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Germ line–inherited H3K27me3 restricts enhancer function during maternal-to-zygotic transition

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Science  14 Jul 2017:
Vol. 357, Issue 6347, pp. 212-216
DOI: 10.1126/science.aam5339
  • Fig. 1 Intergenerational inheritance of H3K27me3 from Drosophila oocyte.

    (A) Schematic of one ovariole in the germ line of a Drosophila. In red, regions of E(z)-KD (arrow). The right side of the cartoon depicts the apposition of maternal and paternal pronuclei. (B) Stage 10 egg chamber of a strain expressing HA-E(z). The H3K27me3 marks the oocyte (arrowhead). HA-E(z) is present in the nucleoplasm and on the chromatin of the wild-type oocyte. See fig. S1E. Scale bars, 10 μm. (C) Antibody to H3K27me3 (α-H3K27me3) and 4′,6-diamidino-2-phenylindole (DAPI) staining of the apposed pronuclei in one-cell wild-type and E(z)-KD embryos. H3K27me3 is also transmitted to the embryo on the maternal gamete in the absence of E(z). See fig. S1B. Scale bars, 2 μm. (D) α-H3K27me3 and DAPI staining of wild-type early embryos at different developmental cycles. For clarity, mitotic chromosomes are shown. See fig. S2, A and B. Scale bar, 2 μm (here and below). (E) α-H3K27me3 and DAPI staining of E(z)-KD embryos. (F) α-HA and DAPI staining of HA-E(z) strain in early embryos at different developmental cycles and at different phases of the cell cycle. (G) α-HA and DAPI staining of E(z)-KD embryos.

  • Fig. 2 Polycomb-mediated chromatin regulation during early embryogenesis, prior to ZGA.

    (A) Cartoon of early embryonic development of Drosophila. The number of cycles corresponds to the mitotic divisions. During cycles 9 to 13, an initial subset of 100 genes is transcribed (blue wave). After 14 mitotic divisions, the embryo is fully transcriptionally competent (green wave). (B) H3K27me3 ChIP-seq tracks. The Hox cluster (right inset) is already decorated with H3K27me3 before cycle 9. Other H3K27me3-marked regions lose the mark during development (left inset). The enriched H3K27me3 domains (EDs, false discovery rate = 0.05) are shown below each track in black. The last track shows the H3K27me3 signal in E(z)-KD embryos at ZGA. See also fig. S2I. (C) Venn diagram showing the total number and overlap of EDs detected at each developmental stage. (D) Box plot showing the size distribution (in kb) of the enriched domains.

  • Fig. 3 Loss of H3K27me3 before zygotic genome activation leads to embryonic lethality and homeotic transformation.

    (A) E(z) mRNA levels were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Before ZGA (cycles 9 to 13), the maternally loaded mRNA (red) is strongly reduced upon E(z)-KD. At ZGA (cycle 14), E(z) mRNA (green) is zygotically transcribed in E(z)-KD embryos (cycles 9 to 13, P = 4.4 × 10−5; cycle 14, P = 0.008; t test). (B) Hatching rate of fertilized control and E(z)-KD eggs (P = 0.0002, Mann-Whitney test). See fig. S3, A to C. (C) Hatching rate of E(z)-TS embryos reared at a permissive temperature, at a nonpermissive temperature, and shifted to a permissive temperature from ZGA onward (P < 0.0001, Mann-Whitney test). (D) Total extracts of stage 17 (end of embryogenesis) embryos were analyzed by Western blot with α-H3K27me3. α-H3 and Ponceau staining were used as loading controls. H3K27me3 is detectable in shifted embryos, confirming that E(z) regains its enzymatic function (lanes 5 and 6). (E) Representative examples of cuticle preparations of embryos reared at a permissive temperature and of embryos shifted to a permissive temperature at ZGA showing homeotic transformations. See fig. S3M. (F) Hatching rate of embryos from fly strains overexpressing H3.3 and H3.3K27M transgenes. (maternal P = 0.008, zygotic P = 0.2; Mann-Whitney test). (G) Total extracts of stage 17 embryos were analyzed by Western blot with α-H3K27me3 upon overexpression of H3.3K27M. Both the maternal and zygotic H3.3K27M mutant transgenes strongly reduced H3K27me3. α-H3 was used as a loading control. (H) Representative examples of cuticle preparations of embryos with maternally overexpressed H3.3 or H3.3K27M. Maternal overexpression of H3.3K27M caused homeotic transformations. (I) Representative examples of cuticle preparations of embryos that express H3.3 or H3.3K27M zygotically (post-ZGA). The embryos do not develop homeotic transformations.

  • Fig. 4 Loss of maternally inherited H3K27me3 triggers increased levels of H3K27ac and precocious expression of regulatory genes.

    (A) Heat maps showing H3K27ac (left two panels) and H3K4me1 (right panel) ChIP signals centered over the union of H3K27ac peaks, as well as log2 fold changes of H3K27me3 upon E(z)-KD (middle panel). See fig. S4C. (B) Violin plot shows log2 fold up-regulation at ZGA in E(z)-KD versus wild-type embryos for the top 200 H3K27ac peak-associated genes (n = 155) (P = 0.00013, Wilcoxon test). See fig. S4H. (C) Genome browser example highlighting that E(z)-KD embryos gain H3K27ac at loci enriched in H3K4me1 and H3K27me3 in wild-type embryos and subsequent Doc1 and Doc2 up-regulation. See fig. S4, D to G. (D) qRT-PCR validation of genes identified as up-regulated in E(z)-KD versus wild-type embryos at ZGA by RNA-seq. As negative controls, we tested the expression of genes that show unchanged levels of H3K27ac between wild-type and E(z)-KD. (E) H3K27me3 ChIP-qPCR at the upstream regions of genes with increased expression in E(z)-KD versus wild-type embryos at ZGA. *P < 0.05, **P < 0.01, ***P < 0.001; t test, n = 3. (GluRIIA and CG14013 share the same regulatory region.) See fig. S4K.

Supplementary Materials

  • Germ line-inherited H3K27me3 restricts enhancer function during maternalto-zygotic transition

    Fides Zenk, Eva Loeser, Rosaria Schiavo, Fabian Kilpert, Ozren Bogdanović, Nicola Iovino

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

    Download Supplement
    • Materials and Methods
    • Figs. S1 to S5
    • Tables S1 to S3
    • Descriptions of databases S1 to S4
    • References

    Additional Data

    Data S1
    ED annotation
    Data S2
    2 Top200 annotation
    Data S3
    3 DESeq
    Data S4
    4 generated datasets

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