Centromeres as universal hotspots of DNA breakage, driving RAD51-mediated recombination during quiescence.

Centromeres are essential for chromosome segregation in most animals and plants yet are among the most rapidly evolving genome elements. The mechanisms underlying this paradoxical phenomenon remain enigmatic. Here, we report that human centromeres innately harbor a striking enrichment of DNA breaks within functionally active centromere regions. Establishing a single-cell imaging strategy that enables comparative assessment of DNA breaks at repetitive regions, we show that centromeric DNA breaks are induced not only during active cellular proliferation but also de novo during quiescence. Markedly, centromere DNA breaks in quiescent cells are resolved enzymatically by the evolutionarily conserved RAD51 recombinase, which in turn safeguards the specification of functional centromeres. This study highlights the innate fragility of centromeres, which may have been co-opted over time to reinforce centromere specification while driving rapid evolution. The findings also provide insights into how fragile centromeres are likely to contribute to human disease.

DNA strand breaks at centromeres: Friend or foe?

Centromeres are large structural regions in the genomic DNA, which are essential for accurately transmitting a complete set of chromosomes to daughter cells during cell division. In humans, centromeres consist of highly repetitive α-satellite DNA sequences and unique epigenetic components, forming large proteinaceous structures required for chromosome segregation. Despite their biological importance, there is a growing body of evidence for centromere breakage across the cell cycle, including periods of quiescence. In this review, we provide an up-to-date examination of the distinct centromere environments at different stages of the cell cycle, highlighting their plausible contribution to centromere breakage. Additionally, we explore the implications of these breaks on centromere function, both in terms of negative consequences and potential positive effects.

Emerging roles of DNA repair factors in the stability of centromeres.

Satellite DNA sequences are an integral part of centromeres, regions critical for faithful segregation of chromosomes during cell division. Because of their complex repetitive structure, satellite DNA may act as a barrier to DNA replication and other DNA based transactions ultimately resulting in chromosome breakage. Over the past two decades, several DNA repair proteins have been shown to bind and function at centromeres. While the importance of these repair factors is highlighted by various structural and numerical chromosome aberrations resulting from their inactivation, their roles in helping to maintain genome stability by solving the intrinsic difficulties of satellite DNA replication or promoting their repair are just starting to emerge. In this review, we summarize the current knowledge on the role of DNA repair and DNA damage response proteins in maintaining the structure and function of centromeres in different contexts. We also report the recent connection between the roles of specific DNA repair factors at these genomic loci with age-related increase of chromosomal instability under physiological and pathological conditions.

Genome-wide Control of Heterochromatin Replication by the Telomere Capping Protein TRF2.

Hard-to-replicate regions of chromosomes (e.g., pericentromeres, centromeres, and telomeres) impede replication fork progression, eventually leading, in the event of replication stress, to chromosome fragility, aging, and cancer. Our knowledge of the mechanisms controlling the stability of these regions is essentially limited to telomeres, where fragility is counteracted by the shelterin proteins. Here we show that the shelterin subunit TRF2 ensures progression of the replication fork through pericentromeric heterochromatin, but not centromeric chromatin. In a process involving its N-terminal basic domain, TRF2 binds to pericentromeric Satellite III sequences during S phase, allowing the recruitment of the G-quadruplex-resolving helicase RTEL1 to facilitate fork progression. We also show that TRF2 is required for the stability of other heterochromatic regions localized throughout the genome, paving the way for future research on heterochromatic replication and its relationship with aging and cancer.

Unexpected conformational variations of the human centromeric chromatin complex.

We combined classical salt fractionation with chromatin immunoprecipitation to recover human centromeric chromatin under native conditions. We found that >85% of the total centromeric chromatin is insoluble under conditions typically used for native chromatin extraction. To map both soluble and insoluble chromatin in situ, we combined CUT&RUN (cleavage under targets and release using nuclease), a targeted nuclease method, with salt fractionation. Using this approach, we observed unexpected structural and conformational variations of centromere protein A (CENP-A)-containing complexes on different α-satellite dimeric units within highly homogenous arrays. Our results suggest that slight α-satellite sequence differences control the structure and occupancy of the associated centromeric chromatin complex.

SUMOylation regulates Lem2 function in centromere clustering and silencing.

Regulation by the small modifier SUMO is heavily dependent on spatial control of enzymes that mediate the attachment and removal of SUMO on substrate proteins. Here, we show that in the fission yeast Schizosaccharomyces pombe, delocalisation of the SUMO protease Ulp1 from the nuclear envelope results in centromeric defects that can be attributed to hyper-SUMOylation at the nuclear periphery. Unexpectedly, we find that although this localised hyper-SUMOylation impairs centromeric silencing, it can also enhance centromere clustering. Moreover, both effects are at least partially dependent on SUMOylation of the inner nuclear membrane protein Lem2. Lem2 has previously been implicated in diverse biological processes, including the promotion of both centromere clustering and silencing, but how these distinct activities are coordinated was unclear; our observations suggest a model whereby SUMOylation serves as a regulatory switch, modulating Lem2 interactions with competing partner proteins to balance its roles in alternative pathways. Our findings also reveal a previously unappreciated role for SUMOylation in promoting centromere clustering.

A New Chromosome-Assigned Mongolian Gerbil Genome Allows Characterization of Complete Centromeres and a Fully Heterochromatic Chromosome.

Chromosome-scale genome assemblies based on ultralong-read sequencing technologies are able to illuminate previously intractable aspects of genome biology such as fine-scale centromere structure and large-scale variation in genome features such as heterochromatin, GC content, recombination rate, and gene content. We present here a new chromosome-scale genome of the Mongolian gerbil (Meriones unguiculatus), which includes the complete sequence of all centromeres. Gerbils are thus the one of the first vertebrates to have their centromeres completely sequenced. Gerbil centromeres are composed of four different repeats of length 6, 37, 127, or 1,747 bp, which occur in simple alternating arrays and span 1-6 Mb. Gerbil genomes have both an extensive set of GC-rich genes and chromosomes strikingly enriched for constitutive heterochromatin. We sought to determine if there was a link between these two phenomena and found that the two heterochromatic chromosomes of the Mongolian gerbil have distinct underpinnings: Chromosome 5 has a large block of intraarm heterochromatin as the result of a massive expansion of centromeric repeats, while chromosome 13 is comprised of extremely large (>150 kb) repeated sequences. In addition to characterizing centromeres, our results demonstrate the importance of including karyotypic features such as chromosome number and the locations of centromeres in the interpretation of genome sequence data and highlight novel patterns involved in the evolution of chromosomes.

Structural basis for centromere maintenance by Drosophila CENP-A chaperone CAL1.

Centromeres are microtubule attachment sites on chromosomes defined by the enrichment of histone variant CENP-A-containing nucleosomes. To preserve centromere identity, CENP-A must be escorted to centromeres by a CENP-A-specific chaperone for deposition. Despite this essential requirement, many eukaryotes differ in the composition of players involved in centromere maintenance, highlighting the plasticity of this process. In humans, CENP-A recognition and centromere targeting are achieved by HJURP and the Mis18 complex, respectively. Using X-ray crystallography, we here show how Drosophila CAL1, an evolutionarily distinct CENP-A histone chaperone, binds both CENP-A and the centromere receptor CENP-C without the requirement for the Mis18 complex. While an N-terminal CAL1 fragment wraps around CENP-A/H4 through multiple physical contacts, a C-terminal CAL1 fragment directly binds a CENP-C cupin domain dimer. Although divergent at the primary structure level, CAL1 thus binds CENP-A/H4 using evolutionarily conserved and adaptive structural principles. The CAL1 binding site on CENP-C is strategically positioned near the cupin dimerisation interface, restricting binding to just one CAL1 molecule per CENP-C dimer. Overall, by demonstrating how CAL1 binds CENP-A/H4 and CENP-C, we provide key insights into the minimalistic principles underlying centromere maintenance.

Plant chromosome engineering - past, present and future.

Spontaneous chromosomal rearrangements (CRs) play an essential role in speciation, genome evolution and crop domestication. To be able to use the potential of CRs for breeding, plant chromosome engineering was initiated by fragmenting chromosomes by X-ray irradiation. With the rise of the CRISPR/Cas system, it became possible to induce double-strand breaks (DSBs) in a highly efficient manner at will at any chromosomal position. This has enabled a completely new level of predesigned chromosome engineering. The genetic linkage between specific genes can be broken by inducing chromosomal translocations. Natural inversions, which suppress genetic exchange, can be reverted for breeding. In addition, various approaches for constructing minichromosomes by downsizing regular standard A or supernumerary B chromosomes, which could serve as future vectors in plant biotechnology, have been developed. Recently, a functional synthetic centromere could be constructed. Also, different ways of genome haploidization have been set up, some based on centromere manipulations. In the future, we expect to see even more complex rearrangements, which can be combined with previously developed engineering technologies such as recombinases. Chromosome engineering might help to redefine genetic linkage groups, change the number of chromosomes, stack beneficial genes on mini cargo chromosomes, or set up genetic isolation to avoid outcrossing.

Germline ecology: managed herds, tolerated flocks, and pest control.

Multicopy sequences evolve adaptations for increasing their copy number within nuclei. The activities of multicopy sequences under constraints imposed by cellular and organismal selection result in a rich intranuclear ecology in germline cells. mtDNA and rDNA are managed as domestic herds subject to selective breeding by the genes of the single-copy genome. Transposable elements lead a peripatetic existence in which they must continually move to new sites to keep ahead of inactivating mutations at old sites and undergo exponential outbreaks when the production of new copies exceeds the rate of inactivation of old copies. Centromeres become populated by repeats that do little harm. Organisms with late sequestration of germ cells tend to evolve more 'junk' in their genomes than organisms with early sequestration of germ cells.

Mega-sized pericentromeric blocks of simple telomeric repeats and their variants reveal patterns of chromosome evolution in ancient Cycadales genomes.

Simple telomeric repeats composed of six to seven iterating nucleotide units are important sequences typically found at the ends of chromosomes. Here we analyzed their abundance and homogeneity in 42 gymnosperm (29 newly sequenced), 29 angiosperm (one newly sequenced), and eight bryophytes using bioinformatics, conventional cytogenetic and molecular biology approaches to explore their diversity across land plants. We found more than 10 000-fold variation in the amounts of telomeric repeats among the investigated taxa. Repeat abundance was positively correlated with increasing intragenomic sequence heterogeneity and occurrence at non-telomeric positions, but there was no correlation with genome size. The highest abundance/heterogeneity was found in the gymnosperm genus Cycas (Cycadaceae), in which megabase-sized blocks of telomeric repeats (i.e., billions of copies) were identified. Fluorescent in situ hybridization experiments using variant-specific probes revealed canonical Arabidopsis-type telomeric TTTAGGG repeats at chromosome ends, while pericentromeric blocks comprised at least four major telomeric variants with decreasing abundance: TTTAGGG>TTCAGGG >TTTAAGG>TTCAAGG. Such a diversity of repeats was not found in the sister cycad family Zamiaceae or in any other species analyzed. Using immunocytochemistry, we showed that the pericentromeric blocks of telomeric repeats overlapped with histone H3 serine 10 phosphorylation signals. We show that species of Cycas have amplified their telomeric repeats in centromeric and telomeric positions on telocentric chromosomes to extraordinary high levels. The ancestral chromosome number reconstruction suggests their occurrence is unlikely to be the product of ancient Robertsonian chromosome fusions. We speculate as to how the observed chromosome dynamics may be associated with the diversification of cycads.

Mutation Load in Sunflower Inversions Is Negatively Correlated with Inversion Heterozygosity.

Recombination is critical both for accelerating adaptation and purging deleterious mutations. Chromosomal inversions can act as recombination modifiers that suppress local recombination in heterozygotes and thus, under some conditions, are predicted to accumulate such mutations. In this study, we investigated patterns of recombination, transposable element abundance, and coding sequence evolution across the genomes of 1,445 individuals from three sunflower species, as well as within nine inversions segregating within species. We also analyzed the effects of inversion genotypes on 87 phenotypic traits to test for overdominance. We found significant negative correlations of long terminal repeat retrotransposon abundance and deleterious mutations with recombination rates across the genome in all three species. However, we failed to detect an increase in these features in the inversions, except for a modest increase in the proportion of stop codon mutations in several very large or rare inversions. Consistent with this finding, there was little evidence of overdominance of inversions in phenotypes that may relate to fitness. On the other hand, significantly greater load was observed for inversions in populations polymorphic for a given inversion compared to populations monomorphic for one of the arrangements, suggesting that the local state of inversion polymorphism affects deleterious load. These seemingly contradictory results can be explained by the low frequency of inversion heterozygotes in wild sunflower populations, apparently due to divergent selection and associated geographic structure. Inversions contributing to local adaptation represent ideal recombination modifiers, acting to facilitate adaptive divergence with gene flow, while largely escaping the accumulation of deleterious mutations.

A near-complete genome assembly of Brassica rapa provides new insights into the evolution of centromeres.

Brassica rapa comprises many important cultivated vegetables and oil crops. However, Chiifu v3.0, the current B. rapa reference genome, still contains hundreds of gaps. Here, we presented a near-complete genome assembly of B. rapa Chiifu v4.0, which was 424.59 Mb with only two gaps, using Oxford Nanopore Technology (ONT) ultralong-read sequencing and Hi-C technologies. The new assembly contains 12 contigs, with a contig N50 of 38.26 Mb. Eight of the ten chromosomes were entirely reconstructed in a single contig from telomere to telomere. We found that the centromeres were mainly invaded by ALE and CRM long terminal repeats (LTRs). Moreover, there is a high divergence of centromere length and sequence among B. rapa genomes. We further found that centromeres are enriched for Copia invaded at 0.14 MYA on average, while pericentromeres are enriched for Gypsy LTRs invaded at 0.51 MYA on average. These results indicated the different invasion mechanisms of LTRs between the two structures. In addition, a novel repetitive sequence PCR630 was identified in the pericentromeres of B. rapa. Overall, the near-complete genome assembly, B. rapa Chiifu v4.0, offers valuable tools for genomic and genetic studies of Brassica species and provides new insights into the evolution of centromeres.

DNA methylation epigenetically silences crossover hot spots and controls chromosomal domains of meiotic recombination in Arabidopsis.

During meiosis, homologous chromosomes undergo crossover recombination, which is typically concentrated in narrow hot spots that are controlled by genetic and epigenetic information. Arabidopsis chromosomes are highly DNA methylated in the repetitive centromeres, which are also crossover-suppressed. Here we demonstrate that RNA-directed DNA methylation is sufficient to locally silence Arabidopsis euchromatic crossover hot spots and is associated with increased nucleosome density and H3K9me2. However, loss of CG DNA methylation maintenance in met1 triggers epigenetic crossover remodeling at the chromosome scale, with pericentromeric decreases and euchromatic increases in recombination. We used recombination mutants that alter interfering and noninterfering crossover repair pathways (fancm and zip4) to demonstrate that remodeling primarily involves redistribution of interfering crossovers. Using whole-genome bisulfite sequencing, we show that crossover remodeling is driven by loss of CG methylation within the centromeric regions. Using cytogenetics, we profiled meiotic DNA double-strand break (DSB) foci in met1 and found them unchanged relative to wild type. We propose that met1 chromosome structure is altered, causing centromere-proximal DSBs to be inhibited from maturation into interfering crossovers. These data demonstrate that DNA methylation is sufficient to silence crossover hot spots and plays a key role in establishing domains of meiotic recombination along chromosomes.

Centromere organization and UU/V sex chromosome behavior in a liverwort.

In 1917, sex chromosomes in plants were discovered in a liverwort with hetermorphic U and V chromosomes. Such heteromorphy is unexpected because, unlike the XY chromosomes in diploid-dominant plants, in haploid-dominant plants the female U and the male V chromosomes experience largely symmetrical potential recombination environments. Here we use molecular cytogenetics and super-resolution microscopy to study Frullania dilatata, a liverwort with one male and two female sex chromosomes. We applied a pipeline to Illumina sequences to detect abundant types of repetitive DNA and developed FISH probes to microscopically distinguish the sex chromosomes. We also determined the phenotypic population sex ratio because biased ratios have been reported from other liverworts with heteromorphic sex chromosomes. Populations had male-biased sex ratios. The sex chromosomes are monocentric, and of 14 probes studied (eight satellites, five transposable elements and one plastid region), four resulted in unique signals that differentiated the sex chromosomes from the autosomes and from each other. One FISH probe selectively marked the centromeres of both U chromosomes, so we could prove that during meiosis each U chromosome associates with one of the opposite telomeres of the V chromosome, resulting in a head-to-head trivalent. The similarity of the two U chromosomes to each other in size and in their centromere FISH signal positions points to their origin via a non-disjunction event (aneuploidy), which would fit with the general picture of sex chromosomes rarely crossing-over and being prone to suffer from non-disjunction.

Comparative Analyses of Gibbon Centromeres Reveal Dynamic Genus-Specific Shifts in Repeat Composition.

Centromeres are functionally conserved chromosomal loci essential for proper chromosome segregation during cell division, yet they show high sequence diversity across species. Despite their variation, a near universal feature of centromeres is the presence of repetitive sequences, such as DNA satellites and transposable elements (TEs). Because of their rapidly evolving karyotypes, gibbons represent a compelling model to investigate divergence of functional centromere sequences across short evolutionary timescales. In this study, we use ChIP-seq, RNA-seq, and fluorescence in situ hybridization to comprehensively investigate the centromeric repeat content of the four extant gibbon genera (Hoolock, Hylobates, Nomascus, and Siamang). In all gibbon genera, we find that CENP-A nucleosomes and the DNA-proteins that interface with the inner kinetochore preferentially bind retroelements of broad classes rather than satellite DNA. A previously identified gibbon-specific composite retrotransposon, LAVA, known to be expanded within the centromere regions of one gibbon genus (Hoolock), displays centromere- and species-specific sequence differences, potentially as a result of its co-option to a centromeric function. When dissecting centromere satellite composition, we discovered the presence of the retroelement-derived macrosatellite SST1 in multiple centromeres of Hoolock, whereas alpha-satellites represent the predominate satellite in the other genera, further suggesting an independent evolutionary trajectory for Hoolock centromeres. Finally, using de novo assembly of centromere sequences, we determined that transcripts originating from gibbon centromeres recapitulate the species-specific TE composition. Combined, our data reveal dynamic shifts in the repeat content that define gibbon centromeres and coincide with the extensive karyotypic diversity within this lineage.

The stem rust fungus Puccinia graminis f. sp. tritici induces centromeric small RNAs during late infection that are associated with genome-wide DNA methylation.

BACKGROUND: Silencing of transposable elements (TEs) is essential for maintaining genome stability. Plants use small RNAs (sRNAs) to direct DNA methylation to TEs (RNA-directed DNA methylation; RdDM). Similar mechanisms of epigenetic silencing in the fungal kingdom have remained elusive. RESULTS: We use sRNA sequencing and methylation data to gain insight into epigenetics in the dikaryotic fungus Puccinia graminis f. sp. tritici (Pgt), which causes the devastating stem rust disease on wheat. We use Hi-C data to define the Pgt centromeres and show that they are repeat-rich regions (~250 kb) that are highly diverse in sequence between haplotypes and, like in plants, are enriched for young TEs. DNA cytosine methylation is particularly active at centromeres but also associated with genome-wide control of young TE insertions. Strikingly, over 90% of Pgt sRNAs and several RNAi genes are differentially expressed during infection. Pgt induces waves of functionally diversified sRNAs during infection. The early wave sRNAs are predominantly 21 nts with a 5' uracil derived from genes. In contrast, the late wave sRNAs are mainly 22-nt sRNAs with a 5' adenine and are strongly induced from centromeric regions. TEs that overlap with late wave sRNAs are more likely to be methylated, both inside and outside the centromeres, and methylated TEs exhibit a silencing effect on nearby genes. CONCLUSIONS: We conclude that rust fungi use an epigenetic silencing pathway that might have similarity with RdDM in plants. The Pgt RNAi machinery and sRNAs are under tight temporal control throughout infection and might ensure genome stability during sporulation.

Characterization of repeat arrays in ultra-long nanopore reads reveals frequent origin of satellite DNA from retrotransposon-derived tandem repeats.

Amplification of monomer sequences into long contiguous arrays is the main feature distinguishing satellite DNA from other tandem repeats, yet it is also the main obstacle in its investigation because these arrays are in principle difficult to assemble. Here we explore an alternative, assembly-free approach that utilizes ultra-long Oxford Nanopore reads to infer the length distribution of satellite repeat arrays, their association with other repeats and the prevailing sequence periodicities. Using the satellite DNA-rich legume plant Lathyrus sativus as a model, we demonstrated this approach by analyzing 11 major satellite repeats using a set of nanopore reads ranging from 30 to over 200 kb in length and representing 0.73× genome coverage. We found surprising differences between the analyzed repeats because only two of them were predominantly organized in long arrays typical for satellite DNA. The remaining nine satellites were found to be derived from short tandem arrays located within LTR-retrotransposons that occasionally expanded in length. While the corresponding LTR-retrotransposons were dispersed across the genome, this array expansion occurred mainly in the primary constrictions of the L. sativus chromosomes, which suggests that these genome regions are favourable for satellite DNA accumulation.

Genetic and epigenetic effects on centromere establishment.

Endogenous chromosomes contain centromeres to direct equal chromosomal segregation in mitosis and meiosis. The location and function of existing centromeres is usually maintained through cell cycles and generations. Recent studies have investigated how the centromere-specific histone H3 variant CENP-A is assembled and replenished after DNA replication to epigenetically propagate the centromere identity. However, existing centromeres occasionally become inactivated, with or without change in underlying DNA sequences, or lost after chromosomal rearrangements, resulting in acentric chromosomes. New centromeres, known as neocentromeres, may form on ectopic, non-centromeric chromosomal regions to rescue acentric chromosomes from being lost, or form dicentric chromosomes if the original centromere is still active. In addition, de novo centromeres can form after chromatinization of purified DNA that is exogenously introduced into cells. Here, we review the phenomena of naturally occurring and experimentally induced new centromeres and summarize the genetic (DNA sequence) and epigenetic features of these new centromeres. We compare the characteristics of new and native centromeres to understand whether there are different requirements for centromere establishment and propagation. Based on our understanding of the mechanisms of new centromere formation, we discuss the perspectives of developing more stably segregating human artificial chromosomes to facilitate gene delivery in therapeutics and research.

Evolutionary Dynamics of Abundant 7-bp Satellites in the Genome of Drosophila virilis.

The factors that drive the rapid changes in abundance of tandem arrays of highly repetitive sequences, known as satellite DNA, are not well understood. Drosophila virilis has one of the highest relative amounts of simple satellites of any organism that has been studied, with an estimated >40% of its genome composed of a few related 7-bp satellites. Here, we use D. virilis as a model to understand technical biases affecting satellite sequencing and the evolutionary processes that drive satellite composition. By analyzing sequencing data from Illumina, PacBio, and Nanopore platforms, we identify platform-specific biases and suggest best practices for accurate characterization of satellites by sequencing. We use comparative genomics and cytogenetics to demonstrate that the highly abundant AAACTAC satellite family arose from a related satellite in the branch leading to the virilis phylad 4.5-11 Ma before exploding in abundance in some species of the clade. The most abundant satellite is conserved in sequence and location in the pericentromeric region but has diverged widely in abundance among species, whereas the satellites nearest the centromere are rapidly turning over in sequence composition. By analyzing multiple strains of D. virilis, we saw that the abundances of two centromere-proximal satellites are anticorrelated along a geographical gradient, which we suggest could be caused by ongoing conflicts at the centromere. In conclusion, we illuminate several key attributes of satellite evolutionary dynamics that we hypothesize to be driven by processes including selection, meiotic drive, and constraints on satellite sequence and abundance.