Hybrid incompatibility caused by an epiallele Todd Blevinsa,b,1,2,3, Jing Wangb,1, David Pfliegerc, Frédéric Pontvianneb,4, and Craig S. Pikaarda,b,d,3 aHoward Hughes Medical Institute, Indiana University, Bloomington, IN 47405; bDepartment of Biology, Indiana University, Bloomington, IN 47405; cInstitut de Biologie Moléculaire des Plantes, CNRS UPR2357, Université de Strasbourg, F-67084 Strasbourg, France; and dDepartment of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405 Edited by Jasper Rine, University of California, Berkeley, CA, and approved February 10, 2017 (received for review January 7, 2017) Hybrid incompatibility resulting from deleterious gene combinations replication, such that newly replicated daughter strands of DNA is thought to be an important step toward reproductive isolation and inherit the methylation status of mother strands. speciation. Here, we demonstrate involvement of a silent epiallele in Multicopy transgenes frequently become methylated and si- hybrid incompatibility. In Arabidopsis thaliana accession Cvi-0, one of lenced, particularly when inserted into the genome as inverted the two copies of a duplicated histidine biosynthesis gene, HISN6A,is repeats that can give rise to double-stranded RNAs (26). Such mutated, making HISN6B essential. In contrast, in accession Col-0, double-stranded RNAs can be diced into small interfering RNAs HISN6A is essential because HISN6B is not expressed. Owing to these (siRNAs) that guide the cytosine methylation of homologous DNA differences, Cvi-0 × Col-0 hybrid progeny that are homozygous for sequences, a process known as RNA-directed DNA methylation both Cvi-0 HISN6A and Col-0 HISN6B do not survive. We show that (RdDM) (27, 28). Known cases of silencing that involve duplicated HISN6B of Col-0 is not a defective pseudogene, but a stably silenced endogenous genes, such as the phosphoribosylanthranilate isom- epiallele. Mutating HISTONE DEACETYLASE 6 (HDA6), or the cytosine erase (PAI) or folate transporter (AtFOLT)genesofA. thaliana, methyltransferase genes MET1 or CMT3, erases HISN6B’s silent locus resemble transgene silencing in that complex sequence rear- identity, reanimating the gene to circumvent hisn6a lethality and rangements coincide with duplication of a gene to create a novel hybrid incompatibility. These results show that HISN6-dependent hy- (nonancestral) locus engendering siRNA production and homol- brid lethality is a revertible epigenetic phenomenon and provide ogy-dependent DNA methylation (29, 30). Silencing of the an- additional evidence that epigenetic variation has the potential to cestral AtFOLT1 gene, directed by siRNAs derived from the novel limit gene flow between diverging populations of a species. AtFOLT2 locus present in some ecotypes, causes reduced fertil- ity in ecotype-specific hybrid combinations (29). This interesting gene silencing | epigenetic inheritance | DNA methylation | case study has shown that naturally occurring RdDM, involving a histone deacetylation | hybrid incompatibility new paralog that inactivates the ancestral paralog in trans,canbea cause of hybrid incompatibility. utations that accumulate in separate subpopulations of a Here, we demonstrate an epigenetic basis for a previously Mspecies can facilitate reproductive isolation by engender- identified case of hybrid incompatibility (31) involving a gene ing hybrid incompatibility, a reduction in fitness observed among pair, HISN6A and HISN6B. The underlying mechanism of hybrid progeny at the F1 or F2 generation (1–3). Bateson (4), HISN6-based hybrid incompatibility was previously unknown. Dobzhansky (5), and Muller (6) independently proposed that gene We show that the HISN6B gene of ecotype Col-0, and hundreds mutations made benign by compensatory mutations in interacting of other A. thaliana natural accessions, is hypermethylated in its genes within isolated subpopulations prove deleterious when promoter region and is epigenetically silent, making its paralog, subpopulations or ecotypes interbreed, thereby contributing to HISN6A, essential. Inheritance of the silent HISN6B epiallele speciation by reducing gene flow. Examples of this include the lethal consequences of a Saccharomyces cerevisiae protein direct- Significance ing improper splicing of essential Saccharomyces bayanus mRNAs × in S. cerevisiae S. bayanus hybrids (3) or the hybrid necrosis that Deleterious mutations in different copies of a duplicated gene pair results from expression of incompatible innate immune receptors have the potential to cause hybrid incompatibility between di- in Arabidopsis thaliana (1). verging subpopulations, contributing to reproductive isolation and Lynch and Force (7) envisioned an alternative scenario by which speciation. This study demonstrates a case of epigenetic gene si- hybrid incompatibilities might arise, as a result of gene duplication. lencing rather than pseudogene creation by mutation, contribut- Gene duplication initially results in gene redundancy, thereby ing to a lethal gene combination on hybridization of two ecotypes relaxing constraints on sequence and functional divergence, but of Arabidopsis thaliana. Our findings provide direct evidence that mutations most often transform one paralog into a nonfunctional naturally occurring epigenetic variation can contribute to in- – pseudogene (8 10). Lynch and Force recognized that asymmetric compatible hybrid genotypes, reducing gene flow between sub- resolution of gene duplicates in this manner could result in either types of the same species. paralog becoming the sole operational copy in different subpopula- tions of a species, such that hybrid progeny inheriting nonfunctional Author contributions: T.B. and C.S.P. designed research; T.B., J.W., D.P., and F.P. performed alleles of both subpopulations would suffer reduced fitness (7). research; T.B. and C.S.P. analyzed data; and T.B. and C.S.P. wrote the paper. Nonmutational (epigenetic) gene silencing could potentially The authors declare no conflict of interest. contribute to hybrid incompatibility via the scenario envisioned by This article is a PNAS Direct Submission. Lynch and Force. In plants, silent epialleles segregating in Mende- Freely available online through the PNAS open access option. lian fashion can be stably inherited over many generations, and are See Commentary on page 3558. known to affect a number of well-studied traits, including flower 1T.B. and J.W. contributed equally to this work. morphology (11, 12), fruit ripening (13), and sex determination (14). 2Present address: Institut de Biologie Moléculaire des Plantes, CNRS UPR2357, Université Silent epialleles are inherited via the perpetuation of repressive de Strasbourg, F-67084 Strasbourg, France. chromatin states, and various plant chromatin-modifying enzymes 3To whom correspondence may be addressed. Email: [email protected] or are known to participate in epigenetic inheritance, including the [email protected]. Rpd3-like histone deacetylase HDA6, the ATP-dependent chro- 4Present address: Laboratoire Génomes et Développement des Plantes, Université de matin remodeler DDM1, and the DNA methyltransferases MET1 Perpignan Via Domitia, CNRS UMR5096, F-66860 Perpignan, France. and CMT3 (15-25). These enzymes are important for maintaining This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. patterns of cytosine methylation following each round of DNA 1073/pnas.1700368114/-/DCSupplemental. 3702–3707 | PNAS | April 4, 2017 | vol. 114 | no. 14 www.pnas.org/cgi/doi/10.1073/pnas.1700368114 Downloaded by guest on October 2, 2021 requires HDA6-, MET1-, and CMT3-dependent cytosine methyl- in the histidine biosynthesis pathway (32, 33) (Fig. 1B). The gene ation, but is unaffected by mutations disrupting RdDM. Although duplication occurred after A. thaliana diverged from a common SEE COMMENTARY methylated HISN6B epialleles can be stably inherited for at least ancestor with Arabidopsis lyrata (34, 35), resulting in duplication of 30 generations, they can revert to an active state if the epigeneti- a segment of chromosome 1 containing HISN6B (At1g71920) on cally silent state is erased by passage through an hda6 mutant chromosome 5, yielding the HISN6A locus, At5g10330 (33, 36). background. This allows HISN6B to now rescue hisn6a null mu- Although HISN6A and HISN6B coding regions are 100% identical tations in ecotype Col-0 and to restore compatibility with ecotype at the amino acid level, homozygous hisn6a null mutations are Cvi-0, in which HISN6A is defective owing to an internal deletion. embryo-lethal in ecotype Col-0, because HISN6B is not expressed Collectively, our results demonstrate that HISN6-dependent hy- (Fig. 1 B and C) (31, 32). This differential expression of HISN6A brid lethality is a previously unrecognized epigenetic phenomenon. and HISN6B can be observed by RT-PCR amplification followed by digestion with RsaI, which cuts only HISN6A cDNA (Fig. 1C). In Results wild-type (WT) Col-0, only HISN6A is expressed (Fig. 1D); however, HISN6A and HISN6B are duplicated, paralogous genes (Fig. 1A in hda6-6 or hda6-7 mutants (37, 38), HISN6B is expressed as well and Fig. S1) encoding histidinol-phosphate aminotransferase, (Fig. 1D),whichisalsotrueinmutantsforMET1 or CMT3,which which converts imidazole-acetol phosphate to histidinol-phosphate encode enzymes responsible for cytosine maintenance methylation A B C DG no RT no RT E F methylated GENETICS Fig. 1. HDA6, CMT3, and MET1 silence HISN6B via promoter region DNA methylation.