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.

Centromere location in Arabidopsis is unaltered by extreme divergence in CENH3 protein sequence

“…Notwithstanding the complementation of essential functions, data from this study and previous research (Maheshwari et al 2015) show that centromeres built on non-native CENH3s missegregate during early embryogenesis in crosses to wild type. Clearly, the non-native CENH3 proteins are lacking a species-specific adaptation that the endogenous A. thaliana CENH3 has evolved.…”

“…Since the non-native CENH3 transgenes are expressed in the background of an A. thaliana cenh3 null mutation, their conserved localization through two generations, hundreds of mitotic, and two meiotic divisions is not guided by the presence of the native A. thaliana CENH3. More likely, centromere localization is faithfully maintained upon being derived from an epigenetic signal inherited from the heterozygous parent that produced the cenh3 − gamete into which the complementing transgene was introduced through floral dip (Maheshwari et al 2015). Taken together, our results suggest that even the highly diverged Z. mays CENH3 does not mislocalize in A. thaliana, despite causing dramatic segregation errors in crosses to wild type.…”

“…From our previous study, we know that functional complementation of cenh3 has no effect on vegetative or reproductive phenotypes, except in crosses to wild type (Maheshwari et al 2015). We had previously shown that centromeres built on L. oleraceum CENH3 are deficient in comparison to wild type, because when outcrossed, they missegregate dramatically.…”

“…Further, haploid production efficiency was increased proportionally to CENH3 divergence. Up to 19% of the progeny lost the genome of the Z. mays CENH3 expressing parent, whereas in the case of CENH3 from the closer relative L. oleraceum, a maximum of 11% haploids was observed (Maheshwari et al 2015).…”

“…In a previous study (Maheshwari et al 2015), we reported that replacement of A. thaliana CENH3 with CENH3 homologs from distant species resulted in apparently perfect complementation of mitotic and meiotic functions. However, in outcrosses to wild type, chromosomes of the complemented parent missegregate during embryonic growth leading to frequent aneuploidy, haploidy, and death.…”