Research ArticleDEVELOPMENTAL GENETICS

Impaired DNA replication derepresses chromatin and generates a transgenerationally inherited epigenetic memory

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Science Advances  16 Aug 2017:
Vol. 3, no. 8, e1701143
DOI: 10.1126/sciadv.1701143
  • 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.

  • 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.

  • 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).

  • 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.

  • 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.

  • Fig. 6 Transgenerational epigenetic inheritance of acquired expression following DNA replication impairment.

    (A) Experimental design. (B) Quantification of daf-21p::mCherry fluorescence intensity in wt (black) and div-1(or148) (red) animals and in their wt descendants following outcrossing of the or148 allele. At each generation, expression was normalized and compared to the control sample that was generated using the same batch of P0 males but crossed to wt ancestors and propagated in parallel in an analogous way. The y axis is in log scale. Sample size is indicated below each box plot. ****P < 0.0001; *P < 0.05; ns (not significant), P > 0.05 (Wilcoxon rank test).

  • Fig. 7 Summary model.

    Impaired DNA replication during early embryonic divisions leads to inefficient retention of modified histones during chromatin replication, resulting in a reduction of heterochromatin-associated histone modifications and transgene derepression. Multiple generations are required to restore the repressed state.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/8/e1701143/DC1

    fig. S1. Expression of the daf-21p::mCherry reporter in the progeny of animals treated with RNAi targeting different subunits of the DNA polymerase complex and its associated proteins.

    fig. S2. Increased transgene expression in div-1 mutants.

    fig. S3. Maternal div-1 deficiency results in elevated transgene expression in the offspring.

    fig. S4. Transgene up-regulation following pole-2 knockdown is suppressed in the mes-2;met-2;set-25 triple-mutant background.

    fig. S5. Impaired DNA replication reduces H3K27me3 levels on multiple loci.

    fig. S6. Global reduction of repressive histone marks and a gain of activating histone marks in late div-1(or148) embryos.

    fig. S7. Knockdown of pole-2 results in reduction of H3K27me3 mark and increase in H3K4me3 in early embryonic chromatin.

    fig. S8. Reduction in H3K9me3 mark in div-1(or148) mutant L1s detected by Western blot.

    fig. S9. Passage of the transgenic array through impaired replication for a single generation is sufficient to trigger a multigenerational effect.

    fig. S10. Quantification of H3K27me3 in interphase nuclei.

    table S1. List of genes whose knockdown results in upregulation of daf- 21p::mCherry transgene.

    table S2. C. elegans strains used in this study.

    table S3. Primers used in qPCR analyses.

    table S4. Transgenes tested for derepression with div-1(RNAi).

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Expression of the daf-21p::mCherry reporter in the progeny of animals treated with RNAi targeting different subunits of the DNA polymerase complex and its associated proteins.
    • fig. S2. Increased transgene expression in div-1 mutants.
    • fig. S3. Maternal div-1 deficiency results in elevated transgene expression in the offspring.
    • fig. S4. Transgene up-regulation following pole-2 knockdown is suppressed in the mes-2;met-2;set-25 triple-mutant background.
    • fig. S5. Impaired DNA replication reduces H3K27me3 levels on multiple loci.
    • fig. S6. Global reduction of repressive histone marks and a gain of activating histone marks in late div-1(or148) embryos.
    • fig. S7. Knockdown of pole-2 results in reduction of H3K27me3 mark and increase in H3K4me3 in early embryonic chromatin.
    • fig. S8. Reduction in H3K9me3 mark in div-1(or148) mutant L1s detected by Western blot.
    • fig. S9. Passage of the transgenic array through impaired replication for a single generation is sufficient to trigger a multigenerational effect.
    • fig. S10. Quantification of H3K27me3 in interphase nuclei.
    • table S1. List of genes whose knockdown results in upregulation of daf-21p::mCherry transgene.
    • table S2. C. elegans strains used in this study.
    • table S3. Primers used in qPCR analyses.
    • table S4. Transgenes tested for derepression with div-1(RNAi).

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