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Title: Point Mutations in Centromeric Histone Induce Post-zygotic Incompatibility and Uniparental Inheritance}
Article:
Abstract:
The centromeric histone 3 variant( CENH3, aka CENP-A) is essential for the segregation of sister chromatids during mitosis and meiosis. To better define CENH3 functional constraints, we complemented a null allele in Arabidopsis with a variety of mutant alleles, each inducing a single amino acid change in conserved residues of the histone fold domain. Many of these transgenic missense lines displayed wild-type growth and fertility on self-pollination, but exhibited frequent post-zygotic death and uniparental inheritance when crossed with wild-type plants. The failure of centromeres marked by these missense mutation in the histone fold domain of CENH3 reproduces the genome elimination syndromes described with chimeric CENH3 and CENH3 from diverged species. Additionally, evidence that a single point mutation is sufficient to generate a haploid inducer provide a simple one-step method for the identification of non-transgenic haploid inducers in existing mutagenized collections of crop species. As proof of the extreme simplicity of this approach to create haploid-inducing lines, we performed an in silico search for previously identified point mutations in CENH3 and identified an Arabidopsis line carrying the A86V substitution within the histone fold domain. This A87V non-transgenic line, while fully fertile on self-pollination, produced postzygotic death and uniparental haploids when crossed to wild type.
Introduction:
Accurate segregation of eukaryotic chromosomes into daughter cells requires the presence of a centromere. Centromeres are, in most species, a region on each chromosome that directs the assembly of the kinetochore during mitosis and meiosis. The kinetochore is a substantial molecular motor, consisting of hundreds of proteins, which regulates and drives the migration of sister chromatids( in mitosis) or homologous chromosomes( in meiosis I) to opposite poles of the cell[1–4]. Centromeres are largely thought to be determined epigenetically by the presence of nucleosomes containing the centromere-specific histone H3 variant CENH3( aka, CENP-A)[5–9]. In stark contrast to conventional histones, which are among the most conserved proteins in eukaryotes, CENH3 is rapidly evolving[10]. CENH3 structure is divided into two domains, a highly variable( in length and sequence) N-terminal tail and the more conserved C-terminal Histone Fold Domain( HFD). Although a handful of amino acids are highly conserved at the N-terminus of the N-terminal tail domain, the rest of the tail is so rapidly evolving that it cannot be aligned even among fairly related clades. For example, among the eudicots known CENH3 N-terminal tails range in length from 23 to 194 amino acids[11]. The HFD is in contrast, relatively well conserved, although it displays signatures of adaptive evolution in some residues[10, 12]. Given the proven role of CENH3 in the specification of the centromere, it is of no surprise that null alleles, though transmissible, are lethal as homozygotes[13–16]. Similarly, defects in the localization of CENH3- either a failure to reload or promiscuous loading to more than one site per chromosome- would be expected to lead to severe genetic abnormalities. Defects in CENH3 loading have been shown to cause chromosomes instability in several organisms, including budding yeast, humans and Arabidopsis[17–19]. Manipulation of CENH3 itself also has dramatic effects on chromosome segregation, an outcome with both basic and applied significance[11, 20–22]. Swapping the CENH3 hypervariable N-terminal tail with that of histone H3. 3-like and concurrent fusion to GFP( “GFP-tailswap”) produces in a partially sterile plants showing meiotic defects. Interestingly, when the GFP-tailswap line is crossed to the wild type, the chromosomes derived from the parent expressing this chimeric protein misseggregate during embryogenesis, resulting in elimination of the corresponding parental genome, producing haploid plants whose chromosomes were derived from only the wild-type CENH3 parent. Maheshwari et al[11], recently demonstrated that transgenic CENH3 genes derived from progressively distant relatives( through the monocot Z. mays), can complement the lethality of a cenh3-/- null mutant of Arabidopsis, and the transgenic plants were fertile. However, when crossed with plants expressing wild-type CENH3, the progeny displayed various degrees of embryonic lethality, aneuploidy and haploidy. Missegregation affected only chromosomes from the parent expressing the distant CENH3. Translation of these discoveries to haploid production in crops would accelerate trait mapping and plant breeding[23–25]. Implementing the GFP-tailswap or transgenic-complementation approach, however, requires two steps. First, a CENH3 knockout( KO) must be obtained, as the haploid induction trait conferred by the variant CENH3 is suppressed by the wild-type CENH3 protein. Second, this KO mutant must be complemented with the chimeric or trans-species transgene, a genetic modification likely to require expensive regulatory approval, which in some cases is unacceptable to the public. These findings pose a basic question. Could a single amino acid change in CENH3 result in the missegregation syndrome, i. e. in a plant which is fertile on self-pollination, but whose centromeres malfunction when confronted zygotically with centromeres determined by wild-type CENH3? To address this, we decided to explore how single amino acid substitutions in CENH3 affect centromere function and chromosome segregation. Here, we show that changes in CENH3 sequence that could be derived naturally( or through simple chemical mutagenesis) can result in haploid induction upon hybridization, apparently without secondary effects on growth and fertility. This finding indicates that single amino acid changes at this rapidly evolving centromeric protein have dramatic consequences on the mutant ability to hybridize. At the same time, it provides a simple, non-transgenic tool for developing haploid inducers in crops.
Discussion:
Our results on the effects of CENH3 single amino acid variation have two major implications, one basic, the other applied. On the basic side, our results reveal that some single amino acid substitutions can be as efficient as large-scale changes in producing haploid inducers. We found that altering single highly conserved amino acid residues in the histone fold domain results in fit and fertile plants that display postzygotic incompatibility and produce haploids when crossed to the wild type. Centromeres determined by point mutations in CENH3 specify efficient chromosome inheritance in self-crosses, but lead to missegregation in an F1 hybrid when confronted with centromeres determined by the wild-type CENH3. As a result, the hybrid embryo undergoes genome elimination, producing frequent abortion( which may be due to aneuploidy-induced failure of the embryo or endosperm), and aneuploidy or haploidy among the surviving seeds. Using human cell lines, Tachiwana et al., 2011[34] have shown that mutations in CENP-A( human CENH3) HFD loop 1 residues R80 and G81 lead to reduced CENP-A retention in the centromere. CENP-A residues L111, L128 and I132 are involved in CENH3/CENH3 interaction[34, 35]. In addition, mutation in CID( Drosophila CENH3) D211 also results reduced dimerization and mislocalization of the protein[36]. Although we do not have complementation data on the corresponding residues in Arabidopsis CENH3, three of our point mutant haploid inducers, P82S, G83E and A86V, are located immediately before the α-N-helix( Fig 1). Based on the crystal structure of CENP-A, Tachiwana et al.[34] proposed that decreased length of the CENP-A α-N-helix compared the homologous region of H3 confers loose conformation to DNA at the entrance and exit of the CENH3 nucleosomes and that the residues corresponding to the At-CENH3 P82, G83, A86 interact with DNA. The loose connection of DNA to CENP-A nucleosome may be important for centromeric function[34]. These mutations may thus alter the fundamental properties of CENH3 nucleosome thus disrupting the normal behavior of centromeric chromatin. Further in the HFD, two of our haploid inducing mutations, A132T and A136T, reside in the CATD domain, which in human CENP-A was shown to interact with HJURP[37], a factor necessary for efficient loading of CENP-A into nucleosomes. Even though the HJURP homolog has not been identified in plants, the KNL2 protein of A. thaliana is related to the factor that recruits HJURP to the centromere[19], suggesting some conservation in CENH3 recruitment to centromeric chromatin. The deleterious post-zygotic defects observed in hybrids of these mutants to wild-type CENH3, are consistent with the possibility of defective loading. Plants carrying the haploid-inducing point mutations described above are fully fertile, thus the gametes produced by these plants obviously carry functional centromeres. However, when encountering centromeres from wild-type plants, the mutant-derived chromosomes missegregate frequently while the wild-type derived chromosomes segregate normally[11, 20, 21]. The striking difference between embryos that inherited the mutant CENH3 from both parents, and those that inherited a mutant and a wild-type allele implies that the mutant-determined centromeres are defective in the context of the wild-type ones. A competition may be set up for some as-of-yet unidentified aspect of centromere specification, kinetochore building, or spindle attachment. Zygotic reloading of CENH3 has been suggested by observation of GFP-tagged CENH3 by Ingouff et al[38]. Defective reloading of CENH3 has been detected in developing embryos in Hordeum crosses leading to natural genome elimination[39]. Accordingly, differential loading rate or density of CENH3 or other centromeric factor between wild-type and “mutant” centromeres could explain why centromeres determined by mutant CENH3 function well in self crosses but fail in out crosses. Our choice of highly conserved amino acids as targets for mutagenesis was largely motivated by our desire to be able to translate our results to crop species. Given the fact that our plants are viable and fully self- fertile, our results raise the question of why these particular amino acids are conserved and what, if at all, is the evolutionary significance of the outcrossing incompatibility determined by the observed changes. Survey of natural variation found that five of the seven changes are conserved. Although no deleterious effect is apparent in our analyses, it is possible that these changes may have hidden or conditional fitness consequences. Four out of the five amino acid substitutions tested result in a penalty on outcrossing, as a large fraction( up to approximately 30%) of outcross progeny spontaneously abort, and the mutant genome is lost from among a smaller fraction of the survivors. It is possible that this outcrossing penalty alone is sufficient to explain the purifying selection of the residues at these particular positions( G83, A86, A132, A136). Two of the residues we tested were not conserved among angiosperms. One of these substitutions( P82S) is a haploid inducer in Arabidopsis, displaying approx. 19% seed death on outcrossing. Nevertheless, this same amino acid change apparently arose and persisted in 4 different clades of angiosperms, ranging from dicots to monocots( S5 Fig, S6 Table)[33]. While we have yet to determine whether this mutation has a reproductively isolating effect in any species other than Arabidopsis, this result suggests that the mutation is well tolerated( as are P82A and P82V). In conclusion, alleles found to be HI-inducing in Arabidopsis have been evolutionarily successful in other plant species. On the applied side, our findings are relevant to plant breeding. Haploids, which can be doubled to produce perfect homozygotes[21, 40–43] greatly accelerate plant breeding[40], genome assembly from sequence reads in heterozygous species[44], the production of recombinant inbred lines[45] and genetic analysis[31], but are not available for many crop species. Haploid induction through the chimeric version of CENH3( GFP-tailswap) has been demonstrated in reverse breeding[46], synthetic clonal reproduction[47] and rapid QTL mapping[45] in the model plant Arabidopsis thaliana, underscoring the potential of this method[48]. The delayed application in crop plants, however, indicates the difficulties in engineering a system that requires combining a chimeric transgene with a knockout of the endogenous gene. A single-step, non-transgenic haploid inducer system such as described here overcomes this shortcoming( Fig 5). To extend its applicability across plant species, this study focused on amino acid residues conserved in angiosperms. It is plausible that single amino acid changes in variable or less conserved residues may have similar effects. The point mutants of CENH3 that can produce uniparental haploids, all G:C to A:T transitions, are readily identified in existing TILLING[49] populations and so can be immediately applied to crop species, or could be induced in a single step by CRISPR-Cas9 mediated changes. Our analysis suggests that there are 47 highly conserved, EMS-mutable targets in the CENH3 histone fold domain, of which 38 are predicted by SIFT to be “not tolerated”. Given the frequency at which we identified haploid inducers among the mutations predicted “not tolerated” by SIFT( 4 out of 5 tested), our results suggest that our list of 38 mutable sites, if found to be able to complement the cenh3-1 null( Fig 1 and S1 Table), would be excellent candidates for haploid induction.
The centromeric histone protein , CENH3 , plays an important role in chromosome segregation during mitosis and meiosis . Here we show that single amino acid changes in CENH3 , while producing no obvious effect on mitosis or meiosis , affect segregation postzygotically upon outcrossing to plants carrying wild-type centromeres . This results in uniparental inheritance among some progeny , and seed death in a larger fraction of progeny . Interestingly , changes competent to induce haploid in Arabidopsis existed in a TILLING population and in unrelated plant species . Our findings have two major consequences . First , uniparental inheritance facilitates the production of haploid plants that can easily be doubled to produce completely homozygous lines in a single generation . Secondly , our findings suggest that natural variation in CENH3 may result in partial reproductive isolation , because chromosomes of the mutant parent from F1 hybrid progeny are culled during embryonic development , while no reproductive defects are observed in self-pollinated plants . We do not know if the same mutations are haploid-inducing in other species , but uniparental chromosome loss , and the seed abortion that accompanies it results in an outcrossing-specific penalty that could potentially be involved in reproductive isolation .