Impaired DNA replication is a hallmark of cancer and a cause of genomic instability. We report that, in addition to causing genetic change, impaired DNA replication during embryonic development can have major epigenetic consequences for a genome. In a genome-wide screen, we identified impaired DNA replication as a cause of increased expression from a repressed transgene in Caenorhabditis elegans. The acquired expression state behaved as an “epiallele,” being inherited for multiple generations before fully resetting. Derepression was not restricted to the transgene but was caused by a global reduction in heterochromatin-associated histone modifications due to the impaired retention of modified histones on DNA during replication in the early embryo. Impaired DNA replication during development can therefore globally derepress chromatin, creating new intergenerationally inherited epigenetic expression states.

Multicopy transgene arrays are subject to epigenetic repression in the Caenorhabditis elegans germ line by the polycomb repressive complex 2 (PRC2) ( 1 ) and additional chromatin- and small RNA–related pathways ( 2 – 4 ). In C. elegans, modified histones and small RNAs are transmitted across generations ( 5 , 6 ), acting as carriers of epigenetic information ( 7 – 10 ). In addition to germline silencing, multicopy transgene arrays also show variation in their somatic expression level, which, at least in some cases, can be epigenetically inherited between generations ( 11 , 12 ).

RESULTS

To identify regulators of the heritable somatic repression of a daf-21::mCherry multicopy transgene array, we performed a genome-wide RNA interference (RNAi) screen (Fig. 1A). First-stage larval animals were fed in 96-well plates with bacteria expressing double-stranded RNA (dsRNA) targeting ~17,000 protein-coding genes, and expression from the array was scored in the adult worms of the same generation and in their larval progeny. Multiple RNAi clones that increased expression from the transgene targeted core components of the DNA replication machinery: DNA polymerase epsilon (pole-1 or pole-2), the polymerase α-primase complex (div-1, pri-2, or Y47D3A.29/POLA1), replication factor C (rfc-1 or rfc-3), and replication protein A (rpa-2) (Fig. 1B, fig. S1, and table S1).

Fig. 1 Impaired DNA replication during embryonic development derepresses a transgene array. (A) Genome-wide RNAi screen to identify repressors of expression from a multicopy transgene array. (B) Expression of the daf-21p::mCherry transgene in F1 progeny when the indicated genes are inhibited by RNAi. (C) Male worms carrying a daf-21p::GFP multicopy transgene were crossed to wt or div-1(or148) mutant hermaphrodites. (D to F) Expression was quantified in F1 embryos by time-lapse microscopy. Quantification in (F) is at t = 300, indicated by the dashed line in (E) [6.2-fold difference, P = 1.4 × 10−46, two-sided t test; n = 33 and 42 for progeny of wt and div-1 hermaphrodites, respectively]. Crossing male div-1 animals to hermaphrodites carrying the daf-21p::mCherry did not result in an elevated transgene expression in the progeny (fig. S3), demonstrating that div-1(or148) heterozygosity in the progeny does not affect transgene expression during embyrogenesis. Effects on additional transgenes are summarized in table S4.

The core replication machinery is mostly encoded by essential genes, but we could confirm the RNAi phenotypes using a hypomorphic allele, or148, of the gene encoding the B subunit of DNA polymerase α-primase, div-1 (fig. S2) (13). This allele is a point mutation that causes delayed embryonic division due to prolonged S phase at 20°C and lethality at 25°C (13).

In C. elegans, the early stages of embryonic development are under maternal control (14). To test whether impaired DNA replication during embryonic development is sufficient to derepress the array, we crossed male animals carrying a daf-21p::GFP multicopy array to hermaphrodites carrying the div-1 mutation (Fig. 1C). In this way, the array is delivered from a wild-type (wt) father into an egg produced by mutant div-1 mothers, that is, containing mutant maternal div-1 mRNA and DIV-1 protein. Expression in the resulting progeny was strongly up-regulated from the onset of zygotic transcription (Fig. 1, D to F). In contrast, crossing mutant div-1 fathers to wt hermaphrodites carrying the array did not result in array derepression (fig. S3). Thus, impaired DNA replication during very early embryonic development results in increased expression from the start of zygotic transcription.

As in mammals, repressed chromatin in C. elegans is associated with specific histone modifications: trimethylation of histone H3 at lysine 27 (H3K27me3) and di- and trimethylation of H3 lysine 9 (H3K9me2/3) (15). In C. elegans, addition of H3K27me3 is catalyzed by the PRC2 (MES-2/3/6) complex (16). Inactivation of mes-2 (Fig. 2, A and B) strongly increased expression from the transgene array. Similarly, inactivation of MET-2, a putative histone methyltransferase required for mono- and dimethylation of H3K9 (3, 17), also strongly increased expression from the array (Fig. 2, A and B), as did inactivation of the putative H3K9me3 methyltransferase SET-25 (Fig. 2, A and B) (3). The very strong reduction in H3K9 methylation in a met-2;set-25 double mutant (3, 18) increased expression more than either single mutant (Fig. 2, A and B), and expression was highest in animals lacking H3K27me3 and H3K9me1/2/3 (mes-2;met-2;set-25 triple mutants; Fig. 2, A and B), consistent with multiple repressive pathways being partially redundantly involved in repression of the array.

Fig. 2 The absence of repressive histone modifications suppresses the effect of impaired replication on expression. (A) Quantification of daf-21p::mCherry fluorescence intensity in adult worms with the indicated genotypes. Sample size: wt, 34; set-25, 37; met-2, 32; mes-2, 43; met-2;set-25, 32; mes-2;met-2;set-25, 29. (B) Representative images of mCherry fluorescence. BF, bright field. (C) Quantification of daf-21p::mCherry fluorescence intensity in L1 larvae with the indicated genotypes when either div-1 or hsp-1 is inhibited by RNAi. Each dot represents one worm. The y axis is in log scale. P values were calculated by two-sided t test. Sample size: wt (control, 153; div-1, 203; hsp-1, 180), mes-2 (control, 173; div-1, 32; hsp-1, 58), met-2;set-25 (control, 145; div-1, 73; hsp-1, 153), mes-2;met-2;set-25 (control, 55; div-1, 112; hsp-1, 97). (D) Representative images of mCherry fluorescence. Bottom: The contrast is adjusted for each genotype so that the change in expression relative to the control RNAi can be visualized. Ctrl, control.

We tested the effects of impaired DNA replication in embryos lacking these histone modifications alone and in combination. Impaired replication still resulted in a strong increase in expression in animals lacking H3K27me3 (Fig. 2, C and D), indicating that the effects of impaired replication are not simply due to altered inclusion of this modification. Similarly, the array was still strongly up-regulated when replication was impaired in animals lacking all H3K9 methylation (Fig. 2, C and D). Thus, the impact of impaired DNA replication cannot be due to alterations in just one of these repressive chromatin pathways. In contrast, the impact of impaired replication was strongly reduced in animals lacking both H3K27me3 and H3K9me1/2/3 (Fig. 2, C and D). This is not due to RNAi insensitivity or any saturation effect because inhibition of the chaperone HSP-1, which triggers a stress response and drives expression through the daf-21 promoter, still strongly increased expression from the array (Fig. 2, C and D). Increased expression from the array after pole-2(RNAi) treatment was also partially suppressed in mes-2;met-2;set-25 triple-mutant animals (fig. S4). This is consistent with impaired replication altering expression from the array by interfering with repression by multiple histone modifications (H3K27me3 and H3K9me1/2/3). In the absence of these modifications, impaired replication has a reduced effect on expression.

To characterize how the chromatin marks of the array are altered when replication is impaired, we first used chromatin immunoprecipitation (ChIP) to compare the levels of H3K27me3 in wt animals and in div-1 mutants. H3K27me3 was reduced on the array in animals with impaired replication (~3- and ~4-fold in the gene body and the promoter, respectively) (P < 0.01) (fig. S5). Impaired replication therefore interferes with the maintenance of H3K27me3 on the array. However, H3K27me3 levels changed similarly on four additional regions of the genome (fig. S5), indicating that the alterations to chromatin are not restricted to the high-copy array.

To investigate this further, we quantified the global levels of H3K27me3 in wt and div-1 chromatin from embryonic nuclei using immunofluorescence. Consistent with the ChIP results, H3K27me3 levels were globally reduced in the nuclei of early div-1 mutant embryos (Fig. 3A; 1.35-fold, P = 0.022). We used the same technique to quantify the levels of H3K9me3 and found that they were also globally depleted in the chromatin of div-1 embryos (Fig. 3B; 1.4-fold, P = 0.025). In contrast, the levels of a transcription activation–associated histone modification, H3K4me3, were increased in the div-1 embryos (Fig. 3C; 1.4-fold, P = 0.0055). All the embryos quantified were from the 2- to 10-cell stage, which is before the onset of major zygotic transcription, indicating that the changes in chromatin are not a secondary consequence of any changes in transcription. We also observed similar changes in late-stage div-1(RNAi) embryos (fig. S6), indicating that the changes in chromatin are maintained after the onset of transcription and during development. Depleting pole-2 confirmed the results obtained with div-1(RNAi) (fig. S7). The globally reduced levels of H3K27me3 and H3K9me3 were also confirmed by Western blotting (Fig. 3D and fig. S8). Impaired DNA replication therefore globally alters the histone modification levels in chromatin, including those before the onset of widespread zygotic transcription (19).

Fig. 3 Impaired DNA replication globally alters histone modifications. (A to C) Representative images and quantification of histone modification levels in wt and div-1 embryos derived from self-fertilizing hermaphrodites. Average of each embryo after subtracting the background is plotted. Fold change relative to wt and P values (two-sided t test) is indicated for each comparison. DAPI, 4′,6-diamidino-2-phenylindole. Scale bars, 50 μm. (D) Western blot analysis showing H3K27me3 and total H3. Samples are three biological replicates from synchronized wt and div-1(or148) L1s from hermaphrodite parents. Quantification is shown on the right (means + SD; two-sided t test). Antibodies used here were validated by us and others (5, 12).

To test whether loci other than the transgene array also have altered expression when replication is impaired, we sequenced RNA from wt and div-1 L1 stage larvae. Consistent with the response of the transgene array, many more genes had increased compared to decreased expression in the div-1 mutants [493 up-regulated genes versus 9 down-regulated genes at a false discovery rate (FDR) of <0.05].

To relate changes in expression to the normal chromatin state of each gene, we used data from the modENCODE consortium (15). Consistent with the response of the array, this revealed widespread up-regulation of genes with normally repressed chromatin states (Fig. 4). This derepression was observed for genes normally characterized by states defined by either high H3K9me2/3 or high H3K27me3 (Fig. 4). In contrast, genes without repressive chromatin states (15) in wt animals were not up-regulated as a group in div-1 mutants (Fig. 4). Together, these results show that impaired DNA replication during early development has a major impact on chromatin and gene expression, globally reducing the levels of repressive histone modifications and causing widespread up-regulation of heterochromatic genes in the resulting animals.

Fig. 4 Impaired DNA replication globally derepresses chromatin. Fold change in expression of genes mapping to different modENCODE chromatin states between div-1 and wt L1 larvae. The number of genes assigned to each state is indicated.

A recent study demonstrated that paternal histones marked with H3K27me3 are transmitted from C. elegans sperm chromatin to the zygote (5). These paternally inherited histones marked with H3K27me3 are then recycled during replication and deposited on the two daughter DNA strands during each of the early embryonic divisions, even in the absence of a functional PRC2 complex (5). We hypothesized that the loss of heterochromatin-associated histone marks might result from reduced retention of heterochromatic histones on the genome during the embryonic cell divisions. To test this, we quantified the decay of paternally inherited histones marked with H3K27me3 in control and div-1(RNAi) embryos that were PRC2-deficient (Fig. 5). In this assay, there is no PRC2 activity in the early embryos because of the maternal mes-2 genotype, and only the paternal genome contributes H3K27me3-marked histones to the zygote. The dilution of these modified histones during the early embryonic cell divisions therefore quantifies the extent to which they are successfully transmitted to and retained on the DNA daughter strands during each replication cycle. Compared to in control embryos, the decay of H3K27me3-modified histones during the early cell cycles was accelerated in div-1(RNAi) animals (Fig. 5, B and C). Thus, impaired DNA replication induces the loss of H3K27me3 by impairing the retention of modified histones on the genome during the replication cycles of the early embryo. However, it is important to note that this assay does not exclude the possibility that impaired replication may also interfere with histone methyltransferase activity.

Fig. 5 Impaired replication interferes with the inheritance of H3K27me3-modified paternal histones. (A) wt males were crossed to mes-2 mutant mothers either on control or div-1(RNAi) plates. Gravid worms were transferred to polylysine slides, gently squashed with a coverslip to allow embryos to extrude, and stained for H3K27me3. Images show H3K27me3 only in the paternal pronucleus. Scale bars, 50 μm. (B) Representative images of two-, four-, and eight-cell embryos from both groups stained with DAPI and an anti-H3K27me3 antibody. Scale bars, 50 μm. (C) Quantification of the H3K27me3 signal originating from the paternally deposited histones. Embryos were grouped according to the developmental stage, and a one-phase decay exponential curve was fitted through the data using Prism software. Decay rate constants for each curve are plotted in the inset. P values were calculated by two-sided t test. Sample size: control 1 (2 to 3, n = 19; 4 to 5, n = 15; 6 to 8, n = 6; 9 to 12, n = 8; 13 to 16, n = 7), control 2 (2 to 3, n = 21; 4 to 5, n = 11; 6 to 8, n = 11; 9 to 12, n = 10; 13 to 16, n = 7), control 3 (2 to 3, n = 16; 4 to 5, n = 17; 6 to 8, n = 7; 9 to 12, n = 2; 13 to 16, n = 1), div-1 1 (2 to 3, n = 16; 4 to 5, n = 23; 6 to 8, n = 15; 9 to 12, n = 9; 13 to 16, n = 5), div-1 2 (2 to 3, n = 36; 4 to 5, n = 27; 6 to 8, n = 16; 9 to 12, n = 22; 13 to 16, n = 5), div-1 3 (2 to 3, n = 28; 4 to 5, n = 22; 6 to 8, n = 10; 9 to 12, n = 4; 13 to 16, n = 1). Bars indicate SD, and the middle dots indicate the means. (D) Summary of the result of the experiment. Impaired DNA replication resulting from div-1 knockdown interferes with efficient transmission of H3K27me3 histones to daughter nuclei.

An important question in epigenetics is the extent to which acquired epigenetic states are transmitted between generations (20). We therefore tested what happens to the expression from the derepressed transgene array after normal DNA replication is restored. If the epigenetic state of the locus is not transmitted between generations, then restoration of normal DNA replication would result in the reestablishment of repression. In contrast, if the derepressed state is transmitted from parent to offspring, then expression would remain high in subsequent generations with normal replication.

To distinguish between these possibilities, we crossed wt males to div-1(or148) hermaphrodites (both carrying the daf-21p::mCherry transgene) and measured mCherry expression in the wt descendants for multiple generations (Fig. 6A). We found that the expression from the array was elevated for five generations after returning to the situation in which both animals and their parents had a wt div-1 genotype (Fig. 6B). Moreover, introducing the div-1(or148) mutation for a single generation before outcrossing was sufficient to induce transgenerationally inherited elevated expression (fig. S9). Thus, impaired DNA replication derepresses the transgene array, and this derepression takes multiple generations to completely reset after normal replication is restored. The return of the transgene expression to the basal level further demonstrates that the effect is epigenetic and not caused by genetic changes.