The variation and evolution of complete human centromeres

Abstract

Human centromeres have been traditionally very difficult to sequence and assemble owing to their repetitive nature and large size1. As a result, patterns of human centromeric variation and models for their evolution and function remain incomplete, despite centromeres being among the most rapidly mutating regions2,3. Here, using long-read sequencing, we completely sequenced and assembled all centromeres from a second human genome and compared it to the finished reference genome4,5. We find that the two sets of centromeres show at least a 4.1-fold increase in single-nucleotide variation when compared with their unique flanks and vary up to 3-fold in size. Moreover, we find that 45.8% of centromeric sequence cannot be reliably aligned using standard methods owing to the emergence of new α-satellite higher-order repeats (HORs). DNA methylation and CENP-A chromatin immunoprecipitation experiments show that 26% of the centromeres differ in their kinetochore position by >500 kb. To understand evolutionary change, we selected six chromosomes and sequenced and assembled 31 orthologous centromeres from the common chimpanzee, orangutan and macaque genomes. Comparative analyses reveal a nearly complete turnover of α-satellite HORs, with characteristic idiosyncratic changes in α-satellite HORs for each species. Phylogenetic reconstruction of human haplotypes supports limited to no recombination between the short (p) and long (q) arms across centromeres and reveals that novel α-satellite HORs share a monophyletic origin, providing a strategy to estimate the rate of saltatory amplification and mutation of human centromeric DNA.

Centromeric chromatin clearings demarcate the site of kinetochore formation

“…This attribute is also reflected in our tomograms where we observe kinetochores of varied size and with varied distribution of kinetochore complexes. Specifically, the finding that CENP-A occupancy is often split between multiple regions within a single centromere (Altemose et al, 2022a; Dubocanin et al, 2023; Gershman et al, 2022; Logsdon et al, 2024; Sacristan et al, 2022) is consistent with our own observation that some kinetochores are comprised of a single chromatin clearing whereas others are comprised of 2-3 clearings. Each of the clearings likely corresponds to a region on the linear DNA sequence with a high local density of CENP-A nucleosomes.…”

“…These DNA sequencing-based results align remarkably well with our findings where tomograms show kinetochore complexes clustered within a distinct clearing space composed of open chromatin. Sequencing maps of human centromeres have also shown that CENP-A occupancy is highly variable in both magnitude and distribution, not only among individuals but also among chromosomes within an individual (Altemose et al, 2022a; Gershman et al, 2022; Logsdon et al, 2024). This attribute is also reflected in our tomograms where we observe kinetochores of varied size and with varied distribution of kinetochore complexes.…”

Comparative genomics of macaques and integrated insights into genetic variation and population history

“…Second, the rDNA array in macaques is only found in chromosome 10, in contrast to humans, where they are located in five chromosomes 15,121 (chr13, chr14, chr15, chr21, and chr22) (Figure 1e). Third, the length and composition of the α-satellite arrays differ between macaques and apes 18,24,49 . Additionally, while the α-satellite arrays are relatively conserved within macaques, they exhibit greater variability in humans 18,24 .…”

“…Third, the length and composition of the α-satellite arrays differ between macaques and apes 18,24,49 . Additionally, while the α-satellite arrays are relatively conserved within macaques, they exhibit greater variability in humans 18,24 . Finally, 91 large-scale genomic differences between macaques and humans have been revealed at single-base resolution, most of which are associated with centromere repositions, inversion, and translocation 47,92,93 (Figure 5).…”

“…Currently, haplotype-resolved genome assemblies can be generated with trio-based, Hi-C-based, or Strand-seq-based approaches 21–23 . These assemblies can then be utilized to construct high-quality pangenome graphs, enhancing our understanding of genetic structure and the evolutionary history of complex genomic regions 24–26 . However, generating haplotype-resolved diploid T2T genome assemblies remains challenging, primarily due to the complexities associated with phasing centromeres, subtelomeres, and SDRs 22 .…”