mitotic chromosome configurations was observed in cells expressing CID-GFP-LacI compared with control GFP-lacI cells and containing the lacO array (Mendiburo, 2011).
The epigenetic model for centromere identity predicts that a functional centromere self-directs the loading of new centromeric marks after each cell division. If CID-GFP-LacI identifies the lacO as a centromere, CID molecules without a LacI fusion should also be recruited to lacO regions. To investigate this, a stable cell line was created containing both an inducible CID-GFP-LacI and a constitutively expressed CID-hemagglutinin (HA) HA construct. Upon pulse induction and targeting of CID-GFP-LacI, a low initial CID-HA recruitment to the lacO (9.7%) was found, increasing by two-fold 7 days later (24%). Higher-resolution analysis using stretched chromatin fibers revealed that at initial time points CID-HA localization is restricted to the lacO region, whereas 7 days after pulse induction, CID-HA or CID is also found spreading into adjacent regions. CID-HA or CID were never found in lacO sequences in the presence of GFP-LacI (Mendiburo, 2011).
CID-GFP-LacI targeting to an ectopic chromosomal locus results in mitotically unstable dicentric chromosomes, hindering the analysis of long-term inheritance of the centromere function. To directly test de novo centromere heritability, an analysis was carried out of whether CID-GFP-LacI targeting confers mitotic stability to a centromere-devoid episomal DNA element carrying lacO sequences and a G418-resistance cassette. This plasmid was transfected into S2 cell lines constitutively expressing low levels of either CID-GFP-LacI or GFP-LacI and kept under selection pressure for 28 days. The initial transfection efficiency was comparable in both cell lines. However, a decline was observed in the amount of replicated plasmids (+DpnI) in GFP-LacI-expressing cells, whereas CID-GFP-LacI cells maintained the plasmid until the end of the experiment. Efficient replication and proper segregation are the two major factors defining the stability of plasmids. For the first 7 days, both cell lines replicated the plasmid with similar efficiency, indicating that correct segregation is the main factor contributing to its maintenance in CID-GFP-LacI cells. This was also observed when selection was removed after 16 days or never applied. Furthermore, CENP-C, NDC80, and microtubule connections were found localizing to CID-GFP-LacI-bound lacO plasmids in mitosis. These plasmids separate earlier than endogenous chromosomes and generally display a symmetric distribution on the spindle poles. GFP-LacI cells grew poorly after transfection, and the few mitotic cells detected showed no colocalization of GFP-positive lacO plasmids with either CENP-C or NDC80. Further characterization of recovered plasmids showed that plasmid size remained stable except for variability in the repetitive lacO array and did not acquire endogenous Drosophila sequences. A minor fraction of concatenated plasmids did not correlate with efficient recruitment of kinetochore proteins (Mendiburo, 2011).
The centromeric mark can self-propagate in the lacO plasmid, as shown by the recruitment of CID-HA to targeted CID-GFP-LacI plasmids and the spreading of both into backbone sequences. To determine whether centromeric chromatin is inherited even after removal of CID-GFP-LacI targeting, the lacO plasmid and a CID-GFP-LacI expression construct were transiently cotransfected to deliver an initial expression pulse that is progressively lost. Episomal lacO plasmids were detected as nonchromosomal CID/CENP-C foci, with or without a GFP signal. Early after transfection, cells were found that contained both GFP-positive and -negative foci and a smaller cell population that had already lost CID-GFP-LacI expression but contained nonchromosomal CID/CENP-C foci. The percentage of these GFP-negative plasmids increased until day 27, when all detected CID/CENP-C foci were devoid of any GFP signal. It is concluded that an initial targeting of CID-GFP-LacI to the lacO plasmid enables the nucleation of centromere function, which is maintained independently of CID-GFP-LacI expression and targeting (Mendiburo, 2011).
In recent years, functional biosynthetic kinetochores have been constructed by targeting kinetochore components and chromatin modifiers to minichromosomes. Even though the possibility of bypassing CENH3 chromatin for the assembly of a kinetochore suggested that CENH3 is dispensable for this function, no evidence of centromere heritability was provided by these studies. Recent reports showing CENP-A-dependent kinetochore formation in vitro or by artificially targeting the CENP-A loading factor in humans (HJURP) further stress the importance of CENH3 in kinetochore assembly. The results presented in this study show that CENH3 behaves as a true centromeric epigenetic mark not only by being sufficient for the recruitment of kinetochore proteins during mitosis but also for directing its own incorporation and maintaining centromere function through each cell division (Mendiburo, 2011).
CENP-A (CID in flies) is the histone H3 variant essential for centromere specification, kinetochore formation, and chromosome segregation during cell division. Recent studies have elucidated major cell cycle mechanisms and factors critical for CENP-A incorporation in mitosis, predominantly in cultured cells. However, the roles, regulation, and cell cycle timing of CENP-A assembly in somatic tissues is not understood in multicellular organisms and in meiosis, the specialized cell division cycle that gives rise to haploid gametes. This study investigated the timing and requirements for CID assembly in mitotic tissues and male and female meiosis in Drosophila, using fixed and live imaging combined with genetic approaches. CID assembly was found to initiate at late telophase and continues during G1 phase in somatic tissues in the organism, later than the metaphase assembly observed in cultured cells. Furthermore, CID assembly occurs at two distinct cell cycle phases during male meiosis: prophase of meiosis I and after exit from meiosis II, in spermatids. CID assembly in prophase I is also conserved in female meiosis. Interestingly, a novel decrease in CID levels was observed after the end of meiosis I and before meiosis II, which correlates temporally with changes in kinetochore organization and orientation. It was also demonstrated that CID is retained on mature sperm despite the gross chromatin remodeling that occurs during protamine exchange. Finally, it was shown that the centromere proteins Chromosome alignment defect 1 (CAL1) and CENP-C are both required for CID assembly in meiosis and normal progression through spermatogenesis. It is concluded that the cell cycle timing of CID assembly in meiosis is different from mitosis and that the efficient propagation of CID through meiotic divisions and on sperm is likely to be important for centromere specification in the developing zygote (Dunleavy, 2012).
This study reveals a surprising diversity of CID assembly timing in mitotic and meiotic tissues in the fruit fly Drosophila melanogaster. During mitosis, CID assembly initiates at late telophase and continues during G1 phase in somatic cells of the larval brain. These results are consistent with the timing and dynamics of CENP-A assembly reported for human cell lines and in general, with centromeric histone deposition outside of S phase, during mitosis and G1 phase. Notably, loading in mitosis was observed to occur at a later mitotic stage (telophase/G1 phase) than previously reported for cultured cells (metaphase) or fly embryos (anaphase). Interestingly, neuroblast stem cells display a subtle difference between cells derived from the same division; the mother cell, which will continue to act as a stem cell, starts CID loading at centromeres 3–6 min earlier than in the daughter cell that is committed to differentiation. It is currently unclear whether this difference in centromere assembly timing is due to differences in the regulation of mitotic exit between stem and daughter cells or is required for or a response to stem cell propagation mechanisms (Dunleavy, 2012).
It is proposed that such differences in timing reflect altered cell cycle regulation in cultured cells compared to animal tissues, and the results emphasize the importance of validating cell culture findings in animal models. It is important to note that despite similarities to the timing observed in human cultured cells (late telophase/G1 phase), the results in Drosophila raise questions about whether the analysis of cultured cells in humans and other species reflects the timing of CENP-A assembly in the organism (Dunleavy, 2012).
These results also show that the cell cycle timing for CID assembly in meiosis differs from mitosis. In male meiosis, there are two phases of CID assembly, at prophase of meiosis I and after exit from meiosis II, and two phases of chromosome segregation, resulting in haploid spermatids with nuclear CID levels equivalent to those observed at the beginning of meiosis. In meiosis in Drosophila females, CID assembly also occurs during prophase of meiosis I. Assembly in prophase provides another example of the restriction of CID assembly to a specific part of the cell cycle outside of S phase, but has not been observed previously in mitotic tissues or cultured cells from other organisms. It is also important to note that meiotic prophase in both male and female Drosophila occurs over days, indicating that CID assembly is gradual over this extended time period. Such slow assembly dynamics are unexpected, given that until now studies in mitotic cells indicate that CENP-A assembly is completed in the order of minutes to hours. How CID assembly is first initiated and then continues over such extended time periods awaits further investigation (Dunleavy, 2012).
It is likely that cell cycle regulators control CID assembly in meiosis as they do in mitosis. For example, a recent study showed that CDK activity inhibits CENP-A assembly in human cells and that blocking CDK activity results in precocious loading in S and G2 phases. Cyclin A is degraded during late prophase of meiosis I. This is consistent with the observed burst in CID assembly during a 10-min time window of late prophase/early prometaphase I, and the previous demonstration that Cyclin A degradation is required for mitotic CID assembly. However, CID assembly also occurs before Cyclin A degradation in meiosis I, implying that other unknown mechanisms initiate and continue assembly prior to late prophase I. Additionally, CID is not loaded between meiosis I and II, even though Cyclin A levels remain low. Instead, the partial degradation of Cyclin B to an intermediate level after meiosis I, which allows for spindle destruction but prevents a second round of DNA synthesis, could inhibit CID assembly between meiosis I and II. Moreover, the slow degradation of Cyclin B at the end of meiosis II could contribute to the gradual CID loading in spermatids, as the second phase of CID assembly after meiotic exit is more similar in terms of cell cycle regulation to the telophase/G1 loading observed in mitotic tissues in the animal and in human cells in culture. However, it was also observed that CID assembly occurs in prophase of meiosis I, when Cyclin B levels are high, but does not occur between meiosis I and II, despite low Cyclin A levels. This suggests that CID assembly in meiosis is regulated by other mechanisms in addition to the inhibition of Cyclin/CDK activities, as proposed for mammalian cells (Dunleavy, 2012).
Another striking observation from this study is that during meiosis I, CID assembly occurs prior to chromosome segregation, whereas most mitotic cells previously studied proceed through most of mitosis with half the maximal amount of CID at centromeres. In addition, a greater than 2-fold increase in CID intensity was observed at centromeres during prophase, even though a 2-fold increase would be sufficient to compensate for CID dilution in premeiotic S phase. What is the role, if any, of an increased level of CID at centromeres during the first meiotic division? In meiosis I, bivalent sister chromatid kinetochores are mono-oriented, instead of bi-oriented as they are in mitosis and meiosis II; combined with the maintenance of sister cohesion at centromeres, this ensures that homologs, and not sisters, segregate during meiosis I. It is speculated that extra CID may be required during the first meiotic division to assemble or maintain mono-oriented kinetochores and microtubule attachments. This hypothesis could also be extended to incorporate the surprising decrease in CID levels observed between the end of meiosis I and the beginning of meiosis II. Loss of CENP-A during normal cell divisions has only previously been observed as accompanying DNA replication and nucleosome segregation in S phase, events that do not occur between meiosis I and II. Thus, it is tempting to speculate that the additional loss of CID after meiosis I could contribute to the currently unknown mechanism responsible for reorganization of kinetochores from mono- to bi-orientation in preparation for meiosis II (Dunleavy, 2012).
Using targeted RNAi depletion of centromeric proteins during Drosophila male meiosis, this study found that both CAL1 and CENP-C are required for CID assembly in prophase of meiosis I. This is consistent with previous observations in mitotic cells, where CAL1, CENP-C, and CID are mutually dependent on each other for centromere localization. It was also found that depletion of CAL1 or CID in larval testes results in CENP-C delocalization from centromeres and sequestration in the nucleolus, again similar to observations in mitosis, possibly because it is no longer in a stable complex with CID or CAL1. The results also show that reduced CAL1 or CENP-C expression results in defective chromosome segregation and that both are required for normal progression through male meiosis. The finding that T1–T3 spermatids depleted for CAL1 or CENP-C have reduced CID at centromeres (although to a lesser extent in the case of CENP-C depletion) also suggests that CAL1 and CENP-C are required for CID assembly during the second phase of loading in spermatids. However, given that cells with reduced CAL1 or CID already show major chromosome segregation defects after meiosis I, meiosis-specific GAL4 drivers active in later stages of meiosis and spermatogenesis, which are currently lacking, are required to directly assay the requirements for CAL1 and CENP-C in the second phase of CID assembly or during fertilization. Requirements for CAL1 and CENP-C in both phases of meiotic CID assembly are surprising, given that centromeric CAL1 levels are greatly reduced during prophase I and at later stages of spermatogenesis and that CENP-C is not localized to centromeres after meiosis II. One intriguing possibility is that CID assembly requires CAL1 and CENP-C removal from centromeres (Dunleavy, 2012).
Another key observation from this study is the retention of CID at centromeres on mature spermatozoa in spite of an extensive period of chromatin remodeling and histone–protamine exchange during spermatocyte maturation. How CID is protected from histone removal prior to protamine exchange at centromeres remains to be investigated. It is possible that the local chromatin environment at centromeres is refractory to protamine exchange or that additional proteins present at centromeres could provide protection. Because fusion of male and female pronuclei does not occur until telophase of the first zygotic division, it is likely that paternal CID at centromeres is required for kinetochore formation and spindle attachment to paternal chromosomes. The amount of paternal CID at centromeres could be critical for the successful epigenetic inheritance of centromere identity and for the viability of the embryo, if paternal CID is diluted during subsequent zygotic divisions. Alternatively, maternal CID could compensate for a reduced level of CID on sperm or establish de novo centromeres on paternal chromosomes. Whatever the mechanism of CID maintenance in the zygote, the regulation of CID assembly on sperm is likely to prove very important in the transmission of epigenetic information and centromere specification into the next generation (Dunleavy, 2012).
The centromere/kinetochore complex plays an essential role in cell and organismal viability by ensuring chromosome movements during mitosis and meiosis. The kinetochore also mediates the spindle attachment checkpoint (SAC), which delays anaphase initiation until all chromosomes have achieved bipolar attachment of kinetochores to the mitotic spindle. CENP-A proteins are centromere-specific chromatin components that provide both a structural and a functional foundation for kinetochore formation. Cells in Drosophila embryos homozygous for null mutations in CENP-A (CID) display an early mitotic delay. This mitotic delay is not suppressed by inactivation of the DNA damage checkpoint and is unlikely to be the result of DNA damage. Surprisingly, mutation of the SAC component BUBR1 partially suppresses this mitotic delay. Furthermore, cid mutants retain an intact SAC response to spindle disruption despite the inability of many kinetochore proteins, including SAC components, to target to kinetochores. It is proposed that SAC components are able to monitor spindle assembly and inhibit cell cycle progression in the absence of sustained kinetochore localization (Blower, 2006; full text of article)
Ahmad, K. and Henikoff, S. (2001). Centromeres are specialized replication domains in heterochromatin. J Cell Biol. 153(1): 101-10. 11285277
Ando, S., et al. (2002). CENP-A, -B, and -C chromatin complex that contains the I-type alpha-satellite array constitutes the prekinetochore in HeLa cells. Mol. Cell. Biol. 22(7): 2229-41. 11884609
Blower, M. D. and Karpen, G. H. (2001). The role of Drosophila CID in kinetochore formation, cell-cycle progression and heterochromatin interactions. Nat. Cell Biol. 3(8): 730-9. 11483958
Blower, M. D., Sullivan, B. A. and Karpen, G. H. (2002). Conserved organization of centromeric chromatin in flies and humans. Developmental Cell 2: 319-330. 11879637
Blower, M. D., Daigle, T., Kaufman, T. and Karpen, G. H. (2006). Drosophila CENP-A mutations cause a BubR1-dependent early mitotic delay without normal localization of kinetochore components. PLoS Genet. 2(7): e110. Medline abstract: 16839185
Buchwitz, B. J., et al. (1999). A histone-H3-like protein in C. elegans. Nature 401(6753): 547-8. 10524621
Carroll, C. W., Milks, K. J. and Straight, A. F. (2010). Dual recognition of CENP-A nucleosomes is required for centromere assembly. J. Cell Biol. 189(7): 1143-55. PubMed Citation: 20566683
Chen, Y., et al. (2000). The N terminus of the centromere H3-like protein Cse4p performs an essential function distinct from that of the histone fold domain. Mol. Cell. Biol. 20(18): 7037-48. 10958698
Collins, C. M., Malacrida, B., Burke, C., Kiely, P. A. and Dunleavy, E. M. (2018). ATP synthase F1 subunits recruited to centromeres by CENP-A are required for male meiosis. Nat Commun 9(1): 2702. PubMed ID: 30006572
Collins, K. A., Furuyama, S. and Biggins, S. (2004). Proteolysis contributes to the exclusive centromere localization of the yeast Cse4/CENP-A histone H3 variant. Curr. Biol. 14: 1968-1972. 15530401
Dalal, Y., Wang, H., Lindsay, S. and Henikoff, S. (2007). Tetrameric structure of centromeric nucleosomes in interphase Drosophila cells. PLoS Biol. 5(8): e218. Medline abstract: 17676993
Dunleavy, E. M., Beier, N. L., Gorgescu, W., Tang, J., Costes, S. V. and Karpen, G. H. (2012). The cell cycle timing of centromeric chromatin assembly in Drosophila meiosis is distinct from mitosis yet requires CAL1 and CENP-C. PLoS Biol 10: e1001460. PubMed ID: 23300382
Erhardt, S., et al. (2008). Genome-wide analysis reveals a cell cycle-dependent mechanism controlling centromere propagation. J. Cell Biol. 183: 805-818. PubMed Citation: 19047461
Fachinetti, D., Diego Folco, H., Nechemia-Arbely, Y., Valente, L. P., Nguyen, K., Wong, A. J., Zhu, Q., Holland, A. J., Desai, A., Jansen, L. E. and Cleveland, D. W. (2013). A two-step mechanism for epigenetic specification of centromere identity and function. Nat Cell Biol 15: 1056-1066. PubMed ID: 23873148
Furuyama, T., Dalal, Y. and Henikoff, S. (2006a). Chaperone-mediated assembly of centromeric chromatin in vitro. Proc. Natl. Acad. Sci. 103(16): 6172-7. Medline abstract: 16601098
Furuyama, T. and Henikoff, S. (2006b). Biotin-tag affinity purification of a centromeric nucleosome assembly complex. Cell Cycle 5(12): 1269-74. Medline abstract: 16775420
Furuyama, T. and Henikoff, S. (2009). Centromeric nucleosomes induce positive DNA supercoils. Cell 138(1): 104-13. PubMed Citation: 19596238
Fujita, Y., et al. (2007). Priming of centromere for CENP-A recruitment by human hMis18alpha, hMis18beta, and M18BP1. Dev. Cell 12(1): 17-30. Medline abstract: 17199038
Glowczewski, L., et al. (2000). Histone-histone interactions and centromere function. Mol. Cell. Biol. 20(15): 5700-11. 10891506
Henikoff, S., Ahmad, K., Platero, J. S. and van Steensel, B. (2000). Heterochromatic deposition of centromeric histone H3-like proteins Proc. Natl. Acad. Sci. 97: 716-721. 10639145
Heun, P., Erhardt, S., Blower, M. D., Weiss, S., Skora, A. D. and Karpen, G. H. (2006). Mislocalization of the Drosophila centromere-specific histone CID promotes formation of functional ectopic kinetochores. Dev. Cell. 10(3): 303-15. 16516834
Hori, T., Shang, W. H., Toyoda, A., Misu, S., Monma, N., Ikeo, K., Molina, O., Vargiu, G., Fujiyama, A., Kimura, H., Earnshaw, W. C. and Fukagawa, T. (2014). Histone H4 Lys 20 monomethylation of the CENP-A nucleosome is essential for kinetochore assembly. Dev Cell 29: 740-749. PubMed ID: 24960696
Howman, E. V., et al. (2000). Early disruption of centromeric chromatin organization in centromere protein A (Cenpa) null mice. Proc. Natl. Acad. Sci. 97(3): 1148-53. 10655499
Hu, H., et al. (2011). Structure of a CENP-A-histone H4 heterodimer in complex with chaperone HJURP. Genes Dev. 25(9): 901-6. PubMed Citation: 21478274
Jager, H., Rauch, M. and Heidmann, S. (2005). The Drosophila melanogaster condensin subunit Cap-G interacts with the centromere-specific histone H3 variant CID. Chromosoma 113(7): 350-61. 15592865
Keith, K. C., et al. (1999). Analysis of primary structural determinants that distinguish the centromere-specific function of histone variant Cse4p from histone H3. Mol. Cell. Biol. 19(9): 6130-9. 10454560
Keith, K. C. and Fitzgerald-Hayes, M. (2000). CSE4 genetically interacts with the Saccharomyces cerevisiae centromere DNA elements CDE I and CDE II but not CDE III. Implications for the path of the centromere DNA around a cse4p variant nucleosome. Genetics 156(3): 973-81. 11063678
Kunitoku, N., et al. (2003). CENP-A phosphorylation by Aurora-A in prophase is required for enrichment of Aurora-B at inner centromeres and for kinetochore function. Dev. Cell 5: 853-864. 14667408
Lagana, A., et al. (2010). A small GTPase molecular switch regulates epigenetic centromere maintenance by stabilizing newly incorporated CENP-A. Nat. Cell Biol. 12(12): 1186-93. PubMed Citation: 21102442
Li, W., Yi, J., Agbu, P., Zhou, Z., Kelley, R. L., Kallgren, S., Jia, S. and He, X. (2017). Replication stress affects the fidelity of nucleosome-mediated epigenetic inheritance. PLoS Genet 13(7): e1006900. PubMed ID: 28749973
Lo, A. W., et al. (2001a). A novel chromatin immunoprecipitation and array (CIA) analysis identifies a 460-kb CENP-A-binding neocentromere DNA. Genome Res. 11(3): 448-57. 11230169
Lo, A. W., et al. (2001b). A 330 kb CENP-A binding domain and altered replication timing at a human neocentromere. EMBO J. 17;20(8): 2087-96. 11296241
Lomonte, P., Sullivan, K. F. and Everett, R. D. (2001). Degradation of nucleosome-associated centromeric histone H3-like protein CENP-A induced by herpes simplex virus type 1 protein ICP0. J. Biol. Chem. 276(8): 5829-35. 11053442
Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F. and Richmond, T. J. (1997). Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature 389: 251-260. PubMed Citation: 9305837
Malik, H. S. and Henikoff, S. (2001) Adaptive evolution of Cid, a centromere-specific histone in Drosophila. Genetics 157: 1293-1298. 11238413
Malik, H. S., Vermaak, D., and Henikoff, S. (2002). Recurrent evolution of DNA-binding motifs in the Drosophila centromeric histone. Proc. Natl. Acad. Sci. 99: 1449-1454. 11805302
Mellone, B. G., et al. (2011). Assembly of Drosophila centromeric chromatin proteins during mitosis. PLoS Genet. 7(5): e1002068. PubMed Citation: 21589899
Meluh, P. B., et al. (1998). Cse4p is a component of the core centromere of Saccharomyces cerevisiae. Cell 94(5): 607-13. 9741625
Mendiburo, M. J., et al (2011). Drosophila CENH3 is sufficient for centromere formation. Science 334(6056): 686-90. PubMed Citation: 22053052
Moreno-Moreno, O., Torras-Llort, M., Azorin, F. (2006). Proteolysis restricts localization of CID, the centromere-specific histone H3 variant of Drosophila, to centromeres. Nucleic Acids Res. 34(21): 6247-55. Medline abstract: 17090596
Oegema, K., et al. (2001). Functional analysis of kinetochore assembly in Caenorhabditis elegans. J. Cell Biol. 153(6): 1209-26. 11402065
Orr, B. and Sunkel, C. E. (2011). Drosophila CENP-C is essential for centromere identity. Chromosoma 120(1): 83-96. PubMed Citation: 20862486
Palmer, D. K., O'Day, K. and Margolis, R. L. (1990). The centromere specific histone CENP-A is selectively retained in discrete foci in mammalian sperm nuclei. Chromosoma 100(1): 32-6. 2101350
Perpelescu, M., Nozaki, N., Obuse, C., Yang, H. and Yoda, K. (2009). Active establishment of centromeric CENP-A chromatin by RSF complex. J. Cell Biol. 185: 397-407. PubMed Citation: 19398759
Rosic, S., Kohler, F. and Erhardt, S. (2014). Repetitive centromeric satellite RNA is essential for kinetochore formation and cell division. J Cell Biol [Epub ahead of print]. PubMed ID: 25365994
Pauleau, A. L. and Erhardt, S. (2011). Centromere regulation: new players, new rules, new questions. Eur J Cell Biol 90: 805-810. PubMed ID: 21684630
Salvany, L., Aldaz, S., Corsetti, E. and Azpiazu, N. (2009). A new role for hth in the early pre-blastodermic divisions in drosophila. Cell Cycle 8: 2748-2755. PubMed ID: 19652544
Schittenhelm, R. B., et al. (2007). Spatial organization of a ubiquitous eukaryotic kinetochore protein network in Drosophila chromosomes. Chromosoma 116(4): 385-402. Medline abstract: 17333235
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 Citation: 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(3): 237-43. Medline abstract: 17222555
Shelby, R. D., Monier, K. and Sullivan, K. F. (2000). Chromatin assembly at kinetochores is uncoupled from DNA replication. J. Cell Biol. 151(5): 1113-8. 11086012
Stoler, S., et al. (1995). A mutation in CSE4, an essential gene encoding a novel chromatin-associated protein in yeast, causes chromosome nondisjunction and cell cycle arrest at mitosis. Genes Dev. 9(5): 573-86. 7698647
Sullivan, B. and Karpen, G. (2001). Centromere identity in Drosophila is not determined in vivo by replication timing. J. Cell Bio. 154: 683-690. 11514585
Sullivan, B. A. and Karpen, G. H. (2004). Centromeric chromatin exhibits a histone modification pattern that is distinct from both euchromatin and heterochromatin. Nat. Struct. Mol. Biol. 11: 1076-1083. 15475964
Takahashi, K., Chen, E. S. and Yanagida, M. (2000). Requirement of Mis6 centromere connector for localizing a CENP-A-like protein in fission yeast. Science 288(5474): 2215-9. 10864871
Usakin, L., Abad, J., Vagin, V. V., de Pablos, B., Villasante, A. and Gvozdev, V. A. (2007). Transcription of the 1.688 satellite DNA family is under the control of RNA interference machinery in Drosophila melanogaster ovaries. Genetics 176: 1343-1349. PubMed ID: 17409066
Van Hooser, A. A., et al. (2001). Specification of kinetochore-forming chromatin by the histone H3 variant CENP-A. J. Cell Sci. 114: 3529-3542. 11682612
Warburton, P. E., et al. (1997). Immunolocalization of CENP-A suggests a distinct nucleosome structure at the inner kinetochore plate of active centromeres. Curr Biol. 7(11): 901-4. 9382805
Yoda, K., et al. (2000). Human centromere protein A (CENP-A) can replace histone H3 in nucleosome reconstitution in vitro. Proc. Natl. Acad. Sci. 97(13): 7266-71. 10840064
Zeitlin, S. G., et al. (2001). Differential regulation of CENP-A and histone H3 phosphorylation in G2/M. J. Cell Sci. 114(Pt 4): 653-61. 11171370
date revised: 12 December 2018
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.