Sperm-inherited H3K27me3 impacts offspring transcription and development in C. elegans

Paternal epigenetic inheritance is gaining attention for its growing medical relevance. However, the form in which paternal epigenetic information is transmitted to offspring and how it influences offspring development remain poorly understood. Here we show that in C. elegans, sperm-inherited chromatin states transmitted to the primordial germ cells in offspring influence germline transcription and development. We show that sperm chromosomes inherited lacking the repressive histone modification H3K27me3 are maintained in that state by H3K36me3 antagonism. Inheritance of H3K27me3-lacking sperm chromosomes results in derepression in the germline of somatic genes, especially neuronal genes, predominantly from sperm-inherited alleles. This results in germ cells primed for losing their germ cell identity and adopting a neuronal fate. These data demonstrate that histone modifications are one mechanism through which epigenetic information from a father can shape offspring gene expression and development.

T here is growing awareness that development and health are influenced by epigenetic information passed from parents to offspring 1,2 . The gametes, sperm and oocyte, are the conduits through which epigenetic information is passed from one generation to the next. Investigating epigenetic inheritance via the maternal line is complicated by the many non-epigenetic factors the oocyte provides to the embryo 1 . For this reason, the field of epigenetic inheritance largely focuses on transmission via sperm. DNA methylation and non-coding RNAs (ncRNAs) are epigenetic carriers that are transmitted by sperm to the embryo and that have been shown to influence gene expression in offspring 3,4 . Modified histones can also be passed, but their role in intergenerational and transgenerational epigenetic inheritance is less clear. In mammals, the sperm genome is primarily packaged with protamines and, while there is current debate about the degree to which the sperm genome retains histones, there is consensus that marked histones are found on developmentally important loci [5][6][7] . Despite this observation and mounting suggestive evidence [8][9][10] , establishing a causal role for sperminherited histone marks in regulating offspring transcription and development has been an ongoing challenge. We took advantage of features of C. elegans to tackle this issue.
We previously showed that C. elegans sperm retain nucleosomes and histone marking genome-wide 11 and that C. elegans Polycomb Repressive Complex 2 (PRC2) maintains inherited states of H3K27me3 during embryogenesis 12 . In wild-type embryos H3K27me3 is enriched over genes that were silent in the parental germline 13,14 . H2K27me3 marking inherited from hermaphrodite parent worms is essential for germline development in offspring, since hermaphrodite parents lacking PRC2 generate offspring in which the primordial germ cells die during early larval development 15 . We thus hypothesize that transmission of chromatin states from parent germ cells via sperm and oocyte to the nascent germ cells in offspring protects germlineappropriate gene expression patterns in the developing and adult germline.
In this work we investigate how chromatin states inherited from parents are maintained in offspring and whether inherited states are important for offspring transcription and development. We elucidate a mechanism through which an inherited H3K27me3(−) state is propagated from parent germ cells (sperm) to offspring germ cells. We show that inheriting a sperm genome lacking the repressive mark H3K27me3 results in derepression of many genes for somatic development, especially neuronal genes, in offspring germlines. This results in germ cells that in a sensitized genetic background lose their germ cell identity and adopt a neuronal fate. Taken together, these findings establish a cause-effect relationship between sperm-inherited histone marks and offspring transcription and development in C. elegans.

Results
Maintenance of an inherited H3K27me3(−) state requires MES-4. When C. elegans embryos inherit some chromosomes with and some chromosomes without H3K27me3, PRC2 maintains inherited states by (1) restoring levels of H3K27me3 on H3K27me3(+) chromosomes after DNA replication and (2) failing to de novo methylate H3K27me3(−) chromosomes 12 . The ability of PRC2 to restore levels of H3K27me3 after genome duplication is likely explained by the EED subunit of PRC2 (MES-6 in worms) binding to H3K27me3 and stimulating the methyltransferase activity of the EZH2 subunit (MES-2 in worms) 16,17 . How PRC2 is prevented from de novo methylating chromosomes inherited lacking H3K27me3 is less clear. One possibility is that chromosomes lacking H3K27me3 are unable to recruit and stimulate PRC2 activity. Another possibility is that these chromosomes bear an opposing mark that antagonizes PRC2 activity. Likely candidates for antagonizing PRC2 activity are histone marks associated with gene expression, hereafter referred to as active marks, and their corresponding histone modifiers 18,19 .
To test if active marks prevent PRC2 from de novo methylating sperm-inherited H3K27me3(−) chromosomes during early embryogenesis, we monitored sperm chromosomes through rounds of cell division in embryos lacking a maternal load of MES-4 or SET-2, which generate the active marks H3K36me2/ 3 13,20 and H3K4me2/3 21 , respectively. We generated embryos that inherited H3K27me3(+) oocyte chromosomes and H3K27me3(−) sperm chromosomes by mating wild-type females with PRC2 mutant males (Fig. 1a). To increase the likelihood that sperm chromosomes completely lacked H3K27me3, we used PRC2 mutant males whose parents also lacked PRC2. We call the offspring of wild-type mothers and PRC2 mutant fathers K27me3 M+P− (M+ for maternal H3K27me3(+), P-for paternal H3K27me3(−)). We assessed whether sperm-inherited chromosomes retained the H3K27me3(−) state or acquired H3K27me3, by tracking the status of H3K27me3 on sperm-inherited chromosomes during early embryogenesis. To facilitate analysis of sperm vs. oocyte chromosomes, we performed this analysis in a temperature-sensitive plk-1 mutant background that keeps sperm-inherited and oocyte-inherited chromosomes in separate nuclei for many divisions 22 (Fig. 1b).
In all 2-cell K27me3 M+P− plk-1 embryos, we observed that one nucleus in each cell contained all H3K27me3(+) chromosomes (oocyte-inherited) and the other nucleus contained H3K27me3(−) chromosomes (sperm-inherited). In control and SET-2-lacking embryos, this pattern persisted in all cells during the early divisions and in the P lineage (germ lineage) throughout embryogenesis (Fig. 1c, d, Supplementary Fig. 1). In contrast, in embryos depleted of MES-4, we observed gradual acquisition of H3K27me3 on the entire set of sperm chromosomes (Fig. 1c, Supplementary Fig. 1). To assess the appearance of H3K27me3 on sperm chromosomes, we analyzed K27me3 M+P-embryos from mothers that were either mutant or wild type for mes-4 (see methods). At the 1-cell stage, all K27me3 M+P− embryos examined contained sperm chromosomes lacking detectable H3K27me3 irrespective of maternal MES-4 (n = 6 with maternal MES-4 and n = 4 without maternal MES-4). In the presence of maternal MES-4, this state was maintained in all embryos up to the 4-cell stage (n = 21) and in most embryos up to the 40-cell stage (n = 15/19). In the absence of maternal MES-4, all 2-cell embryos examined acquired some H3K27me3 on sperm chromosomes (n = 6). The levels of H3K27me3 on sperm and oocyte chromosomes became indistinguishable in embryos by the 4-cell stage (n = 4) and at later stages (n = 15). This gradual acquisition of H3K27me3 matches the gradual loss of H3K36me3 in the absence of maternal MES-4 23 . Thus, MES-4 inhibits PRC2 activity, presumably through MES-4-mediated methylation of H3K36, to maintain the paternal H3K27me3(−) state. These findings show that the H3K27me3(−) state is maintained by opposing PRC2 activity with active marks and also suggest that PRC2 does not require H3K27me3 to seed its activity, as observed in mouse embryonic stem cells 24 .
Sperm chromatin states shape transcription in offspring. Do gamete-inherited chromatin states influence gene expression in offspring? This is a burning question in the field and one that remains elusive, in part because it is difficult to determine cause versus consequence when comparing chromatin states with transcriptional status. To establish a cause-effect relationship   between inherited chromatin and offspring transcription requires that (1) genetically identical individuals display different transcriptional outcomes as a result of inheriting different chromatin states, (2) transcriptional changes must be demonstrated to occur in cis, thus eliminating alternative epigenetic carriers that function in trans (e.g., cytoplasmic ncRNAs), and (3) DNA methylation, which also functions in cis, must be eliminated as a potential mediator of the observed transcriptional changes. We met the third criterion by using the model system C. elegans, which lacks canonical DNA methylation. We met the remaining criteria by comparing transcription from sperm-inherited and oocyte-inherited alleles in genetically identical (hybrid fem-2/+; mes-3/+) worms (Fig. 1a) that inherited the sperm genome with or without H3K27me3, which we call K27me3 M+P+ and K27me3 M+P−. We showed previously that K27me3 M+P− embryos have different chromatin states on their sperm-inherited and oocyteinherited genomes and that those states are maintained in all cells of early embryos 12 . As embryos develop, the gamete-inherited chromatin states are rewritten in somatic cells but maintained through the five cell divisions that generate the two primordial germ cells (PGCs) present in newly hatched larvae 12 (Fig. 1d). When newly hatched larvae begin to feed, the PGCs launch their transcriptional program and begin to proliferate. During larval development, H3K27me3 gradually begins to accumulate on sperm-inherited chromosomes 12 . Absence of H3K27me3 on sperm chromosomes at the onset of germ cell transcription could result in the activation of inappropriate genes. Once activated, a euchromatic state could be propagated at specific loci, sensitizing daughter germ cells to continued expression of inappropriate genes and potentially downstream target genes. We investigated whether global differences in inherited chromatin states in PGCs impact transcription in the developing germline. We used singlenucleotide polymorphisms (SNPs) between parental strains (from Bristol, England, and from Hawaii) to compare transcription from sperm-inherited and oocyte-inherited genomes. This allowed us to determine whether transcriptional changes occurred in cis (from sperm alleles specifically) or in trans (from both sperm and oocyte alleles).
Worms that inherited sperm chromosomes lacking H3K27me3 misexpressed genes in their germlines; 149 genes were upregulated and 116 genes were downregulated (FDR < 0.1) in K27me3 M+P− compared to K27me3 M+P+ worms (Fig. 2a, Supplementary Figs. 2, 3). Of the genes that were upregulated in K27me3 M+P− germlines, 96% are enriched for H3K27me3 in wild-type sperm 11 (Supplementary Fig. 4). While K27me3 M+P− worms were typically fertile, they generated a significant proportion of sterile offspring (Fig. 2b). These findings demonstrate that altering sperm-inherited chromatin states results in changes to germline transcription and function.
Expression of genes assessed by reads overlapping SNPs between Bristol and Hawaii correlated well with expression assessed by using all reads mapping to a gene ( Supplementary  Fig. 3a). Therefore, we could use SNPs to determine, for the 23% of misexpressed genes that contain a SNP, if transcription differences arose from the sperm allele, the oocyte allele, or both (Supplementary Data 1). Our analysis of SNP-containing transcript reads indicated that downregulated genes reflect decreased transcription from both the sperm and oocyte allele. However, upregulated genes reflect increased transcription primarily from the sperm allele (Fig. 2c, d). Importantly, approximately half of these genes were upregulated in cis with increased transcription from the sperm allele specifically (dark red triangles in Fig. 2c). These findings demonstrate that differential marking on sperm-inherited and oocyte-inherited genomes allows sperm-inherited and oocyte-inherited alleles within the same cell to achieve different transcriptional outcomes in offspring. Given that C. elegans lacks canonical DNA methylation, this finding establishes a causal role for sperminherited H3K27me3 in regulating transcription in offspring. It also raises the possibility that environmentally induced changes to gamete marking could lead to transcriptional changes in offspring, with developmental consequences.
The genes upregulated from both sperm and oocyte alleles likely represent downstream targets of an initially misexpressed factor(s). The observation that these genes are more upregulated from sperm alleles than oocyte alleles led us to hypothesize that the presence of H3K27me3 on the oocyte genome acts as a barrier against transcriptional activation of germline-inappropriate genes. H3K27me3 on the oocyte-inherited genome would not be expected to protect it from factors that would shut down expression of germline-appropriate genes. This may explain the observation that downregulated genes were downregulated from both sperm and oocyte alleles. This led us to wonder if the global pattern of misexpression resulting from altered sperm chromatin was a concerted deviation from a germline program. To explore this, we performed gene ontology analysis on upregulated and downregulated genes. We found that downregulated genes are enriched for genes involved in reproduction and that upregulated genes are enriched for genes involved in somatic development, especially neuronal development ( Supplementary Fig. 2b, c). This suggests that K27me3 M+P− germlines are deviating from a germline program of gene expression toward a somatic program, perhaps favoring a neuronal program.
Sperm-inherited H3K27me3 protects germ cell fate in offspring. To test whether germlines in K27me3 M+P− worms that inherited altered sperm chromatin are in fact transitioning toward a neuronal fate, we analyzed worms carrying a panneuronal reporter gene, unc-119::GFP. We compared expression of this reporter in genetically identical worms that inherited sperm chromatin with either a wild-type pattern of H3K27me3, K27me3 M+P+, or lacking H3K27me3, K27me3 M+P−. As noted above, K27me3 M+P− and K27me3 M+P+ worms are fertile; we observed that fertile germlines do not detectably express unc-119::GFP (n > 500) nor do they significantly upregulate unc-119 transcript. Performing this analysis in a sensitized genetic background in which the maternal load of PRC2 was reduced by half (mes-3/+ heterozygous mother) rendered~30% of K27me3 M+P− worms sterile (n = 267/894); <0.3% of K27me3 M+P+ worms were rendered sterile (n = 2/870). In this background, we found that the unc-119::GFP neuronal reporter was expressed in a significant percentage of sterile K27me3 M+P − adult germlines (Supplementary Table 1). Notably, expression of the neuronal reporter increased with age, from 16% in sterile day 1 adults to 31% in sterile day 2 adults. Expression of the neuronal reporter was rarely detected (<0.1%) in germlines from late-stage larvae (L4) (n = 1/146) and never observed in the germlines from the rare sterile K27me3 M+P+ worms. These findings indicate that deviation from a germline program in K27me3 M+P− worms is progressive and strongly suggest that loss of H3K27me3 from the sperm-inherited genome results in a failure to maintain germ cell identity rather than a failure to establish it.
Consistent with K27me3 M+P− germlines adopting a neuronal identity, we readily observed patches of highly concentrated GFP signal in UNC-119::GFP(+) germlines, indicating that the normally syncytial germ cells had cellularized. In some cases, we observed axo-dendritic structures emanating from these bright GFP(+) cells (Fig. 3b, c). Our findings demonstrate that absence of H3K27me3 from the sperm-inherited genome results in the derepression of many somatic genes, especially neuronal genes, predominantly from sperm-inherited alleles. In a sensitized background, lack of H3K27me3 on the sperm-inherited genome results in germlines that transition away from a germline program and toward a neuronal program.  p-values were calculated using a Wilcoxon rank sum test. c Scatter plot comparing sperm-specific and oocyte-specific log 2 fold changes of SNP-containing reads from significantly upregulated genes (in red) and downregulated genes (in blue). Genes upregulated in cis (>1.5-fold increase in reads from the sperm allele; <1.5-fold increase from the oocyte allele) are plotted as dark red triangles. The numbers of misregulated genes above and below the diagonal line are indicated. d Boxplots showing sperm-specific (sp) and oocyte-specific (oo) log 2 fold changes of significantly upregulated genes (34 genes sig up) and significantly downregulated genes (44 sig down). Downregulated genes (blue) are similarly downregulated from sperm and oocyte alleles, while upregulated genes (red) are significantly more upregulated from sperm alleles than oocyte alleles (i.e., pairwise comparisons of transcript reads from sperm vs. oocyte alleles indicate that sperm allele reads are significantly more numerous than oocyte allele reads for upregulated genes but not for downregulated genes). Each box extends from the 25th to the 75th percentile, with the median indicated by the horizontal line; whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range from the box; the individual data point outside of these boundaries is shown as a filled box. The waist indicates the 95% confidence interval for the medians. p-values were generated using the paired student's t-test Discussion Establishment and maintenance of chromatin states are critical during development and aging. During early development, inherited chromatin states must be rewritten to enable differentiation of different lineages. During aging, established chromatin states must be maintained to preserve cell-type-specific gene expression patterns and thus protect cell identity. Defects in these processes can lead to severe developmental defects and cancer. Whether chromatin states established in the parent and transmitted to offspring via sperm or oocyte influence the establishment and/or maintenance of cell fates in those offspring is a critical question in the field. Our findings demonstrate that in C. elegans sperm-inherited chromatin states influence germ cell identity in offspring. Because we observe reprogramming of germ cells toward neuronal cells in adult K27me3 M+P− germlines but only rarely in larval K27me3 M+P− germlines, we conclude that offspring germ cells lose rather than fail to establish their germ cell identity. These findings reveal that in worms, sperm-inherited H3K27me3 is transmitted to offspring germ cells to prevent germline-inappropriate transcription and thus protect germ cell identity. Altogether, our findings point to a model in which gameteinherited patterns of H3K27me3 act as a barrier to inappropriate transcription; in germ cells, this barrier function is particularly important in suppressing transcription of factors that could drive germ cells toward alternative cell identities (Fig. 4). In the germlines of worms that inherited the sperm genome lacking H3K27me3, we speculate that some genes become upregulated as a direct result of diminished H3K27me3 (Factor X in Fig. 4), while other genes may be upregulated as a consequence of being the target of an upregulated gene. An alternative scenario is that Factor X genes are normally expressed in wild-type germ cells but prevented from activating germline-inappropriate target genes by H3K27me3 on those target genes. An example of the first scenario (Fig. 4) is the vab-7 gene, which encodes a neuronal transcription factor 28 ; vab-7 is not expressed in wild-type germlines but becomes upregulated in K27me3 M+P− germlines. Examples of the alternative scenario are genes that encode neuronal specification factors (e.g., die-1 and nsy-7) 28 that are highly expressed in both wild-type and K27me3 M+P− germlines. Regardless of the exact cascade of gene activation, our expression analysis of K27me3 M+P− germlines suggests a programmatic shift toward a neuronal program and supports the hypothesis that an upstream factor(s) is altering gene expression in a concerted manner, especially from the H3K27me3-depleted sperm genome.
Our findings establish a causal role for sperm-inherited histone marks influencing transcription in offspring and reveal their role in protecting cell identity. Together with observations that in mammals, sperm retain modified histones over developmentally important genes 5-7 , this work implicates sperm-inherited chromatin states as one mechanism through which environmentally induced changes to parental epigenomes can impact the development and health of future generations.

Methods
C. elegans growth conditions. Worms were maintained on nematode growth medium (NGM) agar plates spotted with Escherichia coli OP50 and maintained at 15°C (fem-2 and plk-1 containing strains) or 20°C (all other strains). To generate females, fem-2 strains were raised at 24°C until L4.
Analysis of MES-4-depleted and SET-2 mutant embryos. plk-1; fog-1 unc-11 females (Fig. 1b, c) or fem-2(ts) or set-2 fem-2(ts) females ( Supplementary Fig. 1) were mated to N2 (P+) or mes-3 M-Z-; him-8 (P−) males (M-for no maternal load of MES-3, Z-for no zygotic synthesis of MES-3) on plates with mes-4 RNAi, empty vector RNAi, or OP50 ( Supplementary Fig. 1c) and incubated overnight at 23°C (Fig. 1b, c) or 20°C (Supplementary Fig. 1). Embryos were dissected from mated females in egg buffer (25 mM HEPES pH 7.4, 118 mM NaCl, 48 mM KCl, 2 mM CaCl 2 , 2 mM MgCl 2 ) with 1 mM levamisole on polylysine-coated slides. Embryos attached to slides were fixed and stained as described above. Stacks of optical sections were acquired on a Solamere spinning disk confocal system as described above. Stacks were collapsed into maximum intensity projections using Micro-Manager. Single-channel and merge images were generated in Photoshop. To assess the appearance of H3K27me3 on sperm chromosomes in embryos with and without a maternal load of MES-4, optical sections of K27me3 M+P-embryos from fem-2(ts) mothers (with a maternal load of MES-4) or from fem-2(ts); mes-4(bn73) mothers (lacking a maternal load of MES-4) were stained for DNA and H3K27me3 and scored for: 1) undetectable H3K27me3 on some chromosomes, 2) reduced levels of H3K27me3 on some chromosomes relative to other chromosomes, or 3) indistinguishable levels of H3K27me3 on all chromosomes.
Analysis of K27me3 M+P− and M+P+ L1 larvae. fem-2(ts) females were mated to N2 (P+) or mes-3 M-Z-; him-8 (P−) males for~48 h. Gravid females were transferred to 100 µL S Basal (5.85 g NaCl, 1 g K 2 HPO 4 , 6 g KH 2 PO 4 , 1 mL cholesterol (5 mg/mL in ethanol), H 2 0 to 1 L) on gelatin chrom alum-coated slides and incubated overnight in a humid chamber at 20°C. Starved K27me3 M+P− and K27me3 M+P+ L1s were transferred to polylysine-coated slides. L1s attached to slides were fixed and stained as described above. Stacks of optical sections were acquired on a Solamere spinning disk confocal system as described above. Stacks were collapsed into maximum intensity projections using Micro-Manager. Singlechannel and merge images were generated in Photoshop.
Analysis of sensitized K27me3 M+P− and M+P+ germlines. mes-3/hT2-GFP; fem-2(ts) females were mated to either mes-3/hT2-GFP; unc-119 (P+) or mes-3 M +Z−; unc-119 (P−) males to generate K27me3 M+P+ and K27me3 M+P− worms, respectively. 146 L4 K27me3 M+P− larvae were cut at the pharynx to extrude their anterior germline for assessment of UNC-119::GFP expression in germ cells. Sterile adults on day 1 of adulthood (n = 267) were moved to new plates for live analysis to determine whether germ cells expressed UNC-119::GFP. Sterile day 1 adults that lacked germline expression of UNC-119::GFP were allowed to age at 20°C for a repeat analysis of UNC-119::GFP expression in germ cells on day 2 of adulthood. L4s, day 1 sterile adults, and day 2 sterile adults were analyzed for germ cell expression of UNC-119::GFP on a Leica MZ16F microscope equipped with a GFP filter. Germlines were imaged for GFP expression both by live imaging of whole worms and by fixing dissected germlines in egg buffer with 1 mM levamisole and 0.5% tween on polylysine-coated slides and immunostaining for GFP, UNC-64, PGL-1, and/or HTP-3. Stacks of optical sections were acquired on a Solamere spinning disk confocal system controlled by Micro-Manager software as described above. Stacks were collapsed into maximum intensity projections using Micro-Manager and stitched together into composite images using Photoshop. Singlechannel and merge images were generated in Photoshop. In some cases (Fig. 3c), germlines were straightened using ImageJ.

RNA-seq of
Processing and analyzing RNA-seq data. For differential expression analysis, raw sequences were mapped to transcriptome version WS220 using TopHat2 29 . Only reads with one unique mapping were allowed. Otherwise default options were used. Reads mapping to ribosomal RNAs were removed from further analysis. HTSeq 30 was used to obtain read counts per transcript. DESeq2 31 was used to determine differentially expressed genes from HTSeq counts. A Benjamini-Hochberg multiple hypothesis corrected p-value cutoff of 0.1 was used as a significance cutoff to identify significantly differentially expressed genes between K27me3 M+P− and M+P+ germlines.
To identify RNA-sequencing (RNA-seq) reads coming from the Hawaiian or Bristol parent in the hybrid offspring germlines, the annotated single nucleotide polymorphisms (SNPs) of the Hawaiian strain CB4856 genome compared to the Bristol strain N2 WS220 genome were downloaded from WormBase. 126,615 unique SNPs were downloaded, of which 25,980 SNPs mapped to an exon in a total of 9146 coding genes, according to WormBase. Henceforth, we refer to these SNPs as exonic SNPs.
RNA-seq reads from the Bristol strain, Hawaiian strain, and hybrids were mapped to the Bristol (N2) genome version WS220 using TopHat2, once allowing 1 mismatch in a read and a second time not allowing a mismatch. Reads overlapping exonic SNP positions were identified, and HTSeq was used to count these SNP-containing reads for each transcript. Counts per transcript were normalized by the number of total transcript reads, including non-SNPoverlapping reads, for each of the libraries sequenced. An average count per transcript was obtained by averaging the replicates for each of the Bristol, Hawaiian, and hybrid K27me3 M+P− and M+P+ replicate experiments. A further quality control step was imposed. For the Bristol and Hawaiian RNA-seq data, we calculated a distance score between the mismatch 0 and mismatch 1 read counts for each transcript. For each transcript i in the Hawaiian RNA-seq data, a score hz i was calculated: hz i = (hmm1 i − hmm0 i )/(hmm1 i + hmm0 i ) where hmm1 i and hmm0 i denote the normalized read counts for transcript i from mapping with a mismatch 1 and with a mismatch 0 to the Bristol genome, respectively. In the same way, we defined bz i , bmm1 i , and bmm0 i for transcripts from mapping our Bristol RNA-seq data. For Hawaiian transcript reads from exonic SNPs, we expect hz i to be close to 1; all reads should have mapped when allowing 1 mismatch, and none should have mapped when allowing no mismatches. For Bristol transcript reads from exonic SNPs, we expect bz i to be close to 0, as the number of reads from both mappings should have been very similar. We removed transcripts that did not follow these requirements. If a transcript i had a hz i < 0.75 for the Hawaiian RNAseq data, or an absolute value |bz i | > 0.25 for the Bristol RNA-seq data, that transcript was removed from further analysis. These requirements removed 1080 transcripts with exonic SNPs. For the remaining 8066 transcripts we obtained from the hybrid K27me3 M+P− and M+P+ RNA-seq data, we determined the transcript reads from the sperm allele (Bristol) as reads that mapped when allowing 0 mismatches. We determined the transcript reads from the oocyte allele (Hawaii) as reads that mapped when allowing 1 mismatch but that did not map when allowing 0 mismatches. We thus calculated transcript reads from the oocyte allele by subtracting reads that mapped when allowing 0 mismatches (sperm allele reads) from reads that mapped when allowing 1 mismatch (reads from both gamete alleles).
Many genes with exonic SNPs were not expressed in any of our germline RNAseq data. We filtered out these non-expressed transcripts from further analysis by requiring the normalized read counts from the sperm allele or oocyte allele in at least one condition, K27me3 M+P− or M+P+, to be at least 0.5. Because our goal was to use SNP-containing transcript reads as a proxy for total transcript reads, we filtered out transcripts that had a greater than 2.5-fold difference between the log 2 of SNP-containing reads versus the log 2 of total reads for a given transcript in hybrid germlines (Hawaii mother and Bristol father). This requirement removed 190 genes and left 2,606 transcripts for further analysis. To avoid dividing by zero when calculating log fold changes and to reduce the effect of noise on low read counts, we added a pseudo-count of 0.5 to all remaining transcripts from oocyte alleles and sperm alleles in both conditions. GO analysis of significantly misregulated genes. Significantly upregulated genes (361 genes) and downregulated genes (342 genes) in the "wild-type" hybrid vs K27me3 M+P− comparison (FDR < 0.05) were used to perform GO analysis ( Supplementary Fig. 2b, c). GO analysis was performed using the tissue enrichment tool provided by WormBase (version WS266).
Statistics. Sample sizes and statistical tests used are described in the figure legends. Paired student's t-tests and Wilcoxon rank sum tests were performed using R. Four biological replicates of sequencing data were used. No replicates were omitted. Differential expression analysis was performed using DESeq2, which uses negative binomial generalized linear models. Adjusted p-values were calculated using the Benjamini-Hochberg method performed by DESeq2.
Reporting Summary. Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.

Data availability
Raw sequence files and processed data sets generated in this study are available at the Gene Expression Omnibus (GEO) repository, accession GSE123415. Processed data sets available at GEO are as follows: RNA-seq read counts for K27me3 M+P−, K27me3 M+P + and "wild-type" hybrid containing (1) all read counts mapping to a transcript, (2) read counts overlapping SNPs mapped to the Bristol genome allowing 1 mismatch (Hawaii & Bristol reads), (3) read counts overlapping SNPs mapped to the Bristol genome allowing 0 mismatches (Bristol-specific reads), (4) differential expression analysis comparing transcript reads from K27me3 M+P− vs. M+P+, and (5) differential expression analysis comparing transcript reads from K27me3 M+P− vs. "wild-type" hybrid. Supplementary Data 1 consists of sperm-specific and oocyte-specific reads and the log 2 fold changes between K27me3 M+P− and M+P+ for significantly misregulated SNP-containing genes. All other relevant data supporting the key findings of this study are available within the article and its Supplementary Information files or from the corresponding author upon reasonable request. A reporting summary for this Article is available as a Supplementary Information file.