The merging of two divergent genomes in a hybrid is believed to trigger a “genomic shock”, disrupting gene regulation and transposable element (TE) silencing. Here, we tested this expectation by comparing the pattern of expression of transposable elements in their native and hybrid genomic context. For this, we sequenced the transcriptome of the Arabidopsis thaliana genotype Col-0, the A. lyrata genotype MN47 and their F1 hybrid. Contrary to expectations, we observe that the level of TE expression in the hybrid is strongly correlated to levels in the parental species. We detect that at most 1.1% of expressed transposable elements belonging to two speciﬁc subfamilies change their expression level upon hybridization. Most of these changes, however, are of small magnitude. We observe that the few hybrid-speciﬁc modiﬁcations in TE expression are more likely to occur when TE insertions are close to genes. In addition, changes in epigenetic histone marks H3K9me2 and H3K27me3 following hybridization do not coincide with TEs with changed expression. Finally, we further examined TE expression in parents and hybrids exposed to severe dehydration stress. Despite the major reorganization of gene and TE expression by stress, we observe that hybridization does not lead to increased disorganization of TE expression in the hybrid. Although our study did not examine TE transposition activity in hybrids, the examination of the transcriptome shows that TE expression is globally robust to hybridization. The term “genomic shock” is perhaps not appropriate to describe transcrip- tional modiﬁcation in a viable hybrid merging divergent genomes. Key words: hybridization, genomic shock, transposable element, stress, Arabidopsis. Introduction Yet, a recent study of the homoploid hybrid of Arabidopsis Interspeciﬁc hybridization is an important factor in plant thaliana and Arabidopsis lyrata did not report a major disrup- evolution: While fertile allopolyploid hybrids may become tion of the transcriptome and epigenome. Moderate differ- the founders of new species (Soltis and Soltis 2009), the ences in expression of protein-coding genes were reported merging of two divergent genomes in a hybrid can also and the parental pattern of the histone mark H3K27me3 cause mild outbreeding depression or even result in com- appeared to be maintained (Zhu et al. 2017). These species, plete incompatibility (Bomblies and Weigel 2007; Todesco however, differ in both genomic structure and TE content. et al. 2016). At the molecular level, hybridization can lead A recent increase in TE number has been reported for the to a genome-wide misregulation of the transcriptome and A. lyrata genome (Hu et al. 2011). Arabidopsis thaliana TEs epigenome (Lafon-Placette and Ko¨hler 2015). The mobili- are concentrated in pericentromeric regions, rarely venturing zation of up-regulated transposable elements (TE) can fur- in gene rich chromosome arms. By contrast, in A. lyrata TEs ther result in chromosomal rearrangements. Such account for most of the 40% larger genome and are broadly phenomena, ﬁrst described by Barbara McClintock in distributed, occurring in closer vicinity to expressed genes (Hu maize (McClintock 1984) and termed “genomic shock”, et al. 2011). The relatively low frequency of insertion poly- are thought to provide a postzygotic barrier against gene morphisms within species revealed evolutionary tensions on ﬂow between species. insertions in gene rich regions, where permanent TE silencing The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non- commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact email@example.com Genome Biol. Evol. 10(6):1403–1415. doi:10.1093/gbe/evy095 Advance Access publication May 18, 2018 1403 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1403/4999384 by Ed 'DeepDyve' Gillespie user on 20 June 2018 Go ¨ bel et al. GBE can have deleterious consequences on the expression of 1-week intervals. Two micrograms of total RNA were used for neighboring genes (Hollister and Gaut 2009; Lockton and library preparation following the TruSeq Illumina RNA Sample Gaut 2010; Hollister et al. 2011). Finally, several studies Preparation v2 Guide. This includes poly-Aþ RNA selection have observed that A. lyrata TEs tended to be more highly and the use of random primers for reverse-transcription. expressed than A. thaliana alleles in hybrids (He et al. 2012). In Sequencing was performed on Illumina HiSeq2000 following view of these differences in number, distribution, and regula- the manufacturer’s protocols and paired-end 100-bp-long tion of transposable elements, we hypothesized that for reads were obtained. In total, 15–20 million paired reads for hybrids between these species, a genomic shock is more likely the parents and 30–40 million reads for the hybrid were pro- to occur at the level of TE expression. To test this hypothesis, duced (supplementary table 1, Supplementary Material on- we quantiﬁed the impact of parental versus hybrid genomic line). RNA-seq reads were ﬁltered and trimmed for quality background on TE regulation. controlasin He et al. (2016) and mapped on the hybrid ge- We studied transposable elements in the A. thaliana (Col- nome, a concatenation of the A. thaliana Col-0 reference 0) A. lyrata (MN47) hybrid and its respective parental lines, genome (TAIR10, www.arabidopsis.org; last accessed may using RNA-Seq expression data and ChIP-seq data of the his- 25, 2018) and the A. lyrata MN47 reference genome tone marks H3K27me3 and H3K9me2. It is difﬁcult to estab- (Araly1, Nordberg et al. ), using STAR with default lish orthology relationships for TEs, because many of them parameters (Dobin et al. 2013). duplicated after the species separation (Hu et al. 2011; de la Chaux et al. 2012). Thus, our analysis focuses on trans-acting effects experienced by TEs of each genome after hybridiza- tion. Contrary to our primary expectations, we ﬁnd TE silenc- ing to be largely unaffected in the hybrid. No systematic TE and Gene Read Counting relationship could be found between a change of TE expres- sion in the hybrid and the gain or loss of histone marks. We The gene annotations TAIR10 (Col-0, Berardini et al. 2015), further exposed interspeciﬁc hybrids and their parents to se- Alyrata_107_v1.0 (MN47, Hu et al. 2011) and TE annotations vere dehydration stress and conﬁrm that TE regulation (e.g., position and TE family memberships) for A. lyrata and A. remains robust to hybridization in stress conditions. thaliana (Pietzenuk et al. 2016) were merged to form the genome annotation ﬁle used in this analysis. Aligned reads were ﬁltered as follows: for the MN47 genome, only the main Materials and Methods scaffolds 1–8 were considered. Read or fragment alignments Plant Materials and Growth Conditions shorter than 20 or longer than 700 base pairs (bp) were dis- carded. A minimum alignment score of 33 was required in Seeds of A. thaliana accession Col-0 were obtained from the order to accept an RNA-seq match. We allowed multiple Arabidopsis Biological Resource Center (ABRC, USA). RNA-seq read matches on TEs if they were entirely within a Arabidopsis lyrata ssp. lyrata genotype MN47 was obtained single (super)family, following Pietzenuk et al. (2016), and Jin from the lab of D. Weigel (Max Planck Institute for et al. (2015). A read was assigned to a (super)family F on Development, Tu ¨ bingen, Germany). Parental lines were genome G if its primary match was in F on G, and any sec- crossed by pollinating emasculated A. thaliana ﬂowers with ondary matches on G were in F, too. In order to assign a A. lyrata pollen, as described in De Meaux et al. (2006).We match to a (super)family F, a minimum overlap of 50 bp to obtained approximately 40 seeds per silique, 90% of which a member of F was required. Secondary matches on the al- aborted. We did not use embryo rescue to generate more ternative genome were allowed but not counted. Each read hybrids as in Zhu et al. (2017). Reciprocal crosses using thus either contributed a single count to a (super)family or A. thaliana as pollen donor were not successful. was discarded as multiply matching. If a read matched to TE .. TE of family F, the read was deﬁned to contribute 1 n RNA Sampling and Sequencing in Standard and Stress 1/n counts to each of these TEs. Reads from a region of over- Conditions lap a TE and a gene were not discarded, but counted with the Seeds were stratiﬁed for 3 days at 4 C, germinated on soil and TE. These summed fractional per-element counts were the grown for 4 weeks in a growth chamber at 20 C under 14 h ﬁnal output from the counting procedure. They were either 1 2 light/16 C 10 h dark under dim light (100 mmol s m ). A used directly as counts per individual TE or the counts of the dehydration treatment was applied following Seki et al. members of (super)families were summed up to yield aggre- (2002). Plants were removed from the soil and dehydrated gated per-(super)family counts. We implemented the count- on paper for 0 and 3h, in the same growth chamber. The ing procedure in the R programming language. After the aerial part of the plant was sampled, ﬂash-frozen in liquid counting step, annotated TEs without any read count were nitrogen, and RNA was extracted as previously described excluded from the analysis. Genes were counted with the (He et al. 2016). Four biological replicates were collected in same procedure as the TEs. 1404 Genome Biol. Evol. 10(6):1403–1415 doi:10.1093/gbe/evy095 Advance Access publication May 18, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1403/4999384 by Ed 'DeepDyve' Gillespie user on 20 June 2018 Robustness of Transposable Element Regulation GBE Read Count Normalization each bin of the reference sample, the same number of TEs as contained in the bin was randomly selected from the corre- Read counts from genes and TEs were simultaneously nor- sponding distance bin of all TEs. The union of the resampled malized. Each count was ﬁrst multiplied by a factor of 1,000/l, bins then formed one resampled set. The width of the bins where l is the length in base pairs of the respective annotated increased with increasing distance: up to a distance of 1 kb, element (gene, individual TE, or entire TE (super)family). This the bin width was 200 base pairs (bp), between 1 and 5 kb it removes the dependence of the read count on element length was 500 bp, and beyond 5 kb it was 1,000 bp, to avoid having or (super)family size. The total length of a TE (super)family was bins with sparse numbers of TEs. A median, 5% and 95% deﬁned to equal the summed length of all (super)family mem- quantile of each superfamily frequency was extracted from bers considered, discounting any regions of overlap with the 10,000 random samples and compared with the observed other annotated elements. frequency of TEs with hybrid-dependent expression. Differential Expression Analysis The Generalized Linear Models (GLM) support of the R pack- Chromatin Immune-Precipitation Experiments age edgeR (Robinson et al. 2010; McCarthy et al. 2012)was used to test the signiﬁcance of differences in length- Plants were germinated and grown on germination medium normalized counts between the four biological replicates of containing = Murashige and Skoog salts, 3% sucrose, and hybrid and parental transcriptomes. P values were adjusted 0.8% agar. Seeds were stratiﬁed for 5 days at 4 C, and then with the FDR correction (Benjamini and Hochberg 1995). For transferred to a growth chamber under long-day conditions visualization in scatterplots and for the stringent deﬁnition of (14 h cool white light supplemented with red light at 20 C, differential TE expression, length-normalized counts were and 10 h dark at 18 C). One week after germination, plants convertedtoCounts per Million(CPM) andscaled were transferred to soil and grown in the same chamber. (Robinson and Oshlack 2010) using the calcNormFactors Finally, leaves larger than 0.5 cm were sampled from function of the R package edgeR. This transformation mirrors 3-week-old Col-0, and 6-week-old MN47 and F1 hybrids what edgeR does internally when computing differential ex- for chromatin immunoprecipitation (ChIP). Leaves from 10 pression. It removes global count biases due to library size to 15 plants were pooled. Sampling was shifted in the hybrid differences, easing the interpretation of the scatterplots and and the A. lyrata individual, in order to sample material of allowing the set-up of a sample-independent cutoff on the similar development (3 cm rosette diameter). absolute expression value at 0 and 10 CPM. ChIP experiments were performed with the MAGnify Chromatin Immunoprecipitation System (49-2024, Thermo Fisher Scientiﬁc) according to the manufacturer’s instructions, Analysis of Distance Distribution of TEs to Neighboring with the following modiﬁcations. Plant material was ﬁxed in Genes 20 ml Crosslinking Buffer (0.4 M Sucrose, 10 mM Tris–HCl, pH Distances of differential and nondifferential TEs to the nearest 8.0, 10 mM MgCl2, 1% formaldehyde) by applying vacuum gene or gene 5 -end were binned into classes 0–1, 1–2, 2–3, twice for 15 min. Nuclei and chromatin were puriﬁed as in He and 3 kb. (TEs overlapping a gene were not considered.) A et al. (2012). Chromatin was sonicated with a BioRuptor de- pseudocount of 1 was added to each bin count. A saturated vice (Diagenode, Lie ` ge, Belgium) for 30 times 30 s at high log-linear model of the counts was computed using the R setting with 30 s intermittent cooling in ice-water. A DNA frag- glm() function (family ¼ “poisson”), using sum-to-zero con- ment size of 300–600 bp was controlled by running an aliquot trasts. The P value for distance d in supplementary table 1, of decrosslinked and puriﬁed DNA on 1.5% agarose gels. The Supplementary Material online, is the Pr(>jzj) value of the following antibodies were used in immunoprecipitation: anti- interaction coefﬁcient between the factors distance category rat IgG (R9255, Sigma; St. Louis, MO), as well as anti- and signiﬁcance of hybrid-dependent TE expression. H3K27me3, anti-H3K9me2 and anti-H3 (07-449, 07-441, and 07-690, respectively, Millipore; Temecula, CA). To reach Random Expectations of Superfamily Frequencies enough precipitated DNA, the ChIP-DNA precipitation proto- To evaluate the enrichment of TEs changing their expression col was performed independently for eight aliquots of the in hybrid background within each superfamily, we computed collected leaf material, pooled and puriﬁed by Qiagen a random expectation. For this, we used the set of TEs show- MinElute Reaction Cleanup Kit. Finally, we obtained ChIP- ing differential expression in the hybrid background as a ref- DNA samples from Col-0, MN47, and Col-0xMN47 with anti- erence set and performed 10,000 random resamplings of the bodies targeting H3K27me3 and H3K9me2 marks. In addi- same number of TEs from all TEs with evidence of expression. tion, we generated a ChIP-DNA sample targeting Histone 3 To account for the skewed distribution of these TEs in the in a Col-0xMN47 sample to verify that both genomes could be vicinity of non-TE genes, both the reference set and the set of efﬁciently immuno-precipitated and assess ChIP-seq quality all TEs were binned by distance to the closest gene 5 -end. For (see supplementary Methods, Supplementary Material online). Genome Biol. Evol. 10(6):1403–1415 doi:10.1093/gbe/evy095 Advance Access publication May 18, 2018 1405 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1403/4999384 by Ed 'DeepDyve' Gillespie user on 20 June 2018 Go ¨ bel et al. GBE Table 1 Numbers and Fractions of Differentially Expressed TEs at Two Different False-Discovery Rates (FDR 0.01 or0.05) and Two Different Thresholds on the Normalized Counts Per Million (CPM) (all: 0CPM; 10 CPM) FDR £ 0.01 FDR £ 0.05 All 10 CPM All 10 CPM Arabidopsis thaliana 3 þ 2(0.3%) 1 þ 1(0.1%) 5 þ 5(0.6%) 1 þ 2(0.2%) Arabidopsis lyrata 57 þ 2(0.8%) 14 þ 0(0.2%) 73 þ 6(1.1%) 18 þ 1(0.3%) NOTE.—Numbers are given as “number of up-regulations” þ “number of down-regulations.” Fractions were computed relative to all TEs with evidence of expression in standard conditions, discounting members of genome-speciﬁc families. reads were ﬁltered with samtools (Li et al. 2009)for a map- ChIP-Seq Library Preparation and Sequencing ping quality of 3 (-q 3), which discards multimapping reads DNA was sheared with Ion Shear reagents and the Ion Xpress with two equally good mappings but retains those with a Fragment Library Kit (Thermo Fisher Scientiﬁc, Waltham, MA) single best mapping location. Duplicate reads from the am- was used for subsequent library preparation. [AQ10] After pliﬁcation step during library preparation were removed with ligation of the respective Ion Xpress barcode adapters, sam- the MarkDuplicates program from Picard Tools (http://broad- ples were ampliﬁed by 17 or 23 PCR cycles. Ampliﬁed libraries institute.github.io/picard; last accessed May 25, 2018). ChIP- were quantiﬁed on Bioanalyzer DNA High-Sensitivity Chips seq read counts for TEs and non-TE genes were counted fol- (Agilent Technologies, Santa Clara, CA) and diluted to lowing the same pipeline as for RNAseq reads. A ﬁrst analysis 9 pM. Emulsion PCR was performed on the Ion OneTouch of ChIP-seq count distribution conﬁrmed that the distribution System, followed by enrichment for template positive Ion of marks recovered known chromatin domains but revealed Sphere Particles. Sequencing sample was loaded on an Ion 64 genomic regions giving aberrant ChIP signals (see supple- Proton chip and sequenced on the Ion proton sequencer mentary Methods, Supplementary Material online). These 64 (Thermo Fisher Scientiﬁc) according to the manufacturer’s regions were removed from all analyses presented in the instructions. manuscript. ChIP-Seq Read Processing, Mapping and Peak Detection Results Raw data were preprocessed using the TORRENT SUITE ver- Only a Minor Fraction of All Expressed TEs Is Differentially sion 4.0.1 (Thermo Fisher Scientiﬁc) to trim adapter sequen- Expressedinthe Hybrid ces. Using the FASTX-Toolkit (http://hannonlab.cshl.edu/ fastx_toolkit/; last accessed May 25, 2018), reads were then Our annotation contains 10863 TEs on the A. thaliana Col- trimmed from the 3 end to remove low quality bases (phred 0 genome and 30,868 elements on the A. lyrata MN47 <15). Reads shorter than 30 bases and reads of poor overall genome. After excluding genome-speciﬁc TE families, quality (more than half of the bases with phred< 20) were 10,805 and 29,150 elements remained on the Col-0 and discarded. The mapping of the reads was performed with MN47 genomes, respectively. Of these, 1,592 (15%) of the bowtie2 (Langmead and Salzberg 2012), with the end-to- A. thaliana TEs and 7,045 (24%) of the A. lyrata TEs end parameter, without allowing mismatches in the seed (- showed evidence of expression in at least one replicate. N 0), and allowing for up to two alignments per read (-k 2); We quantiﬁed TE expression in normalized Counts Per although then only the best alignment was kept. All samples Million (CPM, see Materials and Methods). TEs contributed were mapped to a hybrid reference genome obtained by but a minor fraction of all reads (on average, only 0.07% in concatenating the genomes of the parent as described above A. thaliana samples and 0.24% in A. lyrata samples). This for RNAseq read mapping. Cross-hybridization between fraction remained comparable in the hybrid transcriptome genomes was negligible (0.5–1% of mapped reads). A sum- (on average 0.06% and 0.30% of A. thaliana and A. lyrata mary of ChIP seq read data are given in supplementary table reads, respectively in the hybrid). Only a small fraction 2, Supplementary Material online. (maximally 2%) of the expressed TEs of both genomes Quality assessment of the assays was performed using were signiﬁcantly differentially expressed between paren- phantompeaktools (Kharchenko et al. 2008; Landt et al. tal and hybrid backgrounds, using different cutoffs to de- 2012) to calculate NSC and RSC metrics (normalized and rel- ﬁne signiﬁcance (table 1). Moreover, this percentage ative strand coefﬁcients, respectively), and the bioconductor dropped below 0.5% when the median expression was ChIPQC package (Carroll et al. 2014) to calculate the square required to be above ten CPM in at least one of the two sum of deviation. NSC was low but RSC metrics were well backgrounds (table 1). These observations show that TE within the recommended range (Landt et al. 2012). Mapped transcription is not severely disturbed in the hybrid. 1406 Genome Biol. Evol. 10(6):1403–1415 doi:10.1093/gbe/evy095 Advance Access publication May 18, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1403/4999384 by Ed 'DeepDyve' Gillespie user on 20 June 2018 Robustness of Transposable Element Regulation GBE FIG.1.—Correlation of TE expression levels, estimated as the median of normalized counts per million over the replicate plants grown in standard conditions, in parent and hybrid for individual TEs, TE families, and superfamilies. TEs or (super)families with signiﬁcant expression change are marked in red. In A. lyrata, the 59 (0.8%) TEs that respond signiﬁcantly to Speciﬁc Rather than Global Effects Act on TE Expression in hybridization at FDR 0.01 were generally induced. In A. the Hybrid thaliana, the 5 (0.3%) affected elements were either induced In order to investigate the effect of hybridization at the levels or repressed (supplementary ﬁg. S1, Supplementary Material of individual elements, families and superfamilies of TEs, we online). SINEs dominated by Sadhu elements (Rangwala and correlated expression of individual TEs and combined expres- Richards 2010) and Harbingers dominated by the ATIS112A sion levels of TE families between parents and hybrids (ﬁg. 1). family (Kapitonov and Jurka 2004) stood out as enriched Although TE transcription in the two genetic backgrounds among the responding A. lyrata TEs (ﬁg. 2). These superfami- correlated well at all levels, the correlation increased from lies were signiﬁcantly overrepresented relative to the set of the individual TEs to the family and superfamily levels. expressed TEs (1 RNA-seq count, permutation test, Spearman’s rank correlation of the median expression of rep- FDR< 0.05). The fact that only a small number of TE families licates in the two backgrounds under control conditions was showed enrichment for differential expressed elements indi- 0.73, 0.89, 0.97 on the three levels of individual TE, family, cates that hybridization does not have a global effect on TE and superfamily, respectively in A. thaliana, and 0.65, 0.86, expression. 0.91, respectively, in A. lyrata. Including four replicates in the RNA-seq analysis was necessary to establish these relation- ships with increased conﬁdence. Using subsets of only two Rare TE Expression Changes Coincide with Alterations in replicates, for example, reduced the parent–hybrid correlation Nearby Gene Expression coefﬁcient of TE expression to 0.53, 0.77, 0.87 on the three levels for A. thaliana, and to 0.51, 0.84, 0.89, respectively, for TEs can change expression of nearby genes by inﬂuencing A. lyrata. local chromatin accessibility. We therefore asked whether The small subset of TEs with signiﬁcantly different expres- hybrid-speciﬁc expression changes of TEs predict a corre- sion level between parent and hybrid was restricted to lower sponding change in neighboring genes. level units (individual TEs and families), indicating that the Compared with TEs with expression not affected by hybrid- differences are case-speciﬁc rather than caused by a global ization, A. lyrata TEs expressed differentially in the hybrid were disruption of TE silencing. depleted in genome regions distant more than 3kb from Genome Biol. Evol. 10(6):1403–1415 doi:10.1093/gbe/evy095 Advance Access publication May 18, 2018 1407 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1403/4999384 by Ed 'DeepDyve' Gillespie user on 20 June 2018 Go ¨ bel et al. GBE FIG.2.—Observed and expected superfamily distribution of differentially expressed TEs (parent vs. hybrid). Colored bars show the observed numbers of TEs showing hybrid-dependent expression in each TE superfamily. Each color corresponds to one superfamily as indicated in the legend. Superfamily names are as given in the annotation of (Pietzenuk et al. 2016). Names with trailing question marks were annotated as deviant/uncertain members of the respective superfamily. Transparent bars indicate expected median count for random sampling within each superfamily. Error bars indicate the 5% and 95% quantile of the 10,000 random draws. Single black stars mark superfamilies whose observed count is signiﬁcantly higher than expected from random expectations. Gray stars indicate depletion (maximally 5% of the universe have counts the observed count of the respective superfamily). Double stars indicate signiﬁcant enrichment or depletion that is robust to FDR correction. Table 2 Distance Distributions to Nearby Genes of Arabidopsis lyrata TEs with Differential Expression in Parent and Hybrid Under Control Conditions Distance to Closest Gene Closest 5 End of a Gene <1 kb 1–2 kb 2–3 kb 3kb <1 kb 1–2 kb 2–3 kb 3kb Number of TEs Diff. exp. PvsH 15 18 13 13 7 24 12 16 Not Diff. Expr. 1,238 1,675 913 3,160 654 1,455 1,078 3,799 P (frequency in distance bin) 0.239 0.539 0.061 4.0e-4 0.554 0.01 0.517 1.5e-4 NOTE.—The table combines distance information of TEs with a signiﬁcant change in expression in the hybrid to either the nearest gene (any orientation) or the nearest 5 of any gene. Numbers of signiﬁcant and nonsigniﬁcant TEs are listed for each distance bin (FDR 0.01). In the lower part, P values are given for the contribution of each distance bin to the difference between the distributions of signiﬁcant and nonsigniﬁcant TEs (see Materials and Methods). Bins with signiﬁcant depletion or enrichment in signiﬁcant TEs are highlighted in blue and orange, respectively. 0 4 the closest gene 5 ends (log-linear model, P 5 10 , preferential location of hybridization-sensitive TEs in euchro- table 2). In addition, counts of hybridization-sensitive TEs matic, gene-rich regions in A. lyrata.In A. thaliana, there were were enriched in the bins 1–2 kb upstream of gene 5 -ends too few differentially expressed TEs in the hybrid background (log-linear model, P 0.01, table 2). This indicates a to reliably compare the distance distributions. 1408 Genome Biol. Evol. 10(6):1403–1415 doi:10.1093/gbe/evy095 Advance Access publication May 18, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1403/4999384 by Ed 'DeepDyve' Gillespie user on 20 June 2018 Robustness of Transposable Element Regulation GBE We further analyzed if changed expression of TEs and their neighboring genes in a hybrid background were correlated. Hybrid-speciﬁc changes in transcription levels of non-TE genes have already been reported (Zhu et al. 2017). We also quan- tiﬁed non-TE gene expression levels in parents and hybrids. Note that in our experiment, the timing of sampling accounted for the developmental differences between the fast developer A. thaliana and both A. lyrata and the hybrid, which are late ﬂowering. Markedly fewer genes were af- fected by hybridization in our experiment (234 in A. thaliana and 395 in A. lyrata), yet, we observed a signiﬁcant overlap in genes reported to change expression in the hybrid back- ground in Zhu et al. (2017) (Hypergeometric test of overlap in genes changing their expression level between hybrid and parents, P< 10 for all comparisons). In A. lyrata 153 TEs with detectable expression were within 2 kb of a gene with hybridization-sensitive expression (FDR 0.01). Among these, 8 (5%) TEs displayed a change in expression in the hybrid background. This percentage dropped to 1% (71/6,892) for TEs with a gene neighbor showing no signiﬁcant change in expression in hybrid versus parent (P< 5 10 ,hypergeo- metric test). In the A. thaliana genome, the number of expressed TEs located in the vicinity of genes was too small to calculate correlations. We conclude that hybrid-speciﬁc TE expression can coincide with a modiﬁcation of the expression of nearby protein-coding genes, but this only affects a very small number of expressed loci. Histone Marks Are Not Modiﬁed in the Hybrid Background Two epigenetic marks are associated with transcriptional silencing in plants: the plastic mark H3K27me3 and the permanent mark H3K9me2 (Kim and Zilberman 2014; Sequeira-Mendes et al. 2014). We proﬁled these two histone modiﬁcations by Chromatin Immuno-Precipitation followed by sequencing (ChIP-Seq) in hybrids and A. thaliana and A. lyrata parents. Although the ChIP-seq H3K27me3 yielded a comparatively lower number of reads, especially for Col-0 (supplementary tables 2, 5,and 6, Supplementary Material online), the distribution of marks on TEs was in agreement with previous reports (ﬁg. 3A, Seymour et al. 2014) and cap- tured well the chromatin domains that were deﬁned in A. thaliana (Sequeira-Mendes et al. 2014, supplementary Methods, Supplementary Material online). In our data, H3K9me2 marks showed a high correlation of normalized read counts between parental and hybrid genomes, both genome-wide (calculated across 10-kb genomic windows) and for individual TEs, TE families, and superfamilies. The FIG.3—Correlation of histone mark levels on TEs in parent and hybrid. genome-wide Spearman correlation coefﬁcients of (A) Genome-wide distribution of H3K9me2 and H3K27me2 on the pa- H3K9me2 read count along the genomes were 0.66 and rental and hybrid genomes. The circular plots represent the hybrid genome used as reference, with the ﬁve A. thaliana chromosomes in green and the eight A. lyrata chromosomes in red. Inner tracks represent gene, TE, FIG.3—Continued H3K9me2 and H3K27me3 densities in 500 kb bins. Top left: Col-0, Top counts. The color code indicates superfamily membership, using the right: MN47, Bottom: hybrid. (B and C) Scatterplots of ChIP-seq read palette of ﬁgure 2. (B) H3K9me2; (C) H3K27me3. Genome Biol. Evol. 10(6):1403–1415 doi:10.1093/gbe/evy095 Advance Access publication May 18, 2018 1409 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1403/4999384 by Ed 'DeepDyve' Gillespie user on 20 June 2018 Go ¨ bel et al. GBE FIG.4—No change in histone mark occupancy is detected for differentially expressed TEs. Shown are scatterplots of each histone marks on each of the two genomes for TEs with signiﬁcantly different expression level in parent versus hybrid under standard conditions. Superfamily membership of the TEs is indicated using the palette of ﬁgure 2. Symbol shapes indicate the direction of gene expression change in hybrid versus parent (upward triangle: up, downward: down). 0.92 for A. thaliana or A. lyrata, respectively. Spearman’s rank Nevertheless, the correlation of H3K27me3 occupancy in correlation coefﬁcients of H3K9me2 for individual TEs, TE- parent and hybrid remained high even for TEs, in A. lyrata families, and superfamilies were 0.76, 0.94, and 0.96 for A. (Spearman’s rank correlation 0.58, 0.78, 0.92 for individual thaliana and 0.87, 0.97, and 0.99 for A. lyrata (ﬁg. 3B and C). TEs, families, and superfamilies). For the A. thaliana/hybrid Although the high correlation values pointed to a largely ad- comparison, the correlation dropped to 0.2, 0.27, 0.21 for ditive inheritance of the mark in the hybrid on all levels of the individual TEs, families, and superfamilies, respectively annotation, the fact that correlation was lowest on the level (ﬁg. 3C). Note however that the Col-0 H3K27me3 ChIP- of individual elements again underscores the role of element- seq experiments yielded a comparatively lower number of speciﬁc responses in the hybrid. reads (supplementary table 2, Supplementary Material While the function of H3K9me2 is to permanently silence online). TEs, H3K27me3 mainly serves to reversibly silence genes We tested then whether changes in histone mark occu- whose expression is restricted to speciﬁc conditions or de- pancy could explain TE expression differences between velopmental time windows (Gan et al. 2015). The distribu- parents and hybrids. TEs with a modiﬁed expression level in tion of H3K27me3 marks was strongly correlated genome- hybrid background generally showed similar H3K27me3 level wide between parental and hybrid backgrounds (Spearman in parent and hybrid (ﬁg. 4). When a departure in epigenetic correlation coefﬁcients: 0.63 and 0.86 for A. thaliana and A. levels was detected between parents and hybrids, the direc- lyrata, respectively, ﬁg. 3A). H3K27me3 is not a typical si- tion of the change in expression of the TE was not necessarily lencing mark of TEs (Sequeira-Mendes et al. 2014). This was in the expected direction (ﬁg. 4). A. lyrata TEs with a modiﬁed most apparent in our data for the LTR superfamily, which expression level in hybrid background also generally showed tended to be associated with the highest level of H3K9me2 similar H3K9me2 mark levels in parents and hybrids. We and the lowest level of H3K27me3 (ﬁg. 3B and C). detected a concordant loss of H3K9me2 marks and increased 1410 Genome Biol. Evol. 10(6):1403–1415 doi:10.1093/gbe/evy095 Advance Access publication May 18, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1403/4999384 by Ed 'DeepDyve' Gillespie user on 20 June 2018 Robustness of Transposable Element Regulation GBE Table 3 Numbers and Fractions of Differentially Expressed TEs in Stress Conditions (3h) and in Response to Stress (3h speciﬁc), at Two Different False-Discovery Rates (FDR 0.01 or 0.05) and Two Different Thresholds on the Normalized Counts Per Million (CPM) (all: 0CPM; 10 CPM) FDR £ 0.01 FDR £ 0.05 All 10 CPM All 10 CPM Arabidopsis thaliana 3h 4 þ 14 (1.1%) 2 þ 4(0.4%) 7 þ 26 (2.1%) 2 þ 5(0.4%) 3h speciﬁc 3 þ 12 (0.9%) 2 þ 3(0.3%) 4 þ 23 (1.7%) 2 þ 4(0.4%) Arabidopsis lyrata 3h 33 þ 18 (0.7%) 4 þ 4(0.1%) 54 þ 37 (1.3%) 10 þ 15 (0.4%) 3h speciﬁc 9 þ 17 (0.4%) 3 þ 4(0.1%) 12 þ 34 (0.7%) 7 þ 14 (0.3%) NOTE.—Fractions were computed relative to all TEs with evidence of expression under stress conditions, discounting members of genome-speciﬁc families. expression in the hybrid for 6 A. lyrata TEs (ﬁg. 4). But there conditions, the correlation of TE expression levels between again, we observed for 2 TEs that the change in H3K9me2 parents and hybrids increased at the family and superfamily occupancy did not reﬂect the change in expression. levels. The Spearman’s rank correlation of the replicates’ me- Altogether, in our data, a differential deposition of dian expression in the two backgrounds was (0.7, 0.81, 0.83) H3K27me3 and H3K9me2 histone methylation marks in the for A. thaliana, and (0.67, 0.91, 0.94) for A. lyrata, for indi- hybrid is rare and generally does not coincide with hybrid- vidual, family and superfamily levels, respectively. dependent changes in TE expression. Interestingly, we observed that more TEs were down- regulated in the hybrids compared with the parents under stress conditions than under standard conditions (ﬁg. 5A and table 3). Only the Harbinger and LINE_L1 superfamiles Robustness of TE Expression to Hybridization Is Maintained were enriched among expressed A. lyrata TEsinstresscon- under Stress Conditions ditions (ﬁg. 5B; permutation test using TEs with 1 RNA-seq It is assumed that transposition activity is induced by stress count as reference, FDR< 0.05). This further corroborates the conditions (McClintock 1984; Cavrak et al. 2014). conclusion that in our two species, TE regulation is robust to Dehydration stress is associated with a vast transcriptome hybridization even under stress conditions. reprogramming (Matsui et al. 2008). We analyzed the depen- dence of transcript abundance upon interspeciﬁc hybridiza- tion for TEs and non-TE genes under strong drought stress Discussion conditions in the same way as described for control condi- No evidence for a genome shock upon hybridization of tions. Having found that TEs with a hybrid-speciﬁc expression A. lyrata and A. thaliana change tend to be close to genes, and given the reprogram- The term “genomic shock,” was initially used to describe the ming of the gene transcriptome by the stress, we asked breakage and large scale rearrangement of chromosomes in whether TE regulation was robust to hybridization even under maize (McClintock 1946, 1984). This term is thus appropriate stress conditions. In our data, 30–40% of the genes of both to describe genome instability following interspeciﬁc hybridi- species changed expression after 3h of dehydration, with sim- zation, that is, unbalanced segregation of homoelogous chro- ilar proportions in the parental and hybrid backgrounds. The mosomes, chromosome loss or translocations (Xiong et al. fraction of stress responsive TEs was much smaller: 3–4% on 2011; Chester et al. 2012; Henry et al. 2014; Spoelhof the A. thaliana genome and 2–3% for A. lyrata (not shown). et al. 2017). Over the years, the term “genome shock” has In addition, we observed no increase in the variance of TE also been used to describe the anticipated alteration in ge- expression in hybrid context (supplementary ﬁg. 3, nome activity immediately following interspeciﬁc hybridiza- Supplementary Material online). Although the fraction of tion or allopolyploidization, although transposon A. thaliana TEs with a signiﬁcant expression difference be- reactivation has seldom been rigorously documented imme- tween parent and hybrid is higher under dehydration stress diately after genome merging (Parisod et al. 2010). In plants, than under control conditions, both the absolute numbers (4 DNA cytosine methylation (mC) and the histone mark up and 14 down) and the fraction (1.1% of all TEs with read H3K9me2 embed TEs in a chromatin context unfavorable count1) remained small (table 3). The fraction of respond- for transcription (Kim and Zilberman 2014). The establishment ing A. lyrata TEs decreased slightly compared with control and maintenance of these marks is not only inherited through conditions, and again the numbers remained small (33 up, DNA replication, it is also guided by small interfering RNAs 18 down, fraction 0.7%). The percentage dropped below (siRNAs) (see Kim and Zilberman 2014 for a review of plant TE 0.5% if an expression level of10 CPM in at least one of silencing mechanisms). Because of their potential to exert the backgrounds was required (table 3). Like under control Genome Biol. Evol. 10(6):1403–1415 doi:10.1093/gbe/evy095 Advance Access publication May 18, 2018 1411 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1403/4999384 by Ed 'DeepDyve' Gillespie user on 20 June 2018 Go ¨ bel et al. GBE genome-wide trans effects via base pair homology of the short 24 nt siRNAs, the population of siRNAs expressed by a genome must be adapted to its gene content to ensure proper gene expression. Interspeciﬁc hybridization, therefore, brings together trans-regulators of TE expression that are not mutually adapted and could deeply disorganize gene regula- tion. Gene expression modiﬁcations following interspeciﬁc hy- bridization or allopolyploidization have been monitored across plant genera as diverse as Gossypium, Senecio, Brassica, Tragopogon, or Oryza (e.g., Hegarty et al. 2006; Buggs et al. 2009;Yoo et al. 2013). Many studies have concluded that epigenome incompatibilities are important contributors to hybrid sterility (Parisod et al. 2009; Ghani et al. 2014; Wang et al. 2014; Xu et al. 2014; Senerchia et al. 2015; Wu et al. 2016). In Aegilops hybrids, for example, parental divergence in TE content was associated with the emergence of new TE copies in hybrids (Senerchia et al. 2015). Allopolyploidy is also believed to be associated with drastic epigenetic reorganiza- tion although evidence for a signiﬁcant reactivation of TE transposition remains scant (Parisod et al. 2010). Our analysis contradicts the conclusions of these previous reports. Comparing the TE expression and epigenetic proﬁle of the A. lyrata and A. thaliana genomes in parental and hy- brid context, we ﬁnd no evidence for a major transcriptomic or epigenomic “shock” driven by inappropriate TE silencing and epigenetic mechanisms. Both TE transcription and the decoration of TEs by the silencing histone mark H3K9me2 are globally unaffected in the hybrid. We observe that only a handful of speciﬁc TEs show a change in expression in hybrids. In addition, the changes in TE expression are generally of small magnitude. We further show that the distribution of silencing epigenetic marks is strongly correlated in parents and hybrids, and its variation does not match the changes in TE expression. Finally, under severe dehydration stress, a FIG.5.—(A) Correlation of TE expression levels in stress conditions, large number of genes change their transcriptional activity estimated as Median normalized counts per million over the replicates, in (Matsui et al. 2008). This broad genome-wide remodeling parent and hybrid for individual TEs, TE families, and superfamilies. TEs or of transcriptional activity, however, far from inducing a sev- (super)families with signiﬁcant expression change are marked in red (sig- ered genome shock, tends to increase the robustness of TE niﬁcant only under control conditions), orange (signiﬁcant both under expression patterns to hybridization. These ﬁndings thus sug- standard and stress conditions), or yellow (signiﬁcant only in stress con- gest that stress does not increase the impact of hybridization, ditions). (B) Observed and expected superfamily distribution of TEs whose further supporting the idea that the control of TE elements is expression is hybrid-dependent speciﬁcally in stress conditions. Colored robust to genome merging. Although these observations par- bars show the observed numbers of TEs showing hybrid-dependent ex- tially contradict previous reports, they are not isolated. In re- pression in each TE superfamily. Each color corresponds to one superfamily cent years, an increasing number of studies, mostly as indicated in the legend. Superfamily names are as given in the annota- conducted in the Brassicaceae family, has begun to report tion of Pietzenuk et al. (2016). Names with trailing question marks were annotated as deviant/uncertain members of the respective superfamily. cases where hybridization does not massively alter gene reg- Transparent bars indicate expected median count for random sampling ulation (Akama et al. 2014; Shi et al. 2015; He et al. 2016; within each superfamily. Error bars indicate the 5% and 95% quantile of Zhang et al. 2016; Zhu et al. 2017). In addition, in tomato, the 10,000 random draws. Single black stars mark superfamilies whose transgressive expression of small RNAs was associated with observed count is signiﬁcantly higher than expected from random expect- the dysregulation of stress responsive genes, but not with that ations. Gray stars indicate depletion (maximally 5% of the universe have of TEs (Shivaprasad et al. 2012). counts the observed count of the respective superfamily). Double stars The contradiction between these studies and previous indicate signiﬁcant enrichment or depletion that is robust to FDR reports may have various explanations. First, it is striking to correction. note that many of the studies that conclude on the absence of 1412 Genome Biol. Evol. 10(6):1403–1415 doi:10.1093/gbe/evy095 Advance Access publication May 18, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1403/4999384 by Ed 'DeepDyve' Gillespie user on 20 June 2018 Robustness of Transposable Element Regulation GBE a global gene expression dysregulation were conducted in the A. thaliana allele of FLC, which is up-regulated by species with well-known genomes, where gene expression FRIGIDA, is one of the alleles most strongly impacted by hy- levels can be quantiﬁed with the best accuracy. They were bridization (supplementary table 3, Supplementary Material not conducted in polyploids with large and imperfectly assem- online; Zhu et al. 2017). Interspeciﬁc differences in trans-act- bled genomes. Second, biological and experimental variance ing factor activity will therefore determine the extent to which between replicates affects the strength of the correlation of gene expression changes upon hybridization. If F1 hybrids are expression in parental versus hybrid backgrounds, and thus fertile, these changes can segregate in subsequent F2 gener- the apparent global impact of hybridization. Finally, it is nec- ations as in, for example, Shivaprasad et al. (2012). essary to contrast diverse sources of information to test Here, focusing on the trans-acting effect of the passage whether hybridization has a broad impact on gene regulation. from a native parental background to a hybrid background on In the allotetraploid A. suecica, modiﬁcation in siRNA levels TE expression, we show that at most 1–2% of expressed TEs was not associated with nonadditive gene expression in newly have expression that is differentially controlled in trans. formed allopolyploids, suggesting that changes in siRNA ex- Several elements indicate that these trans-acting effects may pression were not the result of epigenetic disorganization in be associated with interspeciﬁc differences in mechanisms of the hybrid (Ha et al. 2009). Similarly, in Arabidopsis homo- gene regulation in euchromatic regions. When combined in a ploid hybrids, we observed that hybrid-speciﬁc TE expression hybrid, these trans-acting differences modify the regulatory changes do not coincide with obvious epigenetic changes, a environment of a few TE subfamilies. Indeed, the small pro- pattern also reported for protein-coding genes (Zhu et al. portion of TEs with differential expression in hybrids tend to 2017). Taken together, the different molecular data sets be located in the vicinity of genes. Interspeciﬁc differences in used in this study show that TE expression and the distribution the regulation of euchromatic regions are however likely to be of epigenetic marks, which control gene expression, are not minor, given the small number of elements affected. massively disorganized in hybrids. Interspeciﬁc differences in gene regulation controlled in cis can also create a pattern of genome dominance in hybrids, with a majority of orthologous transcripts showing a transcrip- trans-and cis-Acting Differences between Species Can tion bias towards one parental genome (Yoo et al. 2013; Manifest in F1 Hybrids Cheng et al. 2016; Zhang et al. 2016). For example, in The comparative analysis of gene expression in hybrids and A. thaliana A. lyrata hybrids, a bias towards preferential their parents reﬂects the variability of trans-and cis-regulatory expression of the A. lyrata allele has been reported by inde- mechanisms controlling gene expression in the two species pendent studies (He et al. 2012, 2016; Zhu et al. 2017). (Wittkopp et al. 2004). After hybridization, changes in total Cis-effects have not been examined in this study, because expression will simply reﬂect the number and magnitude of orthologous relationships cannot be established for most trans-regulatory differences between species, whereas the TEs, but for the subset of TEs that are located in orthologous speciﬁc cis-regulatory variant of each transcript will result in positions in both species, the A. lyrata TE copy tended to be allelic expression differences. These differences will manifest more highly expressed than the A. thaliana copy in the hybrid in hybrids on a restricted number of genes or elements, which (He et al. 2012). does not imply that there is a major “genomic shock”. Only trans-acting differences will have nonadditive conse- Incompatibilities Can Also Have an Oligogenic Basis quences on expression. In fungi and cotton allopolyploid, >50% of homeologous genes inherit the expression pattern It may seem at ﬁrst surprising that TE silencing shows so little of the parents (Yoo et al. 2013; Cox et al. 2014). In rice, allelic disruption in interspeciﬁc A. thaliana A. lyrata hybrids, given expression bias in hybrids between Oryza sativa and O. japon- the estimated 6 My of divergence and their vastly different TE ica also reﬂects mostly interspeciﬁc differences. In hybrids be- content (Hu et al. 2011; Hohmann et al. 2015). The proba- tween the diploid O. sativa and the tetraploid O. punctata, bility to observe speciﬁc disruptions in gene expression in a only 16% of the genes showed an expression pattern differ- hybrid background, however, does not necessarily scale with entfrom thatofthe parents (Wu et al. 2016). The analysis of evolutionary distance. The proportion of genes with hybrid- protein-coding gene expression in A. thaliana A.lyrata speciﬁc changes in expression is not greater for hybrids be- hybrids reported a larger proportion of nonadditive gene ex- tween the closely related subspecies O. sativa and O. japonica pression changes for the A. thaliana genome compared with than between O. sativa and the more distant species O. punc- the A. lyrata genome (Zhu et al. 2017). This pattern, like in our tata (Wu et al. 2016). A simple oligogenic combination of study, could not be associated with chromatin modiﬁcations alleles, in fact, can cause what is known as a Dobzhansky– induced by hybridization or any other genome-wide change Muller incompatibility, even in the absence of large in gene regulatory mechanisms (Zhu et al. 2017). However, evolutionary distances between parents. Dobzhansky–Muller the A. thaliana parental genotype Col-0 is missing an active incompatibilities, which can cause major disruption at the allele of the major developmental regulator FRIGIDA.In fact, phenotypic and, sometimes at the transcriptome level, are Genome Biol. Evol. 10(6):1403–1415 doi:10.1093/gbe/evy095 Advance Access publication May 18, 2018 1413 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1403/4999384 by Ed 'DeepDyve' Gillespie user on 20 June 2018 Go ¨ bel et al. GBE Berardini TZ, et al. 2015. The Arabidopsis Information Resource: making not seldom within species, and were even detected within and mining the “gold standard” annotated reference plant genome. populations (Alcazar et al. 2010; Bomblies and Weigel Genesis 53(8):474–485. 2010; Durand et al. 2012; Wright et al. 2013; Chae et al. Bomblies K, Weigel D. 2007. Hybrid necrosis: autoimmunity as a potential 2014). 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Impact of artifact removal on ChIP quality metrics in ChIP-seq and ChIP-exo tion (Rebernig et al. 2015) and will also occasionally affect the data. Front Genet. 5:75. regulation of speciﬁc TEs and favor new bouts of transposi- Cavrak VV, et al. 2014. How a retrotransposon exploits the plant’s heat tion. Without a global robustness of TE regulation to genome stress response for its activation. PLoS Genet. 10(1):e1004115. merging, hybridization and polyploidization would probably Chae E, et al. 2014. Species-wide genetic incompatibility analysis identiﬁes be much less important for plant diversiﬁcation (Alix et al. immune genes as hot spots of deleterious epistasis. Cell 159(6):1341–1351. 2017). We note, however, that the impact of hybridization Cheng F, et al. 2016. Epigenetic regulation of subgenome dominance on TE transposition rate has not been examined in this study. following whole genome triplication in Brassica rapa. New Phytol. 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Genome Biology and Evolution – Oxford University Press
Published: May 18, 2018
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