Centromeres attach chromosomes to spindle microtubules during cell division and, despite this conserved role, show paradoxically rapid evolution and are typified by complex repeats. We used long-read sequencing to generate the Col-CEN Arabidopsis thaliana genome assembly that resolves all five centromeres. The centromeres consist of megabase-scale tandemly repeated satellite arrays, which support CENTROMERE SPECIFIC HISTONE H3 (CENH3) occupancy and are densely DNA methylated, with satellite variants private to each chromosome. CENH3 preferentially occupies satellites that show the least amount of divergence and occur in higher-order repeats. The centromeres are invaded by ATHILA retrotransposons, which disrupt genetic and epigenetic organization. Centromeric crossover recombination is suppressed, yet low levels of meiotic DNA double-strand breaks occur that are regulated by DNA methylation. We propose that Arabidopsis centromeres are evolving through cycles of satellite homogenization and retrotransposon-driven diversification.
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Synthetic maize centromeres transmit chromosomes across generations
Centromeres are long, often repetitive regions of genomes that bind
kinetochore proteins and ensure normal chromosome segregation.
Engineering centromeres that function in vivo has proven to be difficult.
Here we describe a tethering approach that activates functional maize
centromeres at synthetic sequence arrays. A LexA-CENH3 fusion protein
was used to recruit native Centromeric Histone H3 (CENH3) to long arrays of
LexO repeats on a chromosome arm. Newly recruited CENH3 was sufficient
to organize functional kinetochores that caused chromosome breakage,
releasing chromosome fragments that were passed through meiosis and
into progeny. Several fragments formed independent neochromosomes
with centromeres localized over the LexO repeat arrays. The new
centromeres were self-sustaining and transmitted neochromosomes to
subsequent generations in the absence of the LexA-CENH3 activator.
Our results demonstrate the feasibility of using synthetic centromeres
for karyotype engineering applications.
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- Award ID(s):
- 2040218
- NSF-PAR ID:
- 10494278
- Publisher / Repository:
- Nature Plants
- Date Published:
- Journal Name:
- Nature Plants
- Volume:
- 9
- Issue:
- 3
- ISSN:
- 2055-0278
- Page Range / eLocation ID:
- 433 to 441
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Comparative genomics has revealed common occurrences in karyotype evolution such as chromosomal end-to-end fusions and insertions of one chromosome into another near the centromere, as well as many cases of de novo centromeres that generate positional polymorphisms. However, how rearrangements such as dicentrics and acentrics persist without being destroyed or lost remains unclear. Here, we sought experimental evidence for the frequency and timeframe for inactivation and de novo formation of centromeres in maize (Zea mays). The pollen from plants with supernumerary B chromosomes was gamma-irradiated and then applied to normal maize silks of a line without B chromosomes. In ∼8,000 first-generation seedlings, we found many B–A translocations, centromere expansions, and ring chromosomes. We also found many dicentric chromosomes, but a fraction of these show only a single primary constriction, which suggests inactivation of one centromere. Chromosomal fragments were found without canonical centromere sequences, revealing de novo centromere formation over unique sequences; these were validated by immunolocalization with Thr133-phosphorylated histone H2A, a marker of active centromeres, and chromatin immunoprecipitation-sequencing with the CENH3 antibody. These results illustrate the regular occurrence of centromere birth and death after chromosomal rearrangement during a narrow window of one to potentially only a few cell cycles for the rearranged chromosomes to be recognized in this experimental regime.
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Heitman, Joseph (Ed.)ABSTRACT Centromeres are chromosomal regions that are crucial for chromosome segregation during mitosis and meiosis, and failed centromere formation can contribute to chromosomal anomalies. Despite this conserved function, centromeres differ significantly between and even within species. Thus far, systematic studies into the organization and evolution of fungal centromeres remain scarce. In this study, we identified the centromeres in each of the 10 species of the fungal genus Verticillium and characterized their organization and evolution. Chromatin immunoprecipitation of the centromere-specific histone CenH3 (ChIP-seq) and chromatin conformation capture (Hi-C) followed by high-throughput sequencing identified eight conserved, large (∼150-kb), AT-, and repeat-rich regional centromeres that are embedded in heterochromatin in the plant pathogen Verticillium dahliae . Using Hi-C, we similarly identified repeat-rich centromeres in the other Verticillium species. Strikingly, a single degenerated long terminal repeat (LTR) retrotransposon is strongly associated with centromeric regions in some but not all Verticillium species. Extensive chromosomal rearrangements occurred during Verticillium evolution, of which some could be linked to centromeres, suggesting that centromeres contributed to chromosomal evolution. The size and organization of centromeres differ considerably between species, and centromere size was found to correlate with the genome-wide repeat content. Overall, our study highlights the contribution of repetitive elements to the diversity and rapid evolution of centromeres within the fungal genus Verticillium . IMPORTANCE The genus Verticillium contains 10 species of plant-associated fungi, some of which are notorious pathogens. Verticillium species evolved by frequent chromosomal rearrangements that contribute to genome plasticity. Centromeres are instrumental for separation of chromosomes during mitosis and meiosis, and failed centromere functionality can lead to chromosomal anomalies. Here, we used a combination of experimental techniques to identify and characterize centromeres in each of the Verticillium species. Intriguingly, we could strongly associate a single repetitive element to the centromeres of some of the Verticillium species. The presence of this element in the centromeres coincides with increased centromere sizes and genome-wide repeat expansions. Collectively, our findings signify a role of repetitive elements in the function, organization, and rapid evolution of centromeres in a set of closely related fungal species.more » « less
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De novo centromere formation on chromosome fragments with an inactive centromere in maize (Zea mays)more » « less
Abstract The B chromosome of maize undergoes nondisjunction at the second pollen mitosis as part of its accumulation mechanism. Previous work identified 9-Bic-1 (9-B inactivated centromere-1), which comprises an epigenetically silenced B chromosome centromere that was translocated to the short arm of chromosome 9(9S). This chromosome is stable in isolation, but when normal B chromosomes are added to the genotype, it will attempt to undergo nondisjunction during the second pollen mitosis and usually fractures the chromosome in 9S. These broken chromosomes allow a test of whether the inactive centromere is reactivated or whether a de novo centromere is formed elsewhere on the chromosome to allow recovery of fragments. Breakpoint determination on the B chromosome and chromosome 9 showed that mini chromosome B1104 has the same breakpoint as 9-Bic-1 in the B centromere region and includes a portion of 9S. CENH3 binding was found on the B centromere region and on 9S, suggesting both centromere reactivation and de novo centromere formation. Another mini chromosome, B496, showed evidence of rearrangement, but it also only showed evidence for a de novo centromere. Other mini chromosome fragments recovered were directly derived from the B chromosome with breakpoints concentrated near the centromeric knob region, which suggests that the B chromosome is broken at a low frequency due to the failure of the sister chromatids to separate at the second pollen mitosis. Our results indicate that both reactivation and de novo centromere formation could occur on fragments derived from the progenitor possessing an inactive centromere.
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INTRODUCTION Transposable elements (TEs), repeat expansions, and repeat-mediated structural rearrangements play key roles in chromosome structure and species evolution, contribute to human genetic variation, and substantially influence human health through copy number variants, structural variants, insertions, deletions, and alterations to gene transcription and splicing. Despite their formative role in genome stability, repetitive regions have been relegated to gaps and collapsed regions in human genome reference GRCh38 owing to the technological limitations during its development. The lack of linear sequence in these regions, particularly in centromeres, resulted in the inability to fully explore the repeat content of the human genome in the context of both local and regional chromosomal environments. RATIONALE Long-read sequencing supported the complete, telomere-to-telomere (T2T) assembly of the pseudo-haploid human cell line CHM13. This resource affords a genome-scale assessment of all human repetitive sequences, including TEs and previously unknown repeats and satellites, both within and outside of gaps and collapsed regions. Additionally, a complete genome enables the opportunity to explore the epigenetic and transcriptional profiles of these elements that are fundamental to our understanding of chromosome structure, function, and evolution. Comparative analyses reveal modes of repeat divergence, evolution, and expansion or contraction with locus-level resolution. RESULTS We implemented a comprehensive repeat annotation workflow using previously known human repeats and de novo repeat modeling followed by manual curation, including assessing overlaps with gene annotations, segmental duplications, tandem repeats, and annotated repeats. Using this method, we developed an updated catalog of human repetitive sequences and refined previous repeat annotations. We discovered 43 previously unknown repeats and repeat variants and characterized 19 complex, composite repetitive structures, which often carry genes, across T2T-CHM13. Using precision nuclear run-on sequencing (PRO-seq) and CpG methylated sites generated from Oxford Nanopore Technologies long-read sequencing data, we assessed RNA polymerase engagement across retroelements genome-wide, revealing correlations between nascent transcription, sequence divergence, CpG density, and methylation. These analyses were extended to evaluate RNA polymerase occupancy for all repeats, including high-density satellite repeats that reside in previously inaccessible centromeric regions of all human chromosomes. Moreover, using both mapping-dependent and mapping-independent approaches across early developmental stages and a complete cell cycle time series, we found that engaged RNA polymerase across satellites is low; in contrast, TE transcription is abundant and serves as a boundary for changes in CpG methylation and centromere substructure. Together, these data reveal the dynamic relationship between transcriptionally active retroelement subclasses and DNA methylation, as well as potential mechanisms for the derivation and evolution of new repeat families and composite elements. Focusing on the emerging T2T-level assembly of the HG002 X chromosome, we reveal that a high level of repeat variation likely exists across the human population, including composite element copy numbers that affect gene copy number. Additionally, we highlight the impact of repeats on the structural diversity of the genome, revealing repeat expansions with extreme copy number differences between humans and primates while also providing high-confidence annotations of retroelement transduction events. CONCLUSION The comprehensive repeat annotations and updated repeat models described herein serve as a resource for expanding the compendium of human genome sequences and reveal the impact of specific repeats on the human genome. In developing this resource, we provide a methodological framework for assessing repeat variation within and between human genomes. The exhaustive assessment of the transcriptional landscape of repeats, at both the genome scale and locally, such as within centromeres, sets the stage for functional studies to disentangle the role transcription plays in the mechanisms essential for genome stability and chromosome segregation. Finally, our work demonstrates the need to increase efforts toward achieving T2T-level assemblies for nonhuman primates and other species to fully understand the complexity and impact of repeat-derived genomic innovations that define primate lineages, including humans. Telomere-to-telomere assembly of CHM13 supports repeat annotations and discoveries. The human reference T2T-CHM13 filled gaps and corrected collapsed regions (triangles) in GRCh38. Combining long read–based methylation calls, PRO-seq, and multilevel computational methods, we provide a compendium of human repeats, define retroelement expression and methylation profiles, and delineate locus-specific sites of nascent transcription genome-wide, including previously inaccessible centromeres. SINE, short interspersed element; SVA, SINE–variable number tandem repeat– Alu ; LINE, long interspersed element; LTR, long terminal repeat; TSS, transcription start site; pA, xxxxxxxxxxxxxxxx.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1038/s41477-023-01370-8
