Naturally occurring differences in CENH3 affect chromosome segregation in zygotic mitosis of hybrids

Abstract

The point of attachment of spindle microtubules to metaphase chromosomes is known as the centromere. Plant and animal centromeres are epigenetically specified by a centromere-specific variant of Histone H3, CENH3 (a.k.a. CENP-A). Unlike canonical histones that are invariant, CENH3 proteins are accumulating substitutions at an accelerated rate. This diversification of CENH3 is a conundrum since its role as the key determinant of centromere identity remains a constant across species. Here, we ask whether naturally occurring divergence in CENH3 has functional consequences. We performed functional complementation assays on cenh3-1, a null mutation in Arabidopsis thaliana, using untagged CENH3s from increasingly distant relatives. Contrary to previous results using GFP-tagged CENH3, we find that the essential functions of CENH3 are conserved across a broad evolutionary landscape. CENH3 from a species as distant as the monocot Zea mays can functionally replace A. thaliana CENH3. Plants expressing variant CENH3s that are fertile when selfed show dramatic segregation errors when crossed to a wild-type individual. The progeny of this cross include hybrid diploids, aneuploids with novel genetic rearrangements and haploids that inherit only the genome of the wild-type parent. Importantly, it is always chromosomes from the plant expressing the divergent CENH3 that missegregate. Using chimeras, we show that it is divergence in the fast-evolving N-terminal tail of CENH3 that is causing segregation errors and genome elimination. Furthermore, we analyzed N-terminal tail sequences from plant CENH3s and discovered a modular pattern of sequence conservation. From this we hypothesize that while the essential functions of CENH3 are largely conserved, the N-terminal tail is evolving to adapt to lineage-specific centromeric constraints. Our results demonstrate that this lineage-specific evolution of CENH3 causes inviability and sterility of progeny in crosses, at the same time producing karyotypic variation. Thus, CENH3 evolution can contribute to postzygotic reproductive barriers.

Figure 1. Vegetative and reproductive phenotypes of CENH3 complemented lines.

(A) Plants at rosette stage from different complemented lines compared to wild-type Columbia (WT) and GFP-tailswap, a high frequency haploid inducer [20]. The genotype of the endogenous CENH3 locus is indicated in parentheses. LoCENH3 is L. oleraceum CENH3 and BrCENH3 is B. rapa CENH3. AtNTT-LoHFD and LoNTT-AtHFD are chimeric CENH3s described in the key. (B) Anthers stained for viability with Alexander stain. Viable pollen granules stain purple. (C) Measures of fertility based on number of seeds per silique and seed appearance. Bars in different shades of grey represent counts from different T1 lines. For each measurement, seeds from 5 siliques were pooled and counted.

Figure 2. L. oleraceum CENH3 complements meiosis in A. thaliana.

Male meiotic chromosome spreads stained with DAPI for WT Col-0 (A-D, I-L) and L. oleraceum CENH3 cenh3–1/cenh3–1 (T1 family = 19) (E-H, M-P). Scale bar = 10μm.

Figure 3. Characterization of aneuploid genotypes using whole-genome sequencing.

Shown here are pictures of an individual plant alongside its 100kb bin dosage plot and 1 Mb bin SNP analysis across all five chromosomes. The red boxes indicate their relative centromere positions. (A) A diploid Col-0/Ler hybrid individual from a genome elimination cross mediated by LoCENH3. (B–D) The three major aneuploid types represented by examples of each: an individual with a numerical aneuploid chromosome (B), a truncated aneuploid chromosome (C) and a shattered aneuploid chromosome (D). (E–F) Percentage of each type of chromosomal variants of the aneuploids derived from a LoCENH3 (E) and BrCENH3 (F) genome elimination cross.

Figure 4. Analysis of evolutionary divergence in plant CENH3 Histone Fold Domains.

(A) Phylogenetic tree inferred by using the Maximum Likelihood method based on the JTT matrix-based model [52]. The tree with the highest log likelihood (-3935.2849) is shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. (B) Summary of complementation tests of A. thaliana cenh3–1 mutation with CENH3 from increasingly distant plant species.

Figure 5. Identification of sequence motifs in plant CENH3 N-terminal tails.

(A) Schematic representation of CENH3 N-terminal tails from a subset of plant species, in the context of their known phylogenetic relationships. Motifs identified by MEME [51] are represented as different colored blocks. N-terminal tails are drawn to scale with the relative locations of each motif identified. The height of the motif block is proportional to-log(p-value). (B) Motif blocks 4 and 6 in Logos format. All instances where the motifs were identified are included below for comparison.