Centromeric Sites Research Articles

In vivo assembly of bacterial partition condensates on circular supercoiled and linear DNA.

In bacteria, faithful DNA segregation of chromosomes and plasmids is mainly mediated by ParABS systems. These systems, consisting of a ParA ATPase, a DNA binding ParB CTPase, and centromere sites parS, orchestrate the separation of newly replicated DNA copies and their intracellular positioning. Accurate segregation relies on the assembly of a high-molecular-weight complex, comprising a few hundreds of ParB dimers nucleated from parS sites. This complex assembles in a multi-step process and exhibits dynamic liquid-droplet properties. Despite various proposed models, the complete mechanism for partition complex assembly remains elusive. This study investigates the impact of DNA supercoiling on ParB DNA binding profiles invivo, using the ParABS system of the plasmid F. We found that variations in DNA supercoiling does not significantly affect any steps in the assembly of the partition complex. Furthermore, physical modeling, leveraging ChIP-seq data from linear plasmids F, suggests that ParB sliding is restricted to approximately 2 Kbp from parS, highlighting the necessity for additional mechanisms beyond ParB sliding over DNA for concentrating ParB into condensates nucleated at parS. Finally, explicit simulations of a polymer coated with bound ParB suggest a dominant role for ParB-ParB interactions in DNA compaction within ParB condensates.

ISW1a modulates cohesin distribution in centromeric and pericentromeric regions.

Cohesin is a highly conserved, multiprotein complex whose canonical function is to hold sister chromatids together to ensure accurate chromosome segregation. Cohesin association with chromatin relies on the Scc2-Scc4 cohesin loading complex that enables cohesin ring opening and topological entrapment of sister DNAs. To better understand how sister chromatid cohesion is regulated, we performed a proteomic screen in budding yeast that identified the Isw1 chromatin remodeler as a cohesin binding partner. In addition, we found that Isw1 also interacts with Scc2-Scc4. Lack of Isw1 protein, the Ioc3 subunit of ISW1a or Isw1 chromatin remodeling activity resulted in increased accumulation of cohesin at centromeres and pericentromeres, suggesting that ISW1a may promote efficient translocation of cohesin from the centromeric site of loading to neighboring regions. Consistent with the role of ISW1a in the chromatin organization of centromeric regions, Isw1 was found to be recruited to centromeres. In its absence we observed changes in the nucleosomal landscape at centromeres and pericentromeres. Finally, we discovered that upon loss of RSC functionality, ISW1a activity leads to reduced cohesin binding and cohesion defect. Taken together, our results support the notion of a key role of chromatin remodelers in the regulation of cohesin distribution on chromosomes.

Atypical low-copy number plasmid segregation systems, all in one?

Plasmid families harbor different maintenances functions, depending on their size and copy number. Low copy number plasmids rely on active partition systems, organizing a partition complex at specific centromere sites that is actively positioned using NTPase proteins. Some low copy number plasmids lack an active partition system, but carry atypical intracellular positioning systems using a single protein that binds to the centromere site but without an associated NTPase. These systems have been studied in the case of the Escherichia coli R388 and of the Staphylococcus aureus pSK1 plasmids. Here we review these two systems, which appear to be unrelated but share common features, such as their distribution on plasmids of medium size and copy number, certain activities of their centromere-binding proteins, StbA and Par, respectively, as well as their mode of action, which may involve dynamic interactions with the nucleoid-packed chromosome of their hosts.

Karyotyping of commercial cultivars of melon (Cucumis melo L.)

This study on cultivars of melon (Cucumis melo L.) marketed in Brazil was conducted to obtain information to be used in breeding programs of this species. Little is known about the karyotype variability among C. melo L. cultivars targeted at the consumer market. The objective of the present study was to verify the karyotype variability in eight commercial melon cultivars used in the Brazilian market. Slides were stained with 2% Giemsa and assembled with Neomount to perform chromosomal morphometry. GC-rich heterochromatin was observed by CMA3/DAPI staining. 5S rDNA, centromeric satellite DNA (SatDNA), and telomeric sites were visualized using fluorescence in situ hybridization. All images were captured on an Olympus BX41 microscope equipped with a 5M Olympus DP25 digital camera and DP2-BSW software. The cultivars showed symmetrical karyotypes with significant differences in total chromosome length and average chromosome size. Heterochromatic CMA3+ blocks were observed in terminal regions related to satellites (secondary constrictions), as well as in centromeric and pericentromeric regions. A single chromosomal pair of 5S rDNA sites was observed in all cultivars, but at distinct locations. Centromeric satellite sequences, tested for the first time in melon, revealed only centromeric sites. Telomeric sites were observed in all the chromosomes of the cultivars. Karyotype variation was observed in cultivars of melon, which were analyzed for chromosomal morphology and localization of GC-rich heterochromatin, as well centromeric SatDNA, rDNA, and telomeric chromosomal markers. Hence, these cultivars can be used in future breeding programs.

Alpha Satellite Insertion Close to an Ancestral Centromeric Region.

Human centromeres are mainly composed of alpha satellite DNA hierarchically organized as higher-order repeats (HORs). Alpha satellite dynamics is shown by sequence homogenization in centromeric arrays and by its transfer to other centromeric locations, for example, during the maturation of new centromeres. We identified during prenatal aneuploidy diagnosis by fluorescent in situ hybridization a de novo insertion of alpha satellite DNA from the centromere of chromosome 18 (D18Z1) into cytoband 15q26. Although bound by CENP-B, this locus did not acquire centromeric functionality as demonstrated by the lack of constriction and the absence of CENP-A binding. The insertion was associated with a 2.8-kbp deletion and likely occurred in the paternal germline. The site was enriched in long terminal repeats and located ∼10 Mbp from the location where a centromere was ancestrally seeded and became inactive in the common ancestor of humans and apes 20–25 million years ago. Long-read mapping to the T2T-CHM13 human genome assembly revealed that the insertion derives from a specific region of chromosome 18 centromeric 12-mer HOR array in which the monomer size follows a regular pattern. The rearrangement did not directly disrupt any gene or predicted regulatory element and did not alter the methylation status of the surrounding region, consistent with the absence of phenotypic consequences in the carrier. This case demonstrates a likely rare but new class of structural variation that we name “alpha satellite insertion.” It also expands our knowledge on alphoid DNA dynamics and conveys the possibility that alphoid arrays can relocate near vestigial centromeric sites.

Three ParA Dimers Cooperatively Assemble on Type Ia Partition Promoters.

Accurate DNA segregation is essential for faithful inheritance of genetic material. In bacteria, this process is mainly ensured by partition systems composed of two proteins, ParA and ParB, and a centromere site. Auto-regulation of Par operon expression is important for efficient partitioning and is primarily mediated by ParA for type Ia plasmid partition systems. For the F-plasmid, four ParAF monomers were proposed to bind to four repeated sequences in the promoter region. By contrast, using quantitative surface-plasmon-resonance, we showed that three ParAF dimers bind to this region. We uncovered that one perfect inverted repeat (IR) motif, consisting of two hexamer sequences spaced by 28-bp, constitutes the primary ParAF DNA binding site. A similar but degenerated motif overlaps the former. ParAF binding to these motifs is well supported by biochemical and modeling analyses. Molecular dynamics simulations predict that the winged-HTH domain displays high flexibility, which may favor the cooperative ParA binding to the promoter. We propose that three ParAF dimers bind cooperatively to overlapping motifs, thus covering the promoter region. A similar organization is found on closely related and distant plasmid partition systems, suggesting that such promoter organization for auto-regulated Par operons is widespread and may have evolved from a common ancestor.

XerD unloads bacterial SMC complexes at the replication terminus

Bacillus subtilis structural maintenance of chromosomes (SMC) complexes are topologically loaded at centromeric sites adjacent to the replication origin by the partitioning protein ParB. These ring-shaped ATPases then translocate down the left and right chromosome arms while tethering them together. Here, we show that the site-specific recombinase XerD, which resolves chromosome dimers, is required to unload SMC tethers when they reach the terminus. We identify XerD-specific binding sites in the terminus region and show that they dictate the site of unloading in a manner that depends on XerD but not its catalytic residue, its partner protein XerC, or the recombination site dif. Finally, we provide evidence that ParB and XerD homologs perform similar functions in Staphylococcus aureus. Thus, two broadly conserved factors that act at the origin and terminus have second functions in loading and unloading SMC complexes that travel between them.

Physical Modeling of a Sliding Clamp Mechanism for the Spreading of ParB at Short Genomic Distance from Bacterial Centromere Sites.

SummaryBacterial ParB partitioning proteins involved in chromosomes and low-copy-number plasmid segregation are cytosine triphosphate (CTP)-dependent molecular switches. CTP-binding converts ParB dimers to DNA clamps, allowing unidimensional diffusion along the DNA. This sliding property has been proposed to explain the ParB spreading over large distances from parS centromere sites where ParB is specifically loaded. We modeled such a “clamping and sliding” mechanism as a typical reaction-diffusion system, compared it to the F plasmid ParB DNA binding pattern, and found that it can account neither for the long range of ParB binding to DNA nor for the rapid assembly kinetics observed in vivo after parS duplication. Also, it predicts a strong effect on the F plasmid ParB binding pattern from the presence of a roadblock that is not observed in ChIP-sequencing (ChIP-seq). We conclude that although “clamping and sliding” can occur at short distances from parS, another mechanism must apply for ParB recruitment at larger genomic distances.

Formation of the CenH3-Deficient Holocentromere in Lepidoptera Avoids Active Chromatin

Despite the essentiality for faithful chromosome segregation, centromere architectures are diverse among eukaryotes1,2 and embody two main configurations: mono- and holocentromeres, referring, respectively, to localized or unrestricted distribution of centromeric activity. Of the two, some holocentromeres offer the curious condition of having arisen independently in multiple insects, most of which have lost the otherwise essential centromere-specifying factor CenH33 (first described as CENP-A in humans).4-7 The loss of CenH3 raises intuitive questions about how holocentromeres are organized and regulated in CenH3-lacking insects. Here, we report the first chromatin-level description of CenH3-deficient holocentromeres by leveraging recently identified centromere components6,7 and genomics approaches to map and characterize the holocentromeres of the silk moth Bombyx mori, a representative lepidopteran insect lacking CenH3. This uncovered a robust correlation between the distribution of centromere sites and regions of low chromatin activity along B.mori chromosomes. Transcriptional perturbation experiments recapitulated the exclusion of B.mori centromeres from active chromatin. Based on reciprocal centromere occupancy patterns observed along differentially expressed orthologous genes of Lepidoptera, we further found that holocentromere formation in a manner that is recessive to chromatin dynamics is evolutionarily conserved. Our results help us discuss the plasticity of centromeres in the context of a role for the chromosome-wide chromatin landscape in conferring centromere identity rather than the presence of CenH3. Given the co-occurrence of CenH3 loss and holocentricity in insects,7 we further propose that the evolutionary establishment of holocentromeres in insects was facilitated through the loss of a CenH3-specified centromere.

Addressing the role of centromere sites in activation of ParB proteins for partition complex assembly.

The ParB-parS partition complexes that bacterial replicons use to ensure their faithful inheritance also find employment in visualization of DNA loci, as less intrusive alternatives to fluorescent repressor-operator systems. The ability of ParB molecules to interact via their N-terminal domains and to bind to non-specific DNA enables expansion of the initial complex to a size both functional in partition and, via fusion to fluorescent peptides, visible by light microscopy. We have investigated whether it is possible to dispense with the need to insert parS in the genomic locus of interest, by determining whether ParB fused to proteins that bind specifically to natural DNA sequences can still assemble visible complexes. In yeast cells, coproduction of fusions of ParB to a fluorescent peptide and to a TALE protein targeting an endogenous sequence did not yield visible foci; nor did any of several variants of these components. In E.coli, coproduction of fusions of SopB (F plasmid ParB) to fluorescent peptide, and to dCas9 together with specific guide RNAs, likewise yielded no foci. The result of coproducing analogous fusions of SopB proteins with distinct binding specificities was also negative. Our observations imply that in order to assemble higher order partition complexes, ParB proteins need specific activation through binding to their cognate parS sites.

Physical Modeling of a Sliding Clamp Mechanism for the Spreading of ParB at Short Genomic Distance from Bacterial Centromere Sites

Bacterial ParB partitioning proteins involved in chromosomes and low-copy-number plasmid segregation have recently been shown to belong to a new class of CTP-dependent molecular switches. Strikingly, CTP binding and hydrolysis was shown to induce a conformational change enabling ParB dimers to switch between an open and a closed conformation. This latter conformation clamps ParB dimers on DNA molecules, allowing their diffusion in one dimension along the DNA. It has been proposed that this novel sliding property may explain the spreading capability of ParB over more than 10-Kb from parS centromere sites where ParB is specifically loaded. Here, we modeled such a mechanism as a typical reaction-diffusion system and compared this ‘Clamping & sliding’ model to the ParB DNA binding pattern from high-resolution ChIP-sequencing data. We found that this mechanism can not account for all the in vivo characteristics, especially the long range of ParB binding to DNA. In particular, it predicts a strong effect of the presence of a roadblock on the ParB binding pattern that is not observed in ChIP-seq. Moreover, the rapid assembly kinetics observed in vivo after the duplication of parS sites is not easily explained by this mechanism. We propose that ‘Clamping & sliding’ might explain the ParB spreading pattern at short distances from parS but that another mechanism must apply for ParB recruitment at larger genomic distances.

Phosphorylation of Drosophila CENP-A on serine 20 regulates protein turn-over and centromere-specific loading.

Centromeres are specialized chromosomal regions epigenetically defined by the presence of the histone H3 variant CENP-A. CENP-A is required for kinetochore formation which is essential for chromosome segregation during mitosis. Spatial restriction of CENP-A to the centromere is tightly controlled. Its overexpression results in ectopic incorporation and the formation of potentially deleterious neocentromeres in yeast, flies and in various human cancers. While the contribution of posttranslational modifications of CENP-A to these processes has been studied in yeast and mammals to some extent, very little is known about Drosophila melanogaster. Here, we show that CENP-A is phosphorylated at serine 20 (S20) by casein kinase II and that in mitotic cells, the phosphorylated form is enriched on chromatin. Importantly, our results reveal that S20 phosphorylation regulates the turn-over of prenucleosomal CENP-A by the SCFPpa-proteasome pathway and that phosphorylation promotes removal of CENP-A from ectopic but not from centromeric sites in chromatin. We provide multiple lines of evidence for a crucial role of S20 phosphorylation in controlling restricted incorporation of CENP-A into centromeric chromatin in flies. Modulation of the phosphorylation state of S20 may provide the cells with a means to fine-tune CENP-A levels in order to prevent deleterious loading to extra-centromeric sites.

CDK phosphorylation of Xenopus laevis M18BP1 promotes its metaphase centromere localization.

Chromosome segregation requires the centromere, the site on chromosomes where kinetochores assemble in mitosis to attach chromosomes to the mitotic spindle. Centromere identity is defined epigenetically by the presence of nucleosomes containing the histone H3 variant CENP-A. New CENP-A nucleosome assembly occurs at the centromere every cell cycle during G1, but how CENP-A nucleosome assembly is spatially and temporally restricted remains poorly understood. Centromere recruitment of factors required for CENP-A assembly is mediated in part by the three-protein Mis18 complex (Mis18α, Mis18β, M18BP1). Here, we show that Xenopus M18BP1 localizes to centromeres during metaphase-prior to CENP-A assembly-by binding to CENP-C using a highly conserved SANTA domain. We find that Cdk phosphorylation of M18BP1 is necessary for M18BP1 to bind CENP-C and localize to centromeres in metaphase. Surprisingly, mutations which disrupt the metaphase M18BP1/CENP-C interaction cause defective nuclear localization of M18BP1 in interphase, resulting in defective CENP-A nucleosome assembly. We propose that M18BP1 may identify centromeric sites in metaphase for subsequent CENP-A nucleosome assembly in interphase.

A conserved mechanism drives partition complex assembly on bacterial chromosomes and plasmids

Chromosome and plasmid segregation in bacteria are mostly driven by ParABS systems. These DNA partitioning machineries rely on large nucleoprotein complexes assembled on centromere sites (parS). However, the mechanism of how a few parS‐bound ParB proteins nucleate the formation of highly concentrated ParB clusters remains unclear despite several proposed physico‐mathematical models. We discriminated between these different models by varying some key parameters in vivo using the F plasmid partition system. We found that “Nucleation & caging” is the only coherent model recapitulating in vivo data. We also showed that the stochastic self‐assembly of partition complexes (i) is a robust mechanism, (ii) does not directly involve ParA ATPase, (iii) results in a dynamic structure of discrete size independent of ParB concentration, and (iv) is not perturbed by active transcription but is by protein complexes. We refined the “Nucleation & caging” model and successfully applied it to the chromosomally encoded Par system of Vibrio cholerae, indicating that this stochastic self‐assembly mechanism is widely conserved from plasmids to chromosomes.

The TubR-centromere complex adopts a double-ring segrosome structure in Type III partition systems.

In prokaryotes, the centromere is a specialized segment of DNA that promotes the assembly of the segrosome upon binding of the Centromere Binding Protein (CBP). The segrosome structure exposes a specific surface for the interaction of the CBP with the motor protein that mediates DNA movement during cell division. Additionally, the CBP usually controls the transcriptional regulation of the segregation system as a cell cycle checkpoint. Correct segrosome functioning is therefore indispensable for accurate DNA segregation. Here, we combine biochemical reconstruction and structural and biophysical analysis to bring light to the architecture of the segrosome complex in Type III partition systems. We present the particular features of the centromere site, tubC, of the model system encoded in Clostridium botulinum prophage c-st. We find that the split centromere site contains two different iterons involved in the binding and spreading of the CBP, TubR. The resulting nucleoprotein complex consists of a novel double-ring structure that covers part of the predicted promoter. Single molecule data provides a mechanism for the formation of the segrosome structure based on DNA bending and unwinding upon TubR binding.

Plasmid-Encoded RepA Proteins Specifically Autorepress Individual repABC Operons in the Multipartite Rhizobium leguminosarum bv. trifolii Genome.

Rhizobia commonly have very complex genomes with a chromosome and several large plasmids that possess genes belonging to the repABC family. RepA and RepB are members of the ParA and ParB families of partitioning proteins, respectively, whereas RepC is crucial for plasmid replication. In the repABC replicons, partitioning and replication functions are transcriptionally linked resulting in complex regulation of rep gene expression. The genome of R. leguminosarum bv. trifolii TA1 (RtTA1) consists of a chromosome and four plasmids (pRleTA1a-d), equipped with functional repABC genes. In this work, the regulation of transcription of the individual repABC cassettes of the four RtTA1 plasmids was studied. The involvement of the RepA and RepB as well as parS-like centromere sites in this process was depicted, demonstrating some dissimilarity in expression of respective rep regions. RtTA1 repABC genes of individual plasmids formed operons, which were negatively regulated by RepA and RepB. Individual RepA were able to bind to DNA without added nucleotides, but in the presence of ADP, bound specifically to their own operator sequences containing imperfect palindromes, and caused operon autorepression, whereas the addition of ATP stimulated non-specific binding of RepA to DNA. The RepA proteins were able to dimerize/oligomerize: in general dimers formed independently of ATP or ADP, although ATP diminished the concentration of oligomers that were produced. By the comprehensive approach focusing on a set of plasmids instead of individual replicons, the work highlighted subtle differences between the organization and regulation of particular rep operons as well as the structures and specificity of RepA proteins, which contribute to the fine-tuned coexistence of several replicons with similar repABC cassettes in the complex bacterial genome.

Holocentromeres are dispersed point centromeres localized at transcription factor hotspots

Centromeres vary greatly in size and sequence composition, ranging from 'point' centromeres with a single cenH3-containing nucleosome to 'regional' centromeres embedded in tandemly repeated sequences to holocentromeres that extend along the length of entire chromosomes. Point centromeres are defined by sequence, whereas regional and holocentromeres are epigenetically defined by the location of cenH3-containing nucleosomes. In this study, we show that Caenorhabditis elegans holocentromeres are organized as dispersed but discretely localized point centromeres, each forming a single cenH3-containing nucleosome. These centromeric sites co-localize with kinetochore components, and their occupancy is dependent on the cenH3 loading machinery. These sites coincide with non-specific binding sites for multiple transcription factors ('HOT' sites), which become occupied when cenH3 is lost. Our results show that the point centromere is the basic unit of holocentric organization in support of the classical polycentric model for holocentromeres, and provide a mechanistic basis for understanding how centromeric chromatin might be maintained. DOI: http://dx.doi.org/10.7554/eLife.02025.001.

PCGR2 copy number depends on the par locus that forms a ParC-ParB-DNA partition complex in Corynebacterium glutamicum

To characterize the par system of Corynebacterium glutamicum pCGR2 and to manipulate the par components to effectively manipulate plasmid copy number. ParB binds sequence specifically to centromere-binding sites around the parAB operon and serves as an autorepressor. A small ORF (orf4, later named parC) downstream of parAB encodes a protein with 23.7% sequence identity with ParB. ParB is also implicated in the repression of parC transcription. Nonetheless, this ParC protein does not bind to centromere-binding sites and is not essential for plasmid stability. Introduction of a frameshift mutation within ParC implicated the protein in regulation of both parAB and parC. Electrophoretic Mobility Shift Assay confirmed a previously unreported ParC-ParB-parS partition complex. ParC also interacts directly with ParB without the mediation of the centromere sites. Deletion of the par components resulted in different plasmid copy numbers. A previously unreported ParC-ParB-parS partition complex is formed in pCGR2, where interaction of ParC with ParB-parS may affect the level of repression by ParB. Modifying the par components and antisense RNA enables manipulation of plasmid copy number to varying degrees. Genetically manipulating the par components, in combination with deactivation of antisense RNA, is a novel approach to artificially elevate plasmid copy number. This approach can be applied for development of new genetic engineering tools.

De novo centromere formation on a chromosome fragment in maize

The centromere is the part of the chromosome that organizes the kinetochore, which mediates chromosome movement during mitosis and meiosis. A small fragment from chromosome 3, named Duplication 3a (Dp3a), was described from UV-irradiated materials by Stadler and Roman in the 1940s [Stadler LJ, Roman H (1948) Genetics 33(3):273-303]. The genetic behavior of Dp3a is reminiscent of a ring chromosome, but fluoresecent in situ hybridization detected telomeres at both ends, suggesting a linear structure. This small chromosome has no detectable canonical centromeric sequences, but contains a site with protein features of functional centromeres such as CENH3, the centromere specific H3 histone variant, and CENP-C, a foundational kinetochore protein, suggesting the de novo formation of a centromere on the chromatin fragment. To examine the sequences associated with CENH3, chromatin immunoprecipitation was carried out with anti-CENH3 antibodies using material from young seedlings with and without the Dp3a chromosome. A novel peak was detected from the ChIP-Sequencing reads of the Dp3a sample. The peak spanned 350 kb within the long arm of chromosome 3 covering 22 genes. Collectively, these results define the behavior and molecular features of de novo centromere formation in the Dp3a chromosome, which may shed light on the initiation of new centromere sites during evolution.

Repetitive sequences associated with differentiation of W chromosome in Semaprochilodus taeniurus

The possible origins and differentiation of a ZZ/ZW sex chromosome system in Semaprochilodus taeniurus, the only species of the family Prochilodontidae known to possess heteromorphic sex chromosomes, were examined by conventional (C-banding) and molecular (cross-species hybridization of W-specific WCP, Fluorescence in situ hybridization (FISH) with telomere (TTAGGG)n, and Rex1 probes) cytogenetic protocols. Several segments obtained by W-specific probe were cloned, and the sequences localized on the W chromosome were identified by DNA sequencing and search of nucleotide collections of the NCBI and GIRI using BLAST and CENSOR, respectively. Blocks of constitutive heterochromatin in chromosomes of S. taeniurus were observed in the centromere of all autosomal chromosomes and in the terminal, interstitial, and pericentromeric regions of the W chromosome, which did not demonstrate interstitial telomeric sites with FISH of the telomere probe. The Rex1 probe displayed a compartmentalized distribution pattern in some chromosomes and showed signs of invasion of the pericentromeric region in the W chromosome. Chromosomal painting with the W-specific WCP of S. taeniurus onto its own chromosomes showed complete staining of the W chromosome, centromeric sites, and the ends of the Z chromosome, as well as other autosomes. However, cross-species painting using this WCP on chromosomes of S. insignis, Prochilodus lineatus, and P. nigricans did not reveal a proto-W element, but instead demonstrated scattered positive signals of repetitive DNAs. Identification of the W-specific repetitive sequences showed high similarity to microsatellites and transposable elements. Classes of repetitive DNA identified in the W chromosome suggested that the genetic degeneration of this chromosome in S. taeniurus occurred through accumulation of these repetitive DNAs.