Variation in the CENP-A sequence association landscape across diverse inbred mouse strains

Abstract

Centromeres are crucial for chromosome segregation, but their underlying sequences evolve rapidly, imposing strong selection for compensatory changes in centromere-associated kinetochore proteins to assure the stability of genome transmission. While this co-evolution is well documented between species, it remains unknown whether population-level centromere diversity leads to functional differences in kinetochore protein association. Mice (Mus musculus) exhibit remarkable variation in centromere size and sequence, but the amino acid sequence of the kinetochore protein CENP-A is conserved. Here, we apply k-mer-based analyses to CENP-A chromatin profiling data from diverse inbred mouse strains to investigate the interplay between centromere variation and kinetochore protein sequence association. We show that centromere sequence diversity is associated with strain-level differences in both CENP-A positioning and sequence preference along the mouse core centromere satellite. Our findings reveal intraspecies sequence-dependent differences in CENP-A/centromere association and open additional perspectives for understanding centromere-mediated variation in genome stability.

Keywords: CENP-A association; CENP-A positioning; CP: Genomics; Mus musculus; centromere evolution; centromere transcription; centromere variation; genome instability; kinetochore; meiotic drive.

Figure 1.. Consensus-based alignment analysis of strain differences in CENP-A enrichment at centromeres

(A) Experimental design of ChIP-seq and the consensus-guided genomic analyses for CENP-A ChIP sequence enrichment. (B) Boxplots representing the enrichment of reads that mapped to either the major satellite (left) or minor satellite (right) consensus sequences in CENP-A ChIP relative to input samples. Color represents sample identity (AB, antibody). For each boxplot, the horizontal line represents the median, and the vertical line represents the range of values across replicates. (C) Heatmap showing the enrichment of the consensus nucleotide along the minor satellite consensus sequence in CENP-A ChIP compared with input samples from each strain.

Figure 2.. Strain differences in CENP-A positioning along the minor satellite consensus

(A) Schematic depicting the difference in CENP-A and H3 nucleosome coverage between 76-bp single-end reads and trimmed reads. (B) Line plot representing the CENP-A ChIP/input enrichment of read coverage (y axis) at each position along the minor satellite consensus sequence (x axis). Solid (dashed) lines correspond to coverage values calculated using untrimmed (trimmed) reads. The 17-bp CENP-B box motif is marked in gray. (C) Boxplots representing the percent enrichment of CENP-B box motif frequency in ChIP/input samples. For each boxplot, the horizontal line represents the median, and the vertical line represents the range of values. (D) Heatmap presenting pairwise strain Pearson correlation coefficients for the average CENP-A ChIP/input enrichment pattern along the minor satellite consensus sequence. All Pearson correlation coefficients are significant (p < 0.05).

Figure 3.. Shared and unique CENP-A-enriched k-mers across inbred mouse strains

(A) Upset plot representing the extent of k-mer sharing among the top 0.1% most enriched CENP-A-associated k-mers in each strain. Bar height corresponds to the total number of k-mers in each strain set. Each strain set of CENP-A-enriched k-mers is represented by the horizontal bars. The vertical bars represent the number of k-mers that belong to one or more strains, indicated by the dots below. p value was calculated by comparing the observed number of shared k-mers with the distribution of 100,000 bootstrap samples of the data. (B) Bar plots showing the number of CENP-A-enriched 31-mers from each strain with a given number of mismatches (NM) relative to the minor satellite consensus sequence. (C) Line plot depicting where CENP-A-enriched 31-mers map along the minor satellite consensus sequence. The y axis represents the number of 31-mers at a particular position along the minor satellite consensus sequence (x axis). Strains are indicated by line color.

Figure 4.. CENP-A-associated sequences identified by a reference-agnostic, k-mer-based approach form distinct subgroups

Neighbor joining tree constructed from each strain’s top 1,000 CENP-A-associated sequences. Sequences cluster into seven groups. Strain-level contributions to each group are depicted by bar plots. Groups with skewed strain representation are indicated by red Chi-square p values (groups 1, 3, 4, 7). For clades with biased strain representation, Bonferroni post hoc tests were used to identify strains driving the significant signal.

Figure 5.. Relationship between CENP-A-associated sequences across strains

Principal component analysis based on the stylistic similarity of CENP-A-associated sequences in each strain.

Figure 6.. Transcription factor occupancy at centromere satellite DNA

(A) Association of transcription factors at centromere satellite DNA determined from publicly available ChIP-seq datasets. The y axis represents the enrichment of reads that map to the minor satellite consensus sequence in ChIP/input samples for each transcription factor (x axis). Each dot represents the average value of an experiment. The red horizontal line corresponds to ChIP/input value of 1. (B) The frequency of multiple TF motifs among CENP-A-enriched sequences across diverse mouse strains.