Complete genomic and epigenetic maps of human centromeres

Existing human genome assemblies have almost entirely excluded highly repetitive sequences within and near centromeres, limiting our understanding of their sequence, evolution, and essential role in chromosome segregation. Here, we present an extensive study of newly assembled peri/centromeric sequences representing 6.2% (189.9 Mb) of the first complete, telomere-to-telomere human genome assembly (T2T-CHM13). We discovered novel patterns of peri/centromeric repeat organization, variation, and evolution at both large and small length scales. We also found that inner kinetochore proteins tend to overlap the most recently duplicated subregions within centromeres. Finally, we compared chromosome X centromeres across a diverse panel of individuals and uncovered structural, epigenetic, and sequence variation at single-base resolution across these regions. In total, this work provides an unprecedented atlas of human centromeres to guide future studies of their complex and critical functions as well as their unique evolutionary dynamics.

Copy number evolution in simple and complex tandem repeats across the C57BL/6 and C57BL/10 inbred mouse lines

Simple sequence tandem repeats are among the most rapidly evolving compartments of the genome. Some repeat expansions are associated with mammalian disease or meiotic segregation distortion, yet the rates of copy number change across generations are not well known. Here, we use 14 distinct sub-lineages of the C57BL/6 and C57BL/10 inbred mouse strains, which have been evolving independently over about 300 generations, to estimate the rates of copy number changes in genome-wide tandem repeats. Rates of change varied across repeats and across lines. Notably, CAG, whose expansions in coding regions are associated with many neurological and genetic disorders, was highly stable in copy number, likely indicating stabilizing selection. Rates of change were positively correlated with copy number, but the direction and magnitude of changes varied across lines. Some mouse lines experienced consistent losses or gains across most simple repeats, but this did not correlate with copy number changes in complex repeats. Rates of copy number change were similar between simple repeats and the more abundant complex repeats after normalization by copy number. Finally, the Y-specific centromeric repeat had a 4-fold higher rate of change than the homologous centromeric repeat on other chromosomes. Structural differences in satellite complexity, or restriction to the Y chromosome and elevated mutation rates of the male germline, may explain the higher rate of change. Overall, our work underscores the mutational fluidity of long tandem arrays of repeats, and the correlations and constraints between genome-wide tandem repeats which suggest that turnover is not a completely neutral process.

Copy number evolution in simple and complex tandem repeats across the C57BL/6 and C57BL/10 inbred mouse lines

Simple sequence tandem repeats are among the most rapidly evolving compartments of the genome. Some repeat expansions are associated with mammalian disease or meiotic segregation distortion, yet the rates of copy number change across generations are not well known. Here, we use 14 distinct sub-lineages of the C57BL/6 and C57BL/10 inbred mouse strains, which have been evolving independently over about 300 generations, to estimate the rates of copy number changes in genome-wide tandem repeats. Rates of change varied across simple repeats and across lines. Notably, CAG, whose expansions in coding regions are associated with many neurological and other genetic disorders, was highly stable in copy number, likely indicating purifying selection. Rates of change were generally positively correlated with copy number, but the direction and magnitude of changes varied across lines. Some mouse lines experienced consistent losses or gains across most genome-wide simple repeats, but this did not correlate with copy number changes in complex repeats. Rates of copy number change were similar between simple repeats and the much more abundant complex repeats once they were normalized by copy number. Finally, the Y-specific centromeric repeat had a 4-fold higher rate of change than the homologous centromeric repeat on other chromosomes. Structural differences in satellite complexity, or restriction to the Y chromosome and the elevated mutation rate of the male germline, may explain the higher rate of change. Overall, our work underscores the mutational fluidity of long tandem arrays of repeats, and the correlations and constraints between genome-wide tandem repeats which suggest that turnover is not a completely neutral process.

Comparative genomics of Chlamydomonas

Despite its role as a reference organism in the plant sciences, the green alga Chlamydomonas reinhardtii entirely lacks genomic resources from closely related species. We present highly contiguous and well-annotated genome assemblies for three unicellular C. reinhardtii relatives: Chlamydomonas incerta, Chlamydomonas schloesseri, and the more distantly related Edaphochlamys debaryana. The three Chlamydomonas genomes are highly syntenous with similar gene contents, although the 129.2 Mb C. incerta and 130.2 Mb C. schloesseri assemblies are more repeat-rich than the 111.1 Mb C. reinhardtii genome. We identify the major centromeric repeat in C. reinhardtii as a LINE transposable element homologous to Zepp (the centromeric repeat in Coccomyxa subellipsoidea) and infer that centromere locations and structure are likely conserved in C. incerta and C. schloesseri. We report extensive rearrangements, but limited gene turnover, between the minus mating type loci of these Chlamydomonas species. We produce an eight-species core-Reinhardtinia whole-genome alignment, which we use to identify several hundred false positive and missing genes in the C. reinhardtii annotation and >260,000 evolutionarily conserved elements in the C. reinhardtii genome. In summary, these resources will enable comparative genomics analyses for C. reinhardtii, significantly extending the analytical toolkit for this emerging model system.

Comparative genomics of Chlamydomonas

Despite its fundamental role as a model organism in plant sciences, the green alga Chlamydomonas reinhardtii entirely lacks genomic resources for any closely related species, obstructing its development as a study system in several fields. We present highly contiguous and well-annotated genome assemblies for the two closest known relatives of the species, Chlamydomonas incerta and Chlamydomonas schloesseri, and a third more distantly related species, Edaphochlamys debaryana. We find the three Chlamydomonas genomes to be highly syntenous with similar gene contents, although the 129.2 Mb C. incerta and 130.2 Mb C. schloesseri assemblies are more repeat-rich than the 111.1 Mb C. reinhardtii genome. We identify the major centromeric repeat in C. reinhardtii as an L1 LINE transposable element homologous to Zepp (the centromeric repeat in Coccomyxa subellipsoidea) and infer that centromere locations and structure are likely conserved in C. incerta and C. schloesseri. We report extensive rearrangements, but limited gene turnover, between the minus mating-type loci of the Chlamydomonas species, potentially representing the early stages of mating-type haplotype reformation. We produce an 8-species whole-genome alignment of unicellular and multicellular volvocine algae and identify evolutionarily conserved elements in the C. reinhardtii genome. We find that short introns (<~100 bp) are extensively overlapped by conserved elements, and likely represent an important functional class of regulatory sequence in C. reinhardtii. In summary, these novel resources enable comparative genomics analyses to be performed for C. reinhardtii, significantly developing the analytical toolkit for this important model system.

A Game of Thrones at Human Centromeres I. Multifarious structure necessitates a new molecular/evolutionary model

Human centromeres form over arrays of tandemly repeated DNA that are exceptionally complex (repeats of repeats) and long (spanning up to 8 Mbp). They also have an exceptionally rapid rate of evolution. The generally accepted model for the expansion/contraction, homogenization and evolution of human centromeric repeat arrays is a generic model for the evolution of satellite DNA that is based on unequal crossing over between sister chromatids. This selectively neutral model predicts that the sequences of centromeric repeat units will be effectively random and lack functional constraint. Here I used shotgun PacBio SMRT reads from a homozygous human fetal genome (female) to determine and compare the consensus sequences (and levels of intra-array variation) for the active centromeric repeats of all the chromosomes. To include the Y chromosome using the same technology, I used the same type of reads from a diploid male. I found many different forms and levels of conserved structure that are not predicted by –and sometimes contradictory to– the unequal crossing over model. Much of this structure is based on spatial organization of three types of ~170 bp monomeric repeat units that are predicted to influence centromere strength (i.e., the level of outer kinetochore proteins): one with a protein-binding sequence at its 5’ end (a 17 bp b-box that binds CENP-B), a second that is identical to the first except that the b-box is mutated so that it no longer binds CENP-B, and a third lacking a b-box but containing a 19 bp conserved “n-box” sequence near its 5’ end. The frequency and organization of these monomer types change markedly as the number of monomers per repeat unit increases, and also differs between inactive and active arrays. Active arrays are also much longer than flanking, inactive arrays, and far longer than required for cellular functioning. The diverse forms of structure motivate a new hypothesis for the lifecycle of human centromeric sequences. These multifarious levels of structures, and other lines of evidence, collectively indicate that a new model is needed to explain the form, function, expansion/contraction, homogenization and rapid evolution of centromeric sequences.

A Game of Thrones at Human Centromeres II. A new molecular/evolutionary model

Human centromeres are remarkable in four ways: they are i) defined epigenetically by an elevated concentration of the histone H3 variant CENP-A, ii) inherited epigenetically by trans-generational cary-over of nucleosomes containing CENP-A, iii) formed over unusually long and complex tandem repeats (Higher Order Repeats, HORs) that extend over exceptionally long arrays of DNA (up to 8 Mb), and iv) evolve in such a rapid and punctuated manner that most HORs on orthologous chimp and human chromosomes are in different clades. What molecular and evolutionary processes generated these distinctive characteristics? Here I motivate and construct a new model for the formation, expansion/contraction, homogenization and rapid evolution of human centromeric repeat arrays that is based on fork-collapse during DNA replication (in response to proteins bound to DNA and/or collisions between DNA and RNA polymerases) followed by out-of-register re-initiation of replication via Break-Induced Repair (BIR). The model represents a new form of molecular drive. It predicts rapid and sometimes punctuated evolution of centromeric HORs due to a new form of intragenomic competition that is based on two features: i) the rate of tandem copy number expansion, and ii) resistance to invasion by pericentric heterochromatin within a centromere’s HOR array. These features determine which variant array elements will eventually occupy a pivotal region within a centromeric repeat array (switch-point) that gradually expands to populate the entire array. In humans, continuous HOR turnover is predicted due to intra-array competition between three repeat types with an intransitive hierarchy: A < B < C < A, where A = short, single-dimer HORs containing one monomer that binds centromere protein-B (CENP-B) and another that does not, B = moderately longer HORs composed of ≥ 2 dimers, and C = substantially longer HORs that lose their dimeric modular structure. Continuous turnover of proteins that bind centromeric DNA (but these proteins are not constituents of the kinetochore) and polygenic variation influencing position-effect variegation are predicted to cause rapid turnover of centromeric repeats in species lacking HORs and/or CENP-B binding at centromeres. Evolution at centromeres is a molecular ‘Game-of-Thrones’ because centromeric sequences ‘reign’ due to an epigenetic ‘crown’ of CENP-A that is perpetually ‘usurped’ by new sequences that more rapidly assemble large ‘armies’ of tandem repeats and/or resist ‘invasion’ from a surrounding ‘frontier’ of percentric heterochromatin. These ‘regal transitions’ occur in a backdrop of slashing and decapitation (fork-collapse generating truncated sister chromatids) in the context of promiscuous sex that is frequently incestuous (out-of-register BIR between sibling chromatids).

An ion beam–induced Arabidopsis mutant with marked chromosomal rearrangement

Ion beams have been used as an effective tool in mutation breeding for the creation of crops with novel characteristics. Recent analyses have revealed that ion beams induce large chromosomal alterations, in addition to small mutations comprising base changes or frameshifts. In an effort to understand the potential capability of ion beams, we analyzed an Arabidopsis mutant possessing an abnormal genetic trait. The Arabidopsis mutant uvh3-2 is hypersensitive to UVB radiation when photoreactivation is unavailable. uvh3-2 plants grow normally and produce seeds by self-pollination. SSLP and CAPS analyses of F2 plants showed abnormal recombination frequency on chromosomes 2 and 3. PCR-based analysis and sequencing revealed that one-third of chromosome 3 was translocated to chromosome 2 in uvh3-2. FISH analysis using a 180 bp centromeric repeat and 45S ribosomal DNA (rDNA) as probes showed that the 45S rDNA signal was positioned away from that of the 180 bp centromeric repeat in uvh3-2, suggesting the insertion of a large chromosome fragment into the chromosome with 45S rDNA clusters. F1 plants derived from a cross between uvh3-2 and wild-type showed reduced fertility. PCR-based analysis of F2 plants suggested that reproductive cells carrying normal chromosome 2 and uvh3-2–derived chromosome 3 are unable to survive and therefore produce zygote. These results showed that ion beams could induce marked genomic alterations, and could possibly lead to the generation of novel plant species and crop strains.

Integrity of the human centromere DNA repeats is protected by CENP-A, CENP-C, and CENP-T

Centromeres are highly specialized chromatin domains that enable chromosome segregation and orchestrate faithful cell division. Human centromeres are composed of tandem arrays of α-satellite DNA, which spans up to several megabases. Little is known about the mechanisms that maintain integrity of the long arrays of α-satellite DNA repeats. Here, we monitored centromeric repeat stability in human cells using chromosome-orientation fluorescent in situ hybridization (CO-FISH). This assay detected aberrant centromeric CO-FISH patterns consistent with sister chromatid exchange at the frequency of 5% in primary tissue culture cells, whereas higher levels were seen in several cancer cell lines and during replicative senescence. To understand the mechanism(s) that maintains centromere integrity, we examined the contribution of the centromere-specific histone variant CENP-A and members of the constitutive centromere-associated network (CCAN), CENP-C, CENP-T, and CENP-W. Depletion of CENP-A and CCAN proteins led to an increase in centromere aberrations, whereas enhancing chromosome missegregation by alternative methods did not, suggesting that CENP-A and CCAN proteins help maintain centromere integrity independently of their role in chromosome segregation. Furthermore, superresolution imaging of centromeric CO-FISH using structured illumination microscopy implied that CENP-A protects α-satellite repeats from extensive rearrangements. Our study points toward the presence of a centromere-specific mechanism that actively maintains α-satellite repeat integrity during human cell proliferation.

Diverse origins of high copy tandem repeats in grass genomes

In studying genomic architecture, highly repetitive regions have historically posed a challenge when investigating sequence variation and content. High-throughput sequencing has enabled researchers to use whole-genome shotgun sequencing to estimate the abundance of repetitive sequence, and these methodologies have been recently applied to centromeres. Here, we utilize sequence assembly and read mapping to identify and quantify the genomic abundance of different tandem repeat sequences. Previous research has posited that the highest abundance tandem repeat in eukaryotic genomes is often the centromeric repeat, and we pair our bioinformatic pipeline with fluorescent in-situ hybridization data to test this hypothesis. We find that de novo assembly and bioinformatic filters can successfully identify repeats with homology to known tandem repeats. Fluorescent in-situ hybridization, however, shows that de novo assembly fails to identify novel centromeric repeats, instead identifying other potentially important repetitive sequences. Together, our results test the applicability and limitations of using de novo repeat assembly of tandem repeats to identify novel centromeric repeats. Building on our findings of genomic composition, we also set forth a method for exploring the repetitive regions of non-model genomes whose diversity limits the applicability of established genetic resources.