Packaging of DNA into nucleosomes not only helps to store genetic information but also creates diverse means for regulating DNA-templated processes. Attempts to reveal additional functions of the nucleosome have been unsuccessful, owing to cell lethality caused by nucleosome deletion. Taking advantage of the mammalian fertilization process, in which sperm DNA assembles into nucleosomes de novo, we generated nucleosome-depleted (ND) paternal pronuclei by depleting maternal histone H3.3 or its chaperone HIRA in mouse zygotes. We found that the ND pronucleus forms a nuclear envelope devoid of nuclear pore complexes (NPCs). Loss of NPCs is accompanied by defective localization of ELYS, a nucleoporin essential for NPC assembly, to the nuclear rim. Interestingly, tethering ELYS to the nuclear rim of the ND nucleus rescues NPC assembly. Our study thus demonstrates that nucleosome assembly is a prerequisite for NPC assembly during paternal pronuclear formation.
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We thank T. Haraguchi (Osaka University) and T. Kirchhausen (Harvard Medical School) for helpful discussion, F. Aoki (University of Tokyo) for the EGFP-NLS, Flag-H2B and Flag-H4 constructs, and I. Mattaj (European Molecular Biology Laboratory) and G.L. Xu (Chinese Academy of Sciences) for antibodies. We thank T. Haraguchi, G. German, S. Yamaguchi, S. Matoba and D. Cai for critical reading of the manuscript. This work was supported by US National Institutes of Health (U01DK089565 to Y.Z.). A.I. is supported as a research fellow for Research Abroad of the Japan Society for the Promotion of Science. Y.Z. is supported as an Investigator of the Howard Hughes Medical Institute.
The authors declare no competing financial interests.
Integrated supplementary information
(a) Experimental scheme for siRNA-mediated knockdown. GV oocytes were injected with siRNA and cultured for 18 h to reach the MII-stage. Following meiotic maturation, oocytes were fertilized in vitro. (b) RT-qPCR analysis of the Hira mRNA levels in MII oocytes. The relative expression was normalized to GAPDH. The value of Control was set as 1. (c) Representative images of fertilized oocytes stained with anti-HIRA (green) antibody and DAPI (blue) at 2 hrs after fertilization. P; paternal chromatin. M; maternal chromatin. Scale bar, 20 μm. (d) Experimental scheme for siRNA-mediated knockdown of HIRA using the in vitro growth system. Growing oocytes surrounded by granulosa layers are isolated from 12 days old female mice, injected with siRNA, and cultured for 12 days in vitro until they reach the fully grown stage. Following in vitro growth, oocytes are matured and fertilized in vitro. (e) Microinjection into growing oocytes surrounded by granulosa cells. Scale bar, 50 μm. (f) Representative images of oocyte-granulosa cell complex during oocytes growth in vitro of the indicated times. Scale bar, 200 μm. (g) In vitro maturation of cumulus-oocyte complexes following IVG. Cumulus cells surrounding oocytes are expanded during meiotic maturation. Scale bars, 100 μm.
(a) Evaluation of anti-protamine 2 (Pro2) antibody. Surface spread samples of spermatozoa were stained with Pro2 antibody (green) and DAPI (blue). As a negative control, the sample was treated with the secondary antibody only. Scale bar, 10 μm. (b) Surface spread samples of fertilized oocytes were stained with Pro2 (green) and anti-H2AX (red) antibodies and DAPI (blue) at 1 h or 1.5 h post-fertilization (hpf). Note that protamines are still detectable in the condensed region in which H2AX has not been incorporated at 1 h. At 1.5 h, protamines are undetectable in the decondensed paternal DNA in which H2AX is broadly incorporated. A spermatozoon (Sp), which did not enter the oocyte, was used as a positive control for Pro2 staining. Scale bar, 10 μm. (c, d) Representative images of fertilized oocytes stained with Pro2 antibody (white) and DAPI (blue) at 2 hpf. Whole mount samples (c) and surface spread samples (d) were prepared. Note that protamines are undetectable in the paternal DNA of both control and HIRA-depleted zygotes (pointed by arrows). Dotted lines represent the paternal DNA entering the oocytes. The total numbers of zygotes examined were 10-14 and 8-10 per group for the whole mount and surface spread, respectively. Sp; spermatozoa that do not enter the oocyte. P; paternal DNA. M; maternal DNA. PB; polar body. Scale bars, 20 μm.
Supplementary Figure 3 Expression of histone genes and localization of HIRA in H3.3-depleted zygotes.
(a) RT-qPCR analysis of oocytes injected with H3.3 siRNA. GV oocytes were injected with a mixture of siRNAs against H3.3a and H3.3b or control siRNA. Twenty-four hours after microinjection, the oocytes were matured in vitro to reach the MII-stage. RNA was extracted from the MII oocytes and the expression levels of indicated genes were analyzed by RT-qPCR. The relative expression level was normalized to Gapdh. The values of control oocytes were set as 1. The experiment was repeated twice. Error bars indicate as s.d. (b) Representative images of fertilized oocytes stained with anti-HIRA (green), anti-H2AX (red), and DAPI (blue) at 2 hours after fertilization. Note that HIRA localizes to the paternal DNA, where H2AX is not detected, in H3.3-depleted zygotes. A total of 25 zygotes per group were examined. Percentages indicate zygotes showing the depicted phenotype. P; paternal chromatin. M; maternal chromatin. PB; polar body. Scale bar, 20 μm.
Shown are representative images of the paternal and maternal pronucleus in control or H3.3-depleted zygotes. Asterisks indicate nucleolus-like bodies. Scale bars, 2.5 μm.
(a, b) Representative images of the maternal chromatin in control (a) and H3.3-depleted (b) zygotes stained with mAb414 (blue) and DAPI at the indicated times of post-fertilization (hpf). EGFP-ELYS (green) and LBR-RFP (red) mRNAs were injected at GV-stage. Merge = green + red channels. A total of 4-7 and 10-12 of control and H3.3-depleted zygotes, respectively, per time point were examined. Scale bars, 10 μm. (c) Surface spread staining of control zygotes stained with anti-H2AX (red) and Crest (white; centromere marker) antibodies at 2 hpf. The right panel indicates enlarged images of maternal chromosomes, showing that EGFP-ELYS is localized in the rim of maternal chromosome mass.
Supplementary Figure 6 Neither Nup37 nor POM121 is localized to the rim of nucleosome-depleted paternal pronucleus.
(a) Representative images of zygotes stained with mAb414 (Magenta) and DAPI at 8 hrs post-fertilization (hpf). Nup37-EGFP (green) mRNA was injected at GV-stage. A total of 13 or 26 of control or H3.3-depleted zygotes, respectively, were examined. Percentages indicate zygotes showing the depicted phenotype. (b) Representative images of zygotes stained with anti-POM121 (Magenta) and mAb414 (green) antibodies and DAPI at 8 hpf. A total of 7 or 13 of control or H3.3-depleted zygotes, respectively, were examined. P; paternal pronucleus (pointed by arrows). M; maternal pronucleus. Scale bars, 20 μm.
Supplementary Figure 8 Loss of Ran-mediated protein transport does not prevent the localization of ELYS to nuclear rim in zygotes.
(a) Western blot analysis showing the expression of wild-type and Ran mutants at similar levels. GV oocytes were injected with RanWT-mRFP1 (monomeric red fluorescent protein 1), RanT24N–mRFP1, or RanQ69L–mRFP1 mRNA. Following meiotic maturation, extracts of MII oocytes were subjected to Western blot analysis. Tubulin was used as a loading control. Molecular weight (kDa) is shown on the left. Shown is a representative result from two biologically independent experiments. (b) Representative image of the paternal pronucleus in zygotes stained with anti-lamin B1 antibody at 8 hrs post-fertilization (hpf). A total of 7, 10, and 9 zygotes in WT, T24N, and Q69L, respectively, were examined. Percentages indicate zygotes showing the depicted phenotype. Scale bar, 20 μm. (c) Representative images of the paternal and maternal chromatin in zygotes stained with anti-H2AX antibody at 2 hpf. EGFP-ELYS mRNA was co-injected with each form of Ran mRNA. A total of 12, 11, and 11 zygotes in WT, T24N, and Q69L, respectively, were examined. Scale bar, 10 μm. (d) Representative images of EGFP-ELYS and NPC (marked by the mAb414) in the paternal and maternal pronucleus of zygotes at 8 hpf. A total of 15, 20, and 13 zygotes in WT, T24N, and Q69L, respectively, were examined. Scale bar, 10 μm.
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Inoue, A., Zhang, Y. Nucleosome assembly is required for nuclear pore complex assembly in mouse zygotes. Nat Struct Mol Biol 21, 609–616 (2014) doi:10.1038/nsmb.2839
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