Differential effect of aneuploidy on the X chromosome and genes with sex-biased expression in Drosophila

Global analysis of gene expression via RNA sequencing was conducted for trisomics for the left arm of chromosome 2 (2L) and compared with the normal genotype. The predominant response of genes on 2L was dosage compensation in that similar expression occurred in the trisomic compared with the diploid control. However, the male and female trisomic/normal expression ratio distributions for 2L genes differed in that females also showed a strong peak of genes with increased expression and males showed a peak of reduced expression relative to the opposite sex. For genes in other autosomal regions, the predominant response to trisomy was reduced expression to the inverse of the altered chromosomal dosage (2/3), but a minor peak of increased expression in females and further reduced expression in males were also found, illustrating a sexual dimorphism for the response to aneuploidy. Moreover, genes with sex-biased expression as revealed by comparing amounts in normal males and females showed responses of greater magnitude to trisomy 2L, suggesting that the genes involved in dosage-sensitive aneuploid effects also influence sex-biased expression. Each autosomal chromosome arm responded to 2L trisomy similarly, but the ratio distributions for X-linked genes were distinct in both sexes, illustrating an X chromosome-specific response to aneuploidy.Changes in chromosomal dosage have long been known to affect the phenotype or viability of an organism (1–4). Altering the dosage of individual chromosomes typically has a greater impact than varying the whole genome (5–7). This general rule led to the concept of “genomic balance” in that dosage changes of part of the genome produce a nonoptimal relationship of gene products. The interpretation afforded these observations was that genes on the aneuploid chromosome produce a dosage effect for the amount of gene product present in the cell (8).However, when gene expression studies were conducted on aneuploids, it became known that transacting modulations of gene product amounts were also more prevalent with aneuploidy than with whole-genome changes (9–14). Assays of enzyme activities, protein, and RNA levels revealed that any one chromosomal segment could modulate in trans the expression of genes throughout the genome (9–15). These modulations could be positively or negatively correlated with the changed chromosomal segment dosage, but inverse correlations were the most common (10–13). For genes on the varied segment, not only were dosage effects observed, but dosage compensation was also observed, which results from a cancelation of gene dosage effects by inverse effects operating simultaneously on the varied genes (9, 10, 14–18). This circumstance results in “autosomal” dosage compensation (14, 16–18). Studies of trisomic X chromosomes examining selected endogenous genes or global RNA sequencing (RNA-seq) studies illustrate that the inverse effect can also account for sex chromosome dosage compensation in Drosophila (15, 19–21). In concert, autosomal genes are largely inversely affected by trisomy of the X chromosome (15, 19, 21).The dosage effects of aneuploidy can be reduced to the action of single genes whose functions tend to be involved in heterogeneous aspects of gene regulation but which have in common membership in macromolecular complexes (8, 22–24). This fact led to the hypothesis that genomic imbalance effects result from the altered stoichiometry of subunits that affects the function of the whole and that occurs from partial but not whole-genome dosage change (8, 22–25). Genomic balance also affects the evolutionary trajectory of duplicate genes differently based on whether the mode of duplication is partial or whole-genome (22, 23).Here we used RNA-seq to examine global patterns of gene expression in male and female larvae trisomic for the left arm of chromosome 2 (2L). The results demonstrate the strong prevalence of aneuploidy dosage compensation and of transacting inverse effects. Furthermore, because both trisomic males and females could be examined, a sexual dimorphism of the aneuploid response was discovered. Also, the response of the X chromosome to trisomy 2L was found to be distinct from that of the autosomes, illustrating an X chromosome-specific effect. Genes with sex-biased expression, as determined by comparing normal males and females, responded more strongly to trisomy 2L. Collectively, the results illustrate the prevalence of the inverse dosage effect in trisomic Drosophila and suggest that the X chromosome has evolved a distinct response to genomic imbalance as would be expected under the hypothesis that X chromosome dosage compensation uses the inverse dosage effect as part of its mechanism (15).     

Ionizing irradiation-induced radical stress stalls live meiotic chromosome movements by altering the actin cytoskeleton

Meiosis generates haploid cells or spores for sexual reproduction. As a prelude to haploidization, homologous chromosomes pair and recombine to undergo segregation during the first meiotic division. During the entire meiotic prophase of the yeast Saccharomyces cerevisiae, chromosomes perform rapid movements that are suspected to contribute to the regulation of recombination. Here, we investigated the impact of ionizing radiation (IR) on movements of GFP–tagged bivalents in live pachytene cells. We find that exposure of sporulating cultures with >40 Gy (4-krad) X-rays stalls pachytene chromosome movements. This identifies a previously undescribed acute radiation response in yeast meiosis, which contrasts with its reported radioresistance of up to 1,000 Gy in survival assays. A modified 3′-end labeling assay disclosed IR-induced dsDNA breaks (DSBs) in pachytene cells at a linear dose relationship of one IR-induced DSB per cell per 5 Gy. Dihydroethidium staining revealed formation of reactive oxygen species (ROS) in irradiated cells. Immobility of fuzzy-appearing irradiated bivalents was rescued by addition of radical scavengers. Hydrogen peroxide-induced ROS did reduce bivalent mobility similar to 40 Gy X IR, while they failed to induce DSBs. IR- and H₂O₂-induced ROS were found to decompose actin cables that are driving meiotic chromosome mobility, an effect that could be rescued by antioxidant treatment. Hence, it appears that the meiotic actin cytoskeleton is a radical-sensitive system that inhibits bivalent movements in response to IR- and oxidant-induced ROS. This may be important to prevent motility-driven unfavorable chromosome interactions when meiotic recombination has to proceed in genotoxic environments.Exposure to ionizing irradiation (IR) has dire consequences for the cell, because it causes the formation of radicals and reactive oxygen species (ROS) that can oxidize and damage cellular components including proteins and DNA (1), whereas protection from IR-induced radical-mediated protein oxidation can lead to significant radio resistance (2). At the DNA level, IR leads to single-stranded and double-stranded DNA breaks (DSBs), with the latter being a severe threat to cellular survival (3). To cope with DSBs that may arise physiologically and/or by genotoxic environmental impacts such as IR, the cell repairs DSBs by two major pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR), which predominate in the G1 and G2 phase of the cell cycle, respectively, and underlie cell-cycle-dependent sensitivities to IR exposure (4, 5). In the G2 phase and in the first meiotic prophase, DSB repair is mediated by HR, in yeast meiosis addressing ∼150 DSBs (6, 7) that are formed by the Topo2-related endonuclease/transesterase enzyme SPO11 (8). Absence of DSBs and the resulting compromised spore viability (9) can be partially rescued by ionizing irradiation (10). In all, yeast cells exhibit a high resistance to IR (11–13) which is also true for cells in prophase I (10, 14). In the meiosis of numerous species, programmed Spo11-induced DSBs are instrumental for homologous chromosome search and pairing and provide the substrate for HR, generating two outcomes: noncrossovers (NCO) and crossovers (CO) that allow for homolog segregation in the meiosis I division (reviewed by refs. 15 and 16). For CO to occur, homologous chromosomes need to encounter and pair lengthwise (synapse) during first meiotic prophase (see ref. 17). Homolog pairing occurs after completion of premeiotic DNA replication. Live cell studies in the synaptic meiosis of the yeast Saccharomyces cerevisiae (2n = 32) have shown that meiotic telomeres (18), chromosomes, and bivalents undergo a striking mobility throughout the entire prophase I (19–21), which contrasts with the relative immobility of pachytene bivalents in mammalian prophase I (22). It has been found that rapid and continuous telomere and chromosome movements in budding yeast meiocytes depend on actin polymerization (18–20) and an intact meiotic telomere complex (21, 23, 24). Besides a general mobility of chromosomes throughout prophase I, single bivalents are capable to rapidly move away and return to the motile chromosome mass, a behavior termed “maverick” formation (19) or rapid chromosome movements (20, 21).Exposure to ionizing radiation induces a plethora of physicochemical effects in the irradiated cells including DNA damage (1, 3). Extensive research addressing the adverse effects of IR exposure using yeast as a model system had largely been directed toward mutation induction, DSB repair, and cell cycle effects (e.g., 11–13, 25, 26). Meiotic yeast cells exposed to 50–80 krad (500–800 Gy) X or γ irradiation have been shown to exhibit a profound reduction in cell survival, particularly when exposed in the G1 cell cycle phase that lacks a sister chromatid for repair (4). Irradiated meiotic yeast cells exhibit mutations and chromosome missegregation at meiosis I, leading to reduced sporulation (5, 10, 27). While previous studies addressed late deterministic effects in irradiated yeast cells such as DNA repair, mutations, and cell survival, we were interested in the immediate consequences of IR exposure on motile meiotic chromosomes. Bivalent mobility can be expected to promote chromosomal rearrangements, if it continues after the formation of ectopic unregulated DSBs. Chromosomal translocations have, for instance, been observed after irradiation of mitotic budding yeast cells (28) and of meiotic prophase cells of mice (29). Furthermore, meiotic chromosome mobility has been proposed to be involved in regulating (adverse) chromosomal interactions (30). To study the consequences of IR exposure on meiotic chromosome mobility we followed live bivalent movements in X-irradiated and nonirradiated yeast cells expressing the GFP-tagged version of the synaptonemal complex protein ZIP1 (19) undergoing sporulation.     

Insights into ParB spreading from the complex structure of Spo0J and parS

Spo0J (stage 0 sporulation protein J, a member of the ParB superfamily) is an essential component of the ParABS (partition system of ParA, ParB, and parS)-related bacterial chromosome segregation system. ParB (partition protein B) and its regulatory protein, ParA, act cooperatively through parS (partition S) DNA to facilitate chromosome segregation. ParB binds to chromosomal DNA at specific parS sites as well as the neighboring nonspecific DNA sites. Various ParB molecules can associate together and spread along the chromosomal DNA. ParB oligomer and parS DNA interact together to form a high-order nucleoprotein that is required for the loading of the structural maintenance of chromosomes proteins onto the chromosome for chromosomal DNA condensation. In this report, we characterized the binding of parS and Spo0J from Helicobacter pylori (HpSpo0J) and solved the crystal structure of the C-terminal domain truncated protein (Ct-HpSpo0J)-parS complex. Ct-HpSpo0J folds into an elongated structure that includes a flexible N-terminal domain for protein–protein interaction and a conserved DNA-binding domain for parS binding. Two Ct-HpSpo0J molecules bind with one parS. Ct-HpSpo0J interacts vertically and horizontally with its neighbors through the N-terminal domain to form an oligomer. These adjacent and transverse interactions are accomplished via a highly conserved arginine patch: RRLR. These interactions might be needed for molecular assembly of a high-order nucleoprotein complex and for ParB spreading. A structural model for ParB spreading and chromosomal DNA condensation that lead to chromosome segregation is proposed.The integrity of chromosomes and plasmids relies on precise DNA replication and segregation (1, 2). The initiation of DNA replication has to synchronize with the cell cycle to ensure precise chromosome segregation (3). In bacteria, the chromosome-encoded plasmid-partitioning system (Par) (4) and the structural maintenance of chromosomes (SMC) condensation complex (5) are two highly conserved systems associate with chromosome segregation and organization. SMC contributes to the overall stability and organization of genome (6–8). The partition system denoted ParABS is comprised of two proteins (ParA and ParB) and a centromere-like DNA element (parS) (9). ParB binds specifically to parS to form a complex. After binding ATP, ParA can interact with the ParB–parS complex to form a nucleoid–adaptor complex. ParB promotes the ATP hydrolysis activity of the complex to separate the chromosomes (9–13).In the bacterial chromosomal ParABS system, ParB has two functions: one is to regulate chromosome replication and sporulation (8, 12, 14) and the other is to participate in chromosome segregation (5, 15–17). ParB spreads along the chromosomal DNA by binding at specific parS and nonspecific DNA sites to form a high-order partition complex (18–20). This partition complex is required for the loading of SMC onto the chromosomal DNA (5). In addition, the N-terminal domain of ParB can interact with ParA and stimulate its ATPase activity (21). This nucleoid–adaptor complex, ParA–ParB–parS is used to drive chromosome segregation (22, 23). However, the detailed mechanism for this process is still unclear.Members of the ParB superfamily share similar functional domains: an N-terminal domain for protein–protein interactions, a central DNA-binding domain for parS binding, and a C-terminal domain for ParB dimerization (24). Two conserved N-terminal domain residues, Lys3 and Lys7, in the ParB from Bacillus subtilis (BsSpo0J), have been shown to interact with its regulatory protein BsSoj, a member of the ParA superfamily (3). The loss-of-function BsSpo0J R80A mutant was originally discovered by Autret et al. (25) and reportedly has disrupted focus formation by fluorescence microscopy. More recently, Graham et al. (20) showed that BsSpo0J bridges chromosomal DNA using single-molecule experiments. However, its R79A, R80A, and R82A mutants could not spread in vivo and did not bridge DNA in vitro. These highly conserved arginine residues were defined as an arginine patch (20). Furthermore, Broedersz et al. (26) studied the condensation and localization of ParB by computational simulation.The crystal structures of ParB superfamily proteins have been reported for a DNA-free form of TtSpo0J (from Thermus thermophilus, containing the N-terminal and the DNA-binding domains) (10) and three complexes: the RP4–KorB-O_B complex (from plasmid RP4, containing the DNA-binding domain) (27), the P1 ParB–parS complex (from Enterobacteria phage P1, containing the DNA-binding and the C-terminal domains) (28), and the F-SopB–sopC complex (from plasmid F, containing the DNA-binding domain) (29).The Helicobacter pylori ParABS system consists of HpSoj (ParA), HpSpo0J (ParB), and parS DNA (30, 31). Herein, we report the crystal structure of a C-terminal domain truncated HpSpo0J (Ct-HpSpo0J)–parS complex. The N-terminal and the DNA-binding domains are present on Ct-HpSpo0J. The structural details of the complex in combination with results from EMSAs, fluorescence anisotropy assay, and small angle X-ray scattering (SAXS) allow us to propose a model for ParB spreading as it relates to chromosome segregation.     

From the Cover: Bacillus subtilis chromosome organization oscillates between two distinct patterns

Bacterial chromosomes have been found to possess one of two distinct patterns of spatial organization. In the first, called “ori-ter” and exemplified by Caulobacter crescentus, the chromosome arms lie side-by-side, with the replication origin and terminus at opposite cell poles. In the second, observed in slow-growing Escherichia coli (“left-ori-right”), the two chromosome arms reside in separate cell halves, on either side of a centrally located origin. These two patterns, rotated 90° relative to each other, appear to result from different segregation mechanisms. Here, we show that the Bacillus subtilis chromosome alternates between them. For most of the cell cycle, newly replicated origins are maintained at opposite poles with chromosome arms adjacent to each other, in an ori-ter configuration. Shortly after replication initiation, the duplicated origins move as a unit to midcell and the two unreplicated arms resolve into opposite cell halves, generating a left-ori-right pattern. The origins are then actively segregated toward opposite poles, resetting the cycle. Our data suggest that the condensin complex and the parABS partitioning system are the principal driving forces underlying this oscillatory cycle. We propose that the distinct organization patterns observed for bacterial chromosomes reflect a common organization–segregation mechanism, and that simple modifications to it underlie the unique patterns observed in different species.Central to reproduction is the faithful segregation of replicated chromosomes to daughter cells. In eukaryotes, DNA replication, chromosome condensation, and sister chromatid segregation are separated into distinct steps in the cell cycle that are safeguarded by checkpoint pathways. In bacteria, these processes occur concurrently, posing unique challenges to genome integrity and inheritance (1, 2). In the absence of temporal control, bacteria take advantage of spatial organization to promote faithful and efficient chromosome segregation. The organization of the chromosome dictates where the chromosome is replicated, and the factors that organize and compact the newly replicated DNA play a central role in its segregation (1, 2).Studies in different bacteria have revealed strikingly distinct patterns of chromosome organization that appear to arise from different segregation mechanisms. In Caulobacter crescentus and Vibrio cholerae chromosome I, the origin and terminus are located at opposite cell poles, with the two replication arms between them, in a pattern referred to as “ori-ter” (3–5). After replication initiation, one of the sister origins is held in place and the other is actively translocated to the opposite cell pole, regenerating the ori-ter organization in both daughter cells (5–11). By contrast, in slow-growing Escherichia coli, the origin is located in the middle of the nucleoid and the two replication arms reside in opposite cell halves, in a “left-ori-right” pattern (12, 13). Replication initiates at midcell, and the duplicated origins segregate to the quarter positions followed by the left and right arms on either side, regenerating the left-ori-right pattern in the two daughter cells.Although chromosome organization was first analyzed in the Gram-positive bacterium Bacillus subtilis, our understanding of the replication–segregation cycle in this bacterium has remained elusive. In pioneering studies, it was shown that during spore formation, the replicated origins reside at opposite cell poles and the termini at midcell in an ori-ter ter-ori organization (14–18). A similar ori-ter pattern was observed during vegetative growth (15, 19). However, in separate studies, DNA replication was found to initiate at a midcell-localized origin (20, 21). How these disparate patterns fit into a coherent replication–segregation cycle has never been addressed and motivated this study. Our analysis has revealed that the B. subtilis chromosome follows an unexpected and previously unidentified choreography during vegetative growth in which the organization alternates between ori-ter and left-ori-right patterns. Our data further suggest that the highly conserved partitioning system (parABS) and the structural maintenance of chromosomes (SMC) condensin complex, in conjunction with replication initiation, function as the core components for this oscillating cycle. We propose that this cycle enhances the efficiency of DNA replication and sister chromosome segregation and provides a unifying model for the diverse patterns of chromosome organization observed in bacteria.     

Dynamic localization of Mps1 kinase to kinetochores is essential for accurate spindle microtubule attachment

The spindle assembly checkpoint (SAC) is a conserved signaling pathway that monitors faithful chromosome segregation during mitosis. As a core component of SAC, the evolutionarily conserved kinase monopolar spindle 1 (Mps1) has been implicated in regulating chromosome alignment, but the underlying molecular mechanism remains unclear. Our molecular delineation of Mps1 activity in SAC led to discovery of a previously unidentified structural determinant underlying Mps1 function at the kinetochores. Here, we show that Mps1 contains an internal region for kinetochore localization (IRK) adjacent to the tetratricopeptide repeat domain. Importantly, the IRK region determines the kinetochore localization of inactive Mps1, and an accumulation of inactive Mps1 perturbs accurate chromosome alignment and mitotic progression. Mechanistically, the IRK region binds to the nuclear division cycle 80 complex (Ndc80C), and accumulation of inactive Mps1 at the kinetochores prevents a dynamic interaction between Ndc80C and spindle microtubules (MTs), resulting in an aberrant kinetochore attachment. Thus, our results present a previously undefined mechanism by which Mps1 functions in chromosome alignment by orchestrating Ndc80C–MT interactions and highlight the importance of the precise spatiotemporal regulation of Mps1 kinase activity and kinetochore localization in accurate mitotic progression.Faithful distribution of the duplicated genome into two daughter cells during mitosis depends on proper kinetochore–microtubule (MT) attachments. Defects in kinetochore–MT attachments result in chromosome missegregation, causing aneuploidy, a hallmark of cancer (1, 2). To ensure accurate chromosome segregation, cells use the spindle assembly checkpoint (SAC) to monitor kinetochore biorientation and to control the metaphase-to-anaphase transition. Cells enter anaphase only after the SAC is satisfied, requiring that all kinetochores be attached to MTs and be properly bioriented (3, 4). The core components of SAC signaling include mitotic arrest deficient-like 1 (Mad1), Mad2, Mad3/BubR1 (budding uninhibited by benzimidazole-related 1), Bub1, Bub3, monopolar spindle 1 (Mps1), and aurora B. The full SAC function requires the correct centromere/kinetochore localization of all SAC proteins (5).Among the SAC components, Mps1 was identified originally in budding yeast as a gene required for duplication of the spindle pole body (6). Subsequently, Mps1 orthologs were found in various species, from fungi to mammals. The stringent requirement of Mps1 for SAC activity is conserved in evolution (6–13). Human Mps1 kinase (also known as “TTK”) is expressed in a cell-cycle–dependent manner and has highest expression levels and activity during mitosis. Its localization is also dynamic (8, 14). Although the molecular mechanism remains unclear, Mps1 is required to recruit Mad1 and Mad2 to unattached kinetochores, supporting its essential role in SAC activity (15–18). It also is clear that aurora B kinase activity and the outer-layer kinetochore protein nuclear division cycle 80 (Ndc80)/Hec1 are required for Mps1 localization to kinetochores, as evidenced by recent work, including ours (17, 19–24). How Mps1 activates the SAC is now becoming clear. Mps1 recruits Bub1/Bub3 and BubR1/Bub3 to kinetochores through phosphorylation of KNL1, the kinetochore receptor protein of Bub1 and BubR1 (25–30).Despite much progress in understanding Mps1 functions, it remains unclear how Mps1 is involved in regulating chromosome alignment. In budding yeast mitosis, Mps1 regulates mitotic chromosome alignment by promoting kinetochore biorientation independently of Ipl1 (aurora B in humans) (31), but in budding yeast meiosis Mps1 must collaborate with Ipl1 to mediate meiotic kinetochore biorientation (32). In humans, Mps1 regulates chromosomal alignment by modulating aurora B kinase activity (33), but recent chemical biology studies show that Mps1 kinase activity is important for proper chromosome alignment and segregation, independently of aurora B (22, 34–36). Therefore whether Mps1 regulates chromosome alignment through modulation of aurora B kinase activity is still under debate (37).In this study, we reexamined the function of human Mps1 in chromosome alignment. We found that chromosomal alignment is largely achieved in Mps1 knockdown cells, provided that cells are arrested in metaphase in the presence of MG132, a proteasome inhibitor. However, disrupting Mps1 activity via small molecule inhibitors perturbs chromosomal alignment, even in the presence of MG132. This chromosome misalignment is caused by the abnormal accumulation of inactive Mps1 in the kinetochore and the subsequent failure of correct kinetochore–MT attachments. Further, we demonstrate that inactive Mps1 does not depend on the previously reported tetratricopeptide repeat (TPR) domain for localizing to kinetochores, and we identify a previously unidentified region adjacent to the C terminus of the TPR domain that is responsible for localizing inactive Mps1 to kinetochores. Thus, our work highlights that Mps1 kinase activity is necessary in regulating chromosome alignment and that it must be tightly regulated in space and time to ensure proper localization of Mps1 at kinetochores.     

Cell cycle-specific cleavage of Scc2 regulates its cohesin deposition activity

Sister chromatid cohesion (SCC), efficient DNA repair, and the regulation of some metazoan genes require the association of cohesins with chromosomes. Cohesins are deposited by a conserved heterodimeric loading complex composed of the Scc2 and Scc4 proteins in Saccharomyces cerevisiae, but how the Scc2/Scc4 deposition complex regulates the spatiotemporal association of cohesin with chromosomes is not understood. We examined Scc2 chromatin association during the cell division cycle and found that the affinity of Scc2 for chromatin increases biphasically during the cell cycle, increasing first transiently in late G₁ phase and then again later in G₂/M. Inactivation of Scc2 following DNA replication reduces cellular viability, suggesting that this post S-phase increase in Scc2 chromatin binding affinity is biologically relevant. Interestingly, high and low Scc2 chromatin binding levels correlate strongly with the presence of full-length or amino-terminally cleaved forms of Scc2, respectively, and the appearance of the cleaved Scc2 species is promoted in vitro either by treatment with specific cell cycle-staged cellular extracts or by dephosphorylation. Importantly, Scc2 cleavage eliminates Scc2–Scc4 physical interactions, and an scc2 truncation mutant that mimics in vivo Scc2 cleavage is defective for cohesin deposition. These observations suggest a previously unidentified mechanism for the spatiotemporal regulation of cohesin association with chromosomes through cell cycle regulation of Scc2 cohesin deposition activity by Scc2 dephosphorylation and cleavage.Multisubunit, ring-shaped cohesin complexes play key roles in chromosome morphogenesis that are required for faithful chromosome transmission to daughter cells. Newly replicated sister chromatids become tethered together by cohesins during S phase, which promotes chromosome biorientation on mitotic spindles (1). Cohesins also mediate efficient DNA double-strand break repair by homologous recombination (2, 3) and the formation or stabilization of chromatin loops that affect various nuclear processes, such as gene expression and Ig gene rearrangements (reviewed in refs. 4 and 5). Altered gene expression resulting from defective cohesin-mediated chromatin looping is likely responsible for the pathogenesis of Cornelia de Lange Syndrome (CdLS), a dominantly inherited human developmental disorder (6).Sister chromatid cohesion (Scc) proteins form a heterodimeric cohesin deposition complex, but the complex''s activity in deposition is not understood (7). Cohesins copurify with Scc2/Scc4, suggesting that Scc2/Scc4 plays a direct role in deposition (8–11). In the absence of either loader complex subunit, cohesin rings assemble, but fail to be deposited (7, 12, 13). ATP hydrolysis by cohesin’s structural maintenance of chromosome (SMC) subunits is required for cohesin loading, and deposition is inhibited when SMC hinge domains, which mediate Smc1/3 interactions within cohesin, are artificially tethered (8, 14, 15). Thus, Scc2/Scc4 may activate cohesin’s ATPase activity or facilitate a conformational change in cohesin structure that promotes its loading, perhaps by permitting transient hinge opening to allow chromatin to enter cohesin rings or by promoting cohesin oligomerization (14, 16).Factors that regulate Scc2/Scc4 chromatin association are only beginning to be elucidated. Interactions of Scc2 and Scc4 orthologs from Xenopus and humans, and their stable association with chromatin, require the amino termini of both proteins (10, 13, 17, 18). In contrast, the fission yeast Scc2 ortholog alone binds nonchromatinized DNA, but does not exhibit an expected preference for sequences shown to associate with Scc2/Scc4 in vivo (19). Xenopus Scc2/Scc4 chromatin association requires prereplication complexes and Drf1-dependent kinase (DDK) activity (10, 12, 20), although this scenario is not the case in budding yeast (21). Scc2/Scc4 interactions with histone deacetylases and an ATP-dependent chromatin remodeler suggest that underlying chromatin structure also influences Scc2/Scc4 chromatin association (22–26). Whether Scc2/Scc4 plays a role in chromatin remodeling or merely deposits cohesins at remodeled sites is unknown, however.The chromatin association of Scc2/Scc4 and its orthologs is also regulated temporally during the cell cycle, although the specifics of association vary across species. Scc2/Scc4 associates with chromatin in late mitosis of the previous cell cycle in metazoans (12, 13) and in late G₁ in budding yeast, but in all cases, this association precedes DNA replication initiation so that cohesins are deposited in time to tether newly replicated sister chromatids together. Surprisingly, budding yeast Scc2/Scc4 chromatin association is more robust in mitotically arrested cells than in G₁-staged cells. Reduced G₁ Scc2/Scc4 chromatin association is not due to the absence of either loader subunit, because Scc2 and Scc4 protein levels vary little during the cell cycle, or by a lack of assembled cohesin complexes in G₁, because Scc2/Scc4 chromatin association occurs independently of cohesins (18, 27, 28). Scc2/Scc4 removal from chromatin is also regulated and occurs during mitosis in Xenopus and, more specifically, during prophase in humans (12, 13). Although factors responsible for regulating Scc2/Scc4 chromatin association/dissociation during the cell cycle remain enigmatic, evidence that multiple Scc2 orthologs are phosphorylated suggests the intriguing possibility that Scc2 posttranslational modifications regulate Scc2/Scc4 chromatin association (29–31).Here, we describe our efforts to understand how budding yeast Scc2/Scc4 chromatin binding is regulated during the cell cycle. Our results demonstrate the existence of multiple Scc2 protein species in vivo and that a specific cleaved form of Scc2 accumulates at cell cycle periods when Scc2 chromatin binding is weak. The appearance of this cleaved Scc2 species is strongly correlated with Scc2 dephosphorylation, suggesting that the phosphorylation state of Scc2 is critical for the regulation of its stability. Scc2 cleavage is also correlated with the loss of Scc2–Scc4 interactions, and an scc2 truncation mutant that mimics cleaved Scc2 is defective in cohesin deposition. These observations suggest that Scc2–Scc4 interactions and, therefore, the function of the complex in cohesin deposition, may be influenced by dephosphorylation-induced Scc2 cleavage.     

Minimal model for collective kinetochore–microtubule dynamics

Chromosome segregation during cell division depends on interactions of kinetochores with dynamic microtubules (MTs). In many eukaryotes, each kinetochore binds multiple MTs, but the collective behavior of these coupled MTs is not well understood. We present a minimal model for collective kinetochore–MT dynamics, based on in vitro measurements of individual MTs and their dependence on force and kinetochore phosphorylation by Aurora B kinase. For a system of multiple MTs connected to the same kinetochore, the force–velocity relation has a bistable regime with two possible steady-state velocities: rapid shortening or slow growth. Bistability, combined with the difference between the growing and shrinking speeds, leads to center-of-mass and breathing oscillations in bioriented sister kinetochore pairs. Kinetochore phosphorylation shifts the bistable region to higher tensions, so that only the rapidly shortening state is stable at low tension. Thus, phosphorylation leads to error correction for kinetochores that are not under tension. We challenged the model with new experiments, using chemically induced dimerization to enhance Aurora B activity at metaphase kinetochores. The model suggests that the experimentally observed disordering of the metaphase plate occurs because phosphorylation increases kinetochore speeds by biasing MTs to shrink. Our minimal model qualitatively captures certain characteristic features of kinetochore dynamics, illustrates how biochemical signals such as phosphorylation may regulate the dynamics, and provides a theoretical framework for understanding other factors that control the dynamics in vivo.Microtubule (MT) dynamics are critical for cell division. Plus ends of spindle MTs interact with kinetochores, protein complexes that assemble at the centromere of each chromosome, and these dynamic MTs exert forces to move chromosomes. Individual MTs are “dynamically unstable,” spontaneously switching between a polymerizing state and a depolymerizing state (1) with growth, shortening, and switching rates that are regulated by the forces exerted at the MT tips (2–6). For many eukaryotes, however, multiple MTs are connected to each kinetochore, giving rise to collective MT behavior that is not well understood and can be entirely different from the behavior of individual MTs. Here, we develop a model of collective MT dynamics based on the measured force-dependent dynamics of individual MTs.Accurate chromosome segregation depends on correctly biorienting the kinetochore pairs by attaching sister kinetochores to opposite spindle poles. Properly attached kinetochores undergo center-of-mass (CM) and breathing oscillations that are regulated by collective MT dynamics (7–12). Incorrect attachments—such as syntelic attachment of both kinetochores to the same pole—must be corrected (13–17). Tension may cue this process because bioriented kinetochore pairs are under tension while syntelically attached kinetochores are not (7, 9, 15, 17, 18). Error correction is also mediated by Aurora B kinase phosphorylating MT-binding kinetochore proteins (13–17, 19–21). A consistent theory of metaphase kinetochore–MT dynamics should capture CM and breathing oscillations for correctly attached pairs and elucidate the contributions of tension and phosphorylation to syntelic error correction.Several models suggest that chromosome oscillations result from competition between poleward MT-based pulling and antipoleward “polar ejection” forces (22–24). Another model proposes that oscillations occur via a general mechanobiochemical feedback (25). Models of force-dependent MTs interacting with the same object also exhibit cooperative behavior (5, 26–29). However, these models do not explain error correction dynamics. Thus, the underlying physical mechanisms coordinating metaphase chromosome motions are unclear.We address these issues by developing a minimal model for collective MT dynamics based on in vitro measurements of single MTs interacting dynamically with kinetochore proteins (4, 6, 20, 21). In the model, MT polymerization and rescue are promoted by tension and inhibited by compression, whereas depolymerization and catastrophe are enhanced by compression and reduced by tension. With just these features, we find a robust and versatile mechanism by which force-dependent MTs coupled to the same kinetochore may drive metaphase chromosome motions. The force–velocity relation for a MT bundle is fundamentally different from that of a single dynamically unstable MT, exhibiting bistable behavior. Bistability gives rise to kinetochore oscillations and is shifted by phosphorylation to produce error correction. The model qualitatively predicts kinetochore motions in our experiments in which Aurora B is hyperactivated in bioriented kinetochore pairs. Thus, we find that many characteristics of metaphase kinetochore dynamics emerge simply from the force coupling of many MTs to the same kinetochore, and chemical signals such as phosphorylation can regulate this physical mechanism.     

Sequential de novo centromere formation and inactivation on a chromosomal fragment in maize

The ability of centromeres to alternate between active and inactive states indicates significant epigenetic aspects controlling centromere assembly and function. In maize (Zea mays), misdivision of the B chromosome centromere on a translocation with the short arm of chromosome 9 (TB-9Sb) can produce many variants with varying centromere sizes and centromeric DNA sequences. In such derivatives of TB-9Sb, we found a de novo centromere on chromosome derivative 3-3, which has no canonical centromeric repeat sequences. This centromere is derived from a 288-kb region on the short arm of chromosome 9, and is 19 megabases (Mb) removed from the translocation breakpoint of chromosome 9 in TB-9Sb. The functional B centromere in progenitor telo2-2 is deleted from derivative 3-3, but some B-repeat sequences remain. The de novo centromere of derivative 3-3 becomes inactive in three further derivatives with new centromeres being formed elsewhere on each chromosome. Our results suggest that de novo centromere initiation is quite common and can persist on chromosomal fragments without a canonical centromere. However, we hypothesize that when de novo centromeres are initiated in opposition to a larger normal centromere, they are cleared from the chromosome by inactivation, thus maintaining karyotype integrity.The centromere is an important chromosomal region responsible for correct chromosome segregation during cell division. Centromeres are found in the primary constriction region on the chromosome, upon which the kinetochore complex assembles to produce a platform for spindle binding (1). Centromere function is conserved among different species, and several epigenetic markers of active centromeres have been found, including a histone H3 variant referred to as CENH3 in plants (2, 3) or CENP-A in animals (4–6) and phosphorylation of histone H2A at Thr133 in plants (7). Correct loading of CENH3 to the centromere region is a key component of kinetochore assembly (8). Centromeric DNA sequences have experienced rapid evolution (9, 10), and arrangements of DNA sequence in centromere regions differ in species and even in different chromosomes of an individual organism (11). Myriad repeat sequences exist in the centromeres of higher plants. In maize (Zea mays), there are two major types of centromere specific DNA sequences: the simple satellite repeat sequence CentC (12) and centromeric retrotransposon of maize (CRM) (3). Many epigenetic features have been identified in centromeric regions, including DNA methylation levels, histone variants, histone modifications, and RNA components (11). Both epigenetic elements and DNA sequences take part in centromere formation and maintenance, but it is still unknown how genetic and epigenetic factors work together in this process.The centromere is one of the most complex regions on the chromosome, and complete DNA sequencing through the centromeric region is difficult to obtain due to their highly repetitive nature. Centromere sizes, defined by CENP-A/CENH3 binding regions, range from 125 bp in Saccharomyces cerevisiae to 500–1,500 kb in humans and mice (11). In plants, centromere sizes can range to several megabases (Mb) with many repetitive transposable elements, which makes it difficult to study centromere structure and function. For example, the sizes of centromere 2 and 5 in maize are roughly 2 and 7 Mb, respectively.Previous work sought misdivision derivatives of the B chromosome centromere using a translocation between the supernumerary chromosome and the short arm of chromosome 9 (9S) to reduce the size of the centromere for functional studies (13–15). B chromosomes are extra chromosomes that have been found in many plants, animals, and fungi. In maize, a reciprocal translocation between a B chromosome and the short arm of chromosome 9 produced two chromosomes referred to as B-9 and 9-B (13), together referred to as TB-9Sb. Chromosome 9-B contains the long arm of the B chromosome and most of chromosome 9, including its centromere. Correspondingly, chromosome B-9 contains part of the short arm of chromosome 9 and the other part of the B chromosome with the active B centromere. The translocation breakpoint is near Wx1, which is located on 9-B (16). The B centromere of B-9 can undergo misdivision during meiosis, producing many derivatives (14). The first misdivision derivative was a pseudoisochromosome, and subsequently, many telocentric chromosomes and isochromosomes were derived by additional misdivisions (13, 17). Misdivision events can be recognized in crosses of TB-9Sb onto a tester via a fusion-breakage cycle recognized by the behavior of the C1 color marker on the B-9 chromosome. The cycle continues during endosperm development to produce a mosaic phenotype but is “healed” in the embryo, which when grown and analyzed cytologically will reveal the nature of the new chromosomes formed (13, 17). This type of screen was used to assemble a large collection of misdivisions to examine the structural features of the B centromere (18). Centromere sizes of these derivatives were changed and progressively reduced. In these previous studies, molecular analysis of centromere size relied on studying the B centromere-specific DNA repeat before the maize centromere elements, CentC and CRM, were known. The B-specific repeat allows this centromere to be studied against the background of the other centromeres; it surrounds and is interspersed within the active core of the B centromere (15, 19).New functional centromeres formed at ectopic locations rather than native centromeric regions on the chromosomes are called de novo centromeres. Many de novo centromeres have been found in human patients and other organisms (20, 21). There are reports of de novo centromeres in plants, such as barley (Hordeum vulgare) (22), oat (Avena sativa)-maize addition lines (23), and maize (24, 25). The conditions for de novo centromere formation remain unclear (26). Recent research revealed that many de novo centromeres prefer to form near native centromeric regions or in the heterochromatic regions, such as the pericentromere and telomere (27, 28). There are also de novo centromeres in human formed far from native centromeres (20). We have previously described two de novo centromeres in maize: one is near the position of the native centromere (25) and the other is distal to the site of the corresponding native centromere (24). Specific chromatin environments may be required for centromere formation, but the major elements are as yet unknown.DNA sequence alone is insufficient to direct centromere formation, and dicentric chromosomes containing two centromeres are good examples. To be stable, structurally dicentric chromosomes must have one inactive and one active centromere; otherwise, two active centromeres will lead to chromosome breakage during cell division. In maize, many dicentric chromosomes have been reported from B-A translocation chromosome derivatives (29). Dicentric chromosomes can be produced through the process of the chromosome type breakage-fusion-bridge (BFB) cycle, and the inactive centromeres can be reactivated by intrachromosomal recombination (30). The DNA sequences of the active and inactive centromeres of dicentric chromosomes are essentially identical, but the centromere activity states are completely different. We screened several misdivision derivatives using FISH probes specific to maize centromere sequences, CentC and CRM, as well as probes specific to the B centromere repeat sequence (B-repeat) to gain further insight into the nature of the centromeres in this collection. We discovered that one such chromosome, derivative 3-3, lacks detectable CentC and CRM signals, but still has a functional centromere that is not associated with the B-repeat sequence. The results of chromatin immunoprecipitation sequencing (ChIP-seq) using maize CENH3 antibody revealed that a 288-kb region on 9S is involved in the de novo centromere formation. The functional B centromere of progenitor telo2-2 is deleted from derivative 3-3. Further, new derivatives of derivative 3-3 had been selected (31) but there was no change in the B-specific repeat patterns. Here, we found that the de novo centromere of 3-3 has become inactive in all of its derivatives, and in each case a shift to a new de novo centromere position occurred; one of these contains only a 200-kb CENH3 binding region within 9S. The other two are apparently in B chromosome sequences. Thus, sequential de novo centromere formation and exchange of centromere activity occurred in chromosome 3-3 and its derivatives, providing new insight into centromere formation and maintenance.These results help formulate the nature of de novo centromere formation. In all of the examples now documented in maize, the size range is within a few hundred kilobases. In contrast, normal maize centromeres, as noted above, are typically several megabases. The regular occurrence of de novo centromeres found here and previously (24, 25) indicates that they are capable of being formed regularly on chromosomal fragments that are structurally acentric; however, they do not persist in normal chromosomes. The reason might reside in the previous observation in maize (30) and wheat (32) that in functional dicentrics the smaller centromere becomes inactive in a tug of war between large and small. However, in the absence of a normal centromere, the present work illustrates that de novo centromeres can persist. Thus, in normal chromosomes, if a de novo is initiated, it will be as quickly inactivated in opposition to the much larger preexisting centromere; the chromosome will not be affected, and will seldom change structure over evolutionary time despite such a high rate of de novo formation. This hypothesis also suggests that a selective pressure will be placed on the normal centromeres to expand to a size that can regularly inactivate de novo centromeres based on their initial size at formation.     

Assembly,translocation, and activation of XerCD-dif recombination by FtsK translocase analyzed in real-time by FRET and two-color tethered fluorophore motion

The FtsK dsDNA translocase functions in bacterial chromosome unlinking by activating XerCD-dif recombination in the replication terminus region. To analyze FtsK assembly and translocation, and the subsequent activation of XerCD-dif recombination, we extended the tethered fluorophore motion technique, using two spectrally distinct fluorophores to monitor two effective lengths along the same tethered DNA molecule. We observed that FtsK assembled stepwise on DNA into a single hexamer, and began translocation rapidly (∼0.25 s). Without extruding DNA loops, single FtsK hexamers approached XerCD-dif and resided there for ∼0.5 s irrespective of whether XerCD-dif was synapsed or unsynapsed. FtsK then dissociated, rather than reversing. Infrequently, FtsK activated XerCD-dif recombination when it encountered a preformed synaptic complex, and dissociated before the completion of recombination, consistent with each FtsK–XerCD-dif encounter activating only one round of recombination.Understanding how molecular machines assemble and act requires a combination of biochemical, structural, and biophysical approaches. In recent years, single-molecule techniques have allowed the observation of biological reactions in real time, thereby avoiding the averaging of ensemble experiments. These single-molecule studies rely on information gained from existing biochemical characterization to set experimental parameters and to place their results in context. To follow complex multistep reactions involving multiple components, methods capable of tracking several observables simultaneously have been established (1–4). In this work, we push the boundaries of a recently developed technique, and apply it to further our understanding of the Escherichia coli XerCD-dif–FtsK molecular machine, which functions in chromosome segregation and coordinates it with cell division.FtsK is a 1,329-aa DNA translocase, which assembles at the division septum and functions in segregating sister chromosomes during the late stages of the cell cycle in a wide range of bacteria (5–8) by activating site-specific recombination by XerCD at dif (9). The two related Tyr recombinases, XerC and XerD, bind the 28-bp dif site located within the terminus region, ter, on the E. coli chromosome. They unlink catenated chromosomes and resolve chromosome dimers formed by homologous recombination (10–14). Independent of its role in activating XerCD-dif recombination, FtsK appears to play a direct role in the segregation of ter (8). FtsK consists of three domains: an essential 179-aa N-terminal domain that anchors it to the division septum, an ∼500-aa C-terminal motor domain, and an ∼650-aa linker domain (6, 7, 15, 16). The motor domain, FtsK_C, is composed of α-, β-, and γ-subdomains (17). The α- and β-subdomains form a dsDNA translocase, belonging to the RecA family of ATPases (17). The γ-subdomain plays a regulatory role in the recognition of the FtsK orientating polar sequence (KOPS) that guides FtsK translocation toward the dif site at ter (18–20), and in the activation of XerCD-dif recombination (9, 14, 21). Activation of recombination requires direct interaction between the γ-subdomain and XerD (22, 23).Our previous work, using a single tethered fluorophore motion (TFM) reporter, in combination with Förster resonance energy transfer (FRET), determined the conformational transitions in the XerCD-dif complex that occurred as XerD mediated an initial strand exchange to form a Holliday junction (HJ), which was resolved by a subsequent XerC-mediated strand exchange (24). In that work, we indirectly inferred the presence of FtsK_C, taking advantage of protein-induced fluorescence enhancement (PIFE) (25). Here, we have expanded TFM-FRET, using two spectrally distinct TFM reporters (one on FtsK_C and one on DNA), to directly observe FtsK_C as it assembles and translocates, and to correlate its behavior, upon arrival at XerCD, with the progress of the recombination reaction.Previous studies of FtsK_C assembly and translocation have used biochemical methods (26, 27) and single-molecule techniques, including magnetic and optical tweezers (18, 28–30); tethered particle motion (TPM) (18); and, more recently, DNA curtains (31, 32). Optical/magnetic tweezers and TPM experiments have relied on loop extrusion by FtsK_C to observe its action (looping by FtsK shortens the length between two DNA ends, hence displacing the bead used in TPM or optical/magnetic tweezers). Many of the single-molecule assays involved the attachment of FtsK_C to quantum dot (QD) labels or used derivatives that were known to aggregate, thereby potentially confounding the interpretation of data, because multiple motors could be present in the region of analysis. Experiments utilizing DNA curtains have revealed that FtsK_C can push, evict, and bypass proteins bound to DNA as it translocates (32). However, FtsK_C stops at least transiently and/or dissociates at XerCD bound to dif (27, 32). Reversals in translocation direction have been observed to occur spontaneously (18, 28, 30, 31) and in response to XerCD bound to dif (32). The use of a fluorophore label, along with singly tethered DNA, has allowed us to observe FtsK_C without requiring any loop extrusion, and to observe its interaction with synaptic complexes of XerCD, where previous single-molecule work has only dealt with unsynapsed XerCD-dif (32). Using this approach, we have determined that FtsK_C assembles on DNA as a single hexamer, and begins translocating rapidly (∼0.25 s), without extruding a loop of DNA. When it reached XerCD bound to dif, either in a synapsed or unsynapsed conformation, it resided briefly for ∼0.5 s and then dissociated without any evidence of reversal. FtsK_C activated recombination when it met synapsed XerCD-dif complexes, and then dissociated faster than the completion of recombination by XerCD.     

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. We 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.Centromeres are the control centers for chromosomes, and thus are essential for accurate segregation in cell division. Centromeres are the DNA regions with a specialized chromatin structure upon which the kinetochore is built. The kinetochore is a complex of at least 100 proteins that contains the proteins to bind microtubules, motors to move on or destabilize microtubules, as well as checkpoint proteins monitoring kinetochore–microtubule attachment (1). The ability of the kinetochore to control microtubule binding and chromosome movement is essential for proper segregation in both mitosis and meiosis. In meiosis, additional constraints are placed on kinetochore function to ensure that homologs segregate in the first division and that segregation of sister chromatids is deferred until the second division (2). Recent studies indicate that in addition, the centromere itself may influence homolog segregation by controlling homolog pairing and formation of the synaptonemal complex (SC) (3).In prophase of meiosis I, the homologs must pair and ultimately become attached, usually by recombination and crossing-over. By quantifying centromere number through prophase I, it has been observed that centromeres pair in yeast, plants, and Drosophila (3). Perhaps unexpectedly, this pairing can be between nonhomologous centromeres; in yeast, this has been proposed as a mechanism to prevent recombination around the centromere, as centromere pairing resolves from initially being nonhomologous to being homologous (4, 5). Homologous centromere pairing may play a critical role in ensuring segregation of chromosomes that do not undergo crossing-over, possibly by affecting orientation of the kinetochores (3, 6–8).The centromere also regulates synapsis via the formation of the SC. SC formation initiates at the centromere and sites of cross-over formation in yeast, and the centromere is the first site for SC formation in Drosophila prophase I (9, 10). In addition, the SC persists at the centromere in yeast and Drosophila after the SC present along the chromosome arms has disassembled late in prophase I (7, 9, 11). Although SC assembly does not begin at centromeres in mouse meiosis, it persists at the centromeres and appears to promote proper segregation (12, 13).Another centromere property has been observed in Drosophila oocytes. In most organisms, the centromeres are clustered together at one site at the onset of meiosis, likely a remnant of their configuration in mitosis, but this clustering breaks down as centromeres arrange in pairs (3, 4). In Drosophila, however, the centromeres remain clustered until exit from prophase I at oocyte maturation (9, 10, 14). Although an essential role for centromere clustering has not been demonstrated, it may facilitate homolog pairing, synapsis, or accurate segregation, particularly given that the homologous telomeres do not pair into a bouquet formation in Drosophila meiosis (15, 16). Components of the SC are necessary for centromere clustering, as is the cohesion protein ORD (9, 14).The studies on centromere pairing and clustering define centromere geography within the meiotic nucleus, but they did not test whether centromere structure or function was involved. Centromeres have specialized nucleosomes with a histone H3 variant, centromere protein-A (CENP-A) (17). Incorporation of CENP-A into centromere chromatin is regulated precisely, although it occurs at distinct cell cycle times in different cell types, varying between late mitosis and G1 (17). In vertebrates, a complex of 15 proteins, the constitutive centromere-associated network (CCAN), is present on the CENP-A chromatin throughout the cell cycle and is crucial for assembling kinetochore proteins (1). In Drosophila, the entire CCAN complex has not been identified, although the CENP-C protein is present (18). Another Drosophila protein, CAL1, binds to CENP-A (called CID in Drosophila) in a prenucleosomal complex, and CAL1 is required for loading CID (19–22). CAL1 interacts with both CID and CENP-C, and all three proteins show interdependency for centromere localization (21, 23).Little is known about the activities of these centromere proteins in meiosis. In fission yeast, CENP-C has been demonstrated to be critical for kinetochore–microtubule binding in meiosis and also to control kinetochore orientation in meiosis I (24). The timing of assembly of kinetochore and centromere proteins onto meiotic chromosomes has been examined in mouse spermatocytes (25) and in Drosophila spermatocytes and sperm (26, 27). RNAi studies have shown that CAL1 and CENP-C (the latter to a lesser extent) are needed for CID localization in Drosophila male meiosis, with reduction in the levels of any of these three proteins being associated with meiotic segregation errors (26). Drosophila males differ from most organisms in not undergoing recombination or forming an SC, and centromere clustering does not occur (28). A question of particular interest that has yet to be addressed is whether centromere architecture and function are required for centromere clustering and pairing in meiosis.     

Male-specific lethal complex in Drosophila counteracts histone acetylation and does not mediate dosage compensation

Dosage compensation is achieved in male Drosophila by a twofold up-regulation of the single X chromosome to reach the level of the two X chromosomes in females. A popular hypothesis to explain this phenomenon is that the male-specific lethal (MSL) complex, which is present at high levels on the male X, mediates this modulation of gene expression. One member of the complex, MOF, a histone acetyltransferase, acetylates lysine 16 of histone H4 and another, MSL2, which is only expressed in males, triggers its assembly. Here, we find that when a GAL4-MOF fusion protein is targeted to an upstream-activating sequence linked to a miniwhite reporter, up-regulation occurs in females but down-regulation in males, even though in the latter the whole MSL complex is recruited to the reporter genes and produces an increased histone acetylation. The expression of a GAL4-MSL2 fusion protein does not cause dosage compensation of X and autosomal reporters in females, although its expression causes the organization of the MSL complex on the reporter genes, leading to increased histone acetylation. RNAseq analysis of global endogenous gene expression in females with ectopic expression of MSL2 to coat the X chromosomes shows no evidence of increased expression compared with normal females. These data from multiple approaches indicate that the MSL complex does not mediate dosage compensation directly, but rather its activity overrides the high level of histone acetylation and counteracts the potential overexpression of X-linked genes to achieve the proper twofold up-regulation in males.Dosage compensation is achieved in male Drosophila by an approximate twofold up-regulation of the X chromosome to equal the two X chromosomes in females (1, 2). For several decades, it has been proposed that the components of the male-specific lethal (MSL) complex are present on the male X chromosome and produce the twofold modulation (3, 4). One member of the complex, MOF, a histone acetyltransferase, acetylates lysine 16 of histone H4 (5) and another, MSL2, which is male-specific, triggers its X chromosome assembly (6). The MSL2 protein is not expressed in females because it is blocked at translation by the female-specific sex-lethal (SXL) protein (7). Gene-expression data taken in support of the MSL hypothesis have normalized X expression to autosomal expression and found a reduced X/A ratio that was interpreted as a loss of compensation when the MSL complex was dissociated (3, 8–10). However, when gene expression is assayed phenotypically or in absolute terms, rather than as a ratio of X to autosomal expression, data from ectopic assembly of the complex in females failed to demonstrate up-regulation of the X chromosomes (11, 12) and the dissolution of the complex does not eliminate compensation (11–15). Many assayed autosomal genes were increased in expression in the maleless (mle) mutant males (11, 13, 14), providing an explanation for the reduced ratios when X values were normalized to the autosomes.The retention of dosage compensation and elevated autosomal expression conforms to the prediction that the X monosomic state triggers an inverse dosage effect that is commonly found in aneuploids (16–23). We hypothesize that this general reaction to genomic imbalance appears to have been modified and selected for the process of dosage compensation, which could account for the twofold up-regulation in males. However, because in normal males the autosomes are similarly expressed to those of females, it was hypothesized that the MSL complex present only in males sequesters MOF from the autosomes to mute any autosomal inverse effect, and counteracts the action of the resulting very high levels of histone acetylation on the X to allow the proper twofold modulation for compensation (11, 14). To examine further the function of the MSL complex, MOF and MSL2 were targeted to reporter genes to determine their impact on gene expression and dosage compensation and a global study of gene expression was conducted in females with ectopic MSL complex on their X chromosomes.     

Spontaneous slow replication fork progression elicits mitosis alterations in homologous recombination-deficient mammalian cells

Homologous recombination deficient (HR⁻) mammalian cells spontaneously display reduced replication fork (RF) movement and mitotic extra centrosomes. We show here that these cells present a complex mitotic phenotype, including prolonged metaphase arrest, anaphase bridges, and multipolar segregations. We then asked whether the replication and the mitotic phenotypes are interdependent. First, we determined low doses of hydroxyurea that did not affect the cell cycle distribution or activate CHK1 phosphorylation but did slow the replication fork movement of wild-type cells to the same level than in HR⁻ cells. Remarkably, these low hydroxyurea doses generated the same mitotic defects (and to the same extent) in wild-type cells as observed in unchallenged HR⁻ cells. Reciprocally, supplying nucleotide precursors to HR⁻ cells suppressed both their replication deceleration and mitotic extra centrosome phenotypes. Therefore, subtle replication stress that escapes to surveillance pathways and, thus, fails to prevent cells from entering mitosis alters metaphase progression and centrosome number, resulting in multipolar mitosis. Importantly, multipolar mitosis results in global unbalanced chromosome segregation involving the whole genome, even fully replicated chromosomes. These data highlight the cross-talk between chromosome replication and segregation, and the importance of HR at the interface of these two processes for protection against general genome instability.DNA is continuously subjected to injury by exogenous and endogenous sources. The faithful transmission of genetic material relies on the DNA damage response (DDR), which coordinates a network of pathways, including DNA replication-repair-recombination, the cell cycle checkpoint, and chromosome segregation. A defect in any of these pathways causes genetic instability and cancer predisposition. Strikingly, both spontaneous DDR activation as a consequence of endogenous replication stress and centrosome abnormalities, which cause uneven chromosome segregation, have been reported in precancerous and early-stage malignancies (1–10). Therefore, endogenous stresses must play a key role in spontaneous chromosome instability and in cancer etiology.Homologous recombination (HR) is an evolutionarily conserved process that controls the balance between genetic stability and diversity. Specifically, HR plays a pivotal role in the reactivation of replication forks that have been blocked, contributing to DNA replication accuracy (11–16). Replication forks are routinely inactivated by endogenous stress (17, 18); therefore, HR should play an essential role to protect cells against these types of stresses, and HR deficiency should reveal endogenous replication stress. Remarkably, unchallenged HR-deficient (HR⁻) cells display both a genome-wide decrease in replication fork speed (19) and a spontaneous increase in the frequency of cells containing extra centrosomes (20–28). Two hypotheses may account for these two phenotypes. First, replication stress leads to chromosome alteration at incomplete replicated regions and chromosome rearrangements (29). However, centrosomes do not contain DNA, and if extra centrosomes at mitosis [mitotic extra centrosome (MEC)] are active, unbalanced chromosome segregation should lead to global chromosome instability, even for fully replicated chromosomes. Second, HR proteins are associated with supernumerary centrosomes; therefore, centrosome duplication defects may directly result from HR misregulation (30, 31).In this study, we addressed whether spontaneous MECs result from slow replication fork movement in HR⁻ cells. The data presented here underline the importance of HR at the molecular interface between replication and chromosome segregation to protect against spontaneous genomic instability.     

Site-specific genetic engineering of the Anopheles gambiae Y chromosome

Despite its function in sex determination and its role in driving genome evolution, the Y chromosome remains poorly understood in most species. Y chromosomes are gene-poor, repeat-rich and largely heterochromatic and therefore represent a difficult target for genetic engineering. The Y chromosome of the human malaria vector Anopheles gambiae appears to be involved in sex determination although very little is known about both its structure and function. Here, we characterize a transgenic strain of this mosquito species, obtained by transposon-mediated integration of a transgene construct onto the Y chromosome. Using meganuclease-induced homologous repair we introduce a site-specific recombination signal onto the Y chromosome and show that the resulting docking line can be used for secondary integration. To demonstrate its utility, we study the activity of a germ-line–specific promoter when located on the Y chromosome. We also show that Y-linked fluorescent transgenes allow automated sex separation of this important vector species, providing the means to generate large single-sex populations. Our findings will aid studies of sex chromosome function and enable the development of male-exclusive genetic traits for vector control.Mosquito species of the Anopheles gambiae complex represent the principal vectors of human malaria, and they pose an enormous burden on global health and economies. Every year, 300–500 million people are infected by malaria and more than 1 million people die as a consequence of Plasmodium parasite infections (1). The malaria mosquito A. gambiae has two pairs of autosomes, termed 2 and 3, and a pair of heteromorphic sex chromosomes X and Y, XX in females and XY in males (2). Extensive nonpairing regions exist between the X and the degenerate Y chromosome. and evidence points to a factor located on the Y chromosome that primarily determines the sex in Anopheles (3). Current models suggest that the evolutionary differentiation of Y chromosomes begins with the acquisition of a male determining factor on a proto-Y chromosome (4, 5). This event is followed by a progressive suppression of recombination between the still largely homomorphic proto-sex chromosomes, a process attributed to the acquisition of sexually antagonistic mutations, which are beneficial to the heterogametic sex but detrimental to the homogametic sex (6–8). The lack of recombination, together with the male-limited transmission, leads to the degeneration of the Y chromosome, which involves accumulation of deleterious mutations, spread of transposable elements, and silencing of all or most of the genes present on the proto-Y (9–11). As a result, Y chromosomes of many species appear to be strongly heterochromatic and harbor only few genes often involved in male fertility (12–17). The accumulation of repetitive sequences, many of which are also present on other chromosomes, hampers the assembly of Y chromosome contigs following shotgun sequencing. Indeed, despite the completion of the A. gambiae genome project (18), and the knowledge that the primary signal is likely to be associated with the inheritance of the Y (3, 19), no assembly of the Anopheles Y chromosome has been achieved. At present, public databases host only a few hundred kilobases of A. gambiae sequences attributed to the Y, a chromosome that is estimated to comprise 10% of the genome and to be at least 20 Mb in size. None of these Y-specific scaffolds have been physically mapped, because the Y chromosome does not polytenize. The exploration of the Y chromosome will improve our understanding of the evolutionary forces involved in driving chromosome evolution and may enable the manipulation of the molecular pathways that control sex determination and sexual differentiation in mosquitoes. In a number of organisms, Y chromosome genes have been found to be essential for male fertility or sex determination. Recently, a number of excellent candidate genes potentially involved in these processes have been identified on the Y chromosome of anopheline mosquitoes (20, 21). Because interfering with male fertility is an essential part of vector control strategies such as the sterile insect technique, the identification of such genes is of particular interest to mosquito biologists. In this paper, we demonstrate the targeted molecular manipulation of the Y chromosome in A. gambiae, thus opening up a number of ways to explore one of the most fascinating of evolution’s upshots and to harness this genetic tool for vector control.     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.