The genetic and epigenetic landscape of the Arabidopsis centromeres
A closer look at centromeres
Centromeres are key for anchoring chromosomes to the mitotic spindle, but they have been difficult to sequence because they can contain many repeating DNA elements. These repeats, however, carry regularly spaced, distinctive sequence markers because of sequence heterogeneity between the mostly, but not completely, identical DNA sequence repeats. Such differences aid sequence assembly. Naish et al. used ultra-long-read DNA sequencing to establish a reference assembly that resolves all five centromeres in the small mustard plant Arabidopsis. Their view into the subtly homogenized world of centromeres reveals retrotransposons that interrupt centromere organization and repressive DNA methylation that excludes centromeres from meiotic crossover repair. Thus, Arabidopsis centromeres evolve under the opposing forces of sequence homogenization and retrotransposon disruption. —PJH
Structured Abstract
INTRODUCTION
The centromeres of eukaryotic chromosomes assemble the multiprotein kinetochore complex and thereby position attachment to the spindle microtubules, allowing chromosome segregation during cell division. The key function of the centromere is to load nucleosomes containing the CENTROMERE SPECIFIC HISTONE H3 (CENH3) histone variant [also known as centromere protein A (CENPA)], which directs kinetochore formation. Despite their conserved function during chromosome segregation, centromeres show radically diverse organization between species at the sequence level, ranging from single nucleosomes to megabase-scale satellite repeat arrays, which is termed the centromere paradox. Centromeric satellite repeats are variable in sequence composition and length when compared between species and show a high capacity for evolutionary change, both at the levels of primary sequence and array position along the chromosome. However, the genetic and epigenetic features that contribute to centromere function and evolution are incompletely understood, in part because of the challenges of centromere sequence assembly and functional genomics of highly repetitive sequences. New long-read DNA sequencing technologies can now resolve these complex repeat arrays, revealing insights into centromere architecture and chromatin organization.
RATIONALE
Arabidopsis thaliana is a model plant species; its genome was first sequenced in 2000, yet the centromeres, telomeres, and ribosomal DNA repeats have remained unassembled, owing to their high repetition and similarity. Genomic repeats are difficult to assemble from fragmented sequencing reads, with longer, high-identity repeats being the most challenging to correctly assemble. As sequencing reads have become longer and more accurate, eukaryotic de novo genome assemblies have captured an increasingly complete picture of the repetitive component of the genome, including the centromeres. For example, Oxford Nanopore Technologies (ONT) reads have become longer and more accurate, now reaching >100 kilo–base pairs (kbp) in length with 95 to 99% modal accuracy. PacBio high-fidelity (HiFi) reads, although shorter (~15 kbp), are >99% accurate. Using ONT and HiFi reads, it is possible to bridge across interspersed unique marker sequences and accurately assemble centromere sequences. In this study, we used long-read DNA sequencing to generate a genome assembly of the A. thaliana accession Columbia (Col-CEN) that resolves all five centromeres. We use the Col-CEN assembly to derive insights into the chromatin and recombination landscapes within the Arabidopsis centromeres and how these regions evolve.
RESULTS
The Col-CEN assembly reveals that the Arabidopsis centromeres consist of megabase-scale tandemly repeated satellite arrays, which support high CENH3 (the centromere-specific histone variant that recruits kinetochores) occupancy and are densely DNA methylated. We show patterns of higher-order repetition within centromeres and that many satellite variants are private to each chromosome, which has implications for the recombination pathways acting in the centromeres. CENH3 preferentially occupies the satellites with the least amount of divergence and that show higher-order repetition. The Arabidopsis centromeres are mainly composed of satellite repeats that are ~178 bp in length, termed the CEN180 satellites. Arabidopsis centromeres have also been invaded by ATHILA long terminal repeat–class retrotransposons, which disrupt the genetic and epigenetic organization of the centromeres. Using chromatin immunoprecipitation sequencing (ChIP-seq) and immunofluorescence, we demonstrate that the centromeres show a hybrid chromatin state that is distinct from euchromatin and heterochromatin. We show that crossover recombination is suppressed within the centromeres, yet low levels of meiotic double-strand breaks occur, which are regulated by DNA methylation. Together, our Col-CEN assembly reveals the genetic and epigenetic landscapes within the Arabidopsis centromeres.
CONCLUSION
Our Col-CEN assembly and functional genomics analysis have implications for understanding centromere sequence evolution in eukaryotes. We propose that a recombination-based homogenization process, occurring between allelic or nonallelic locations on the same chromosome, maintains the CEN180 library close to the consensus optimal for CENH3 recruitment. The advantage conferred to ATHILA retrotransposons by integration within the centromeres is presently unclear. They may be engaged in centromere drive, supporting the hypothesis that centromere satellite homogenization acts as a mechanism to purge driving elements. Each Arabidopsis centromere appears to represent different stages in cycles of satellite homogenization and ATHILA-driven diversification. These opposing forces provide both a capacity for homeostasis and a capacity for change during centromere evolution. In the future, assembly of centromeres from multiple Arabidopsis accessions and closely related species may further clarify how centromeres form and the evolutionary dynamics of CEN180 and ATHILA repeats.
Assembly of the Arabidopsis centromeres.
The structure of Arabidopsis centromere 1 is shown by fluorescence in situ hybridization (top) [upper-arm bacterial artificial chromosomes (BACs) (green), ATHILA (purple), CEN180 (blue), the telomeric repeat (green), and bottom-arm BACs (yellow)] and a long-read genome assembly (middle). The density of centromeric histone CENH3 binding measured by ChIP-seq is shown (black), alongside the frequency of CEN180 centromere satellite repeats. Red and blue represent forward- and reverse-strand satellites, respectively. The heatmap (bottom) shows patterns of sequence identity across the centromere between nonoverlapping 5-kbp windows. Chr, chromosome 1.
Abstract
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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Supplementary Materials
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Table S6
MDAR Reproducibility Checklist
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Information & Authors
Information
Published In
Science
Volume 374 | Issue 6569
12 November 2021
12 November 2021
Copyright
Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
This is an article distributed under the terms of the Science Journals Default License.
Submission history
Received: 31 March 2021
Accepted: 27 September 2021
Published in print: 12 November 2021
Acknowledgments
This paper is dedicated to Simon Chan. We thank I. Thompson for ATHILA analysis, S. Henikoff for the generous gift of CENH3 antibodies, A. Shumate for help with gene Liftoff interpretation, B. Fischer for advice on high–molecular weight DNA isolation, and M. Pouch for assistance designing FISH probes.
Funding: This work was supported by BBSRC grants BB/S006842/1, BB/S020012/1, and BB/V003984/1 to I.R.H.; European Research Council Consolidator Award ERC-2015-CoG-681987 “SynthHotSpot” to I.R.H.; Marie Curie International Training Network “MEICOM” to I.R.H.; Human Frontier Science Program award RGP0025/2021 to T.K., M.C.S., and I.R.H.; US National Institutes of Health grant S10OD028632-01; US National Science Foundation grants DBI-1350041 and IOS-1732253 to M.C.S.; Royal Society awards UF160222 and RGF/R1/180006 to A.B.; the Czech Science Foundation grant no. 21-03909S to M.A.L.; the Gregor Mendel Institute to F.B.; grants Fonds zur Förderung der wissenschaftlichen Forschung (FWF) P26887, P28320, P30802, P32054, and TAI304 to F.B. and chromatin dynamics W1238 to A.S. and B.J.; Leverhulme Trust Research Leadership grant RL-2012-042 to J.T.; and grants from the Howard Hughes Medical Institute and US National Institutes of Health (RO1GM067014) to R.A.M.
Author contributions: M.N. sequenced DNA; performed genome assembly and analysis, ChIP-seq, and DNA methylation analysis; and wrote the manuscript. M.A. performed genome assembly, polishing, validation, annotation, and analysis and wrote the manuscript. P.W. performed satellite repeat annotation and genome analysis and wrote the manuscript. A.J.T. performed short-read alignment and genome analysis and wrote the manuscript. B.W.A. sequenced DNA, performed optical mapping, and contributed to the assembly. A.S. performed chromatin immunofluorescence analysis. B.J. provided ChIP-seq data. C.L. and P.K. performed immunocytology. N.Y. generated the DMC1 epitope-tagged line. N.H. and K.C. sequenced DNA and contributed to the assembly. L.M.S., J.T., and K.S. performed PacBio sequencing. T.K. and R.A.M. provided intellectual input. T.M. and M.A.L. performed FISH. F.B. supervised ChIP-seq and immunofluorescence analysis and wrote the manuscript. A.B. performed ATHILA annotation and genome analysis and wrote the manuscript. T.P.M. supervised DNA sequencing and genome assembly and analysis and wrote the manuscript. M.C.S. supervised genome assembly, validation, annotation, and analysis and wrote the manuscript. I.R.H. supervised DNA sequencing, genome assembly, validation, annotation, and analysis and wrote the manuscript.
Competing interests: The authors have no competing interests.
Data and materials availability: The ONT sequencing reads used for assembly are available for download at ArrayExpress accession E-MTAB-10272 (www.ebi.ac.uk/arrayexpress/). The PacBio HiFi reads are available for download at European Nucleotide Archive accession number PRJEB46164 (www.ebi.ac.uk/ena/browser/view/PRJEB46164). All data, code and materials are available in the manuscript or the supplementary materials and at https://github.com/schatzlab/Col-CEN.
Authors
Funding Information
National Science Foundation: DBI-1350041
National Science Foundation: IOS-1732253
National Institutes of Health: S10OD028632-01
H2020 European Research Council: ERC-2015-C
H2020 European Research Council: G-681987
Biotechnology and Biological Sciences Research Council: BB/S006842/1
Biotechnology and Biological Sciences Research Council: BB/V003984/1
Czech Science Foundation: 21-03909S
Marie Curie International Training Network MEICOM to IH Human Frontier Science Program: RGP0025/2021
Howard Hughes Medical Institute and National Institutes of Health: RO1GM067014
Human Frontier Science Program: RGP0025/2021
Biotechnology and Biological Sciences Research Council: BB/S020012/1
Austrian Science Fund: P26887
Austrian Science Fund: P28320
Austrian Science Fund: P30802
Austrian Science Fund: P32054
Austrian Science Fund: TAI304
Czech Science Foundation: 21-03909S
Leverhulme Trust: RL-2012-042
Royal Society: UF160222
Royal Society: RGF/R1/180006
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