Chromatin Modifier EP400 Regulates Oocyte Quality and Zygotic Genome Activation in Mice

Abstract Epigenetic modifiers that accumulate in oocytes, play a crucial role in steering the developmental program of cleavage embryos and initiating life. However, the identification of key maternal epigenetic regulators remains elusive. In the findings, the essential role of maternal Ep400, a chaperone for H3.3, in oocyte quality and early embryo development in mice is highlighted. Depletion of Ep400 in oocytes resulted in a decline in oocyte quality and abnormalities in fertilization. Preimplantation embryos lacking maternal Ep400 exhibited reduced major zygotic genome activation (ZGA) and experienced developmental arrest at the 2‐to‐4‐cell stage. The study shows that EP400 forms protein complex with NFYA, occupies promoters of major ZGA genes, modulates H3.3 distribution between euchromatin and heterochromatin, promotes transcription elongation, activates the expression of genes regulating mitochondrial functions, and facilitates the expression of rate‐limiting enzymes of the TCA cycle. This intricate process driven by Ep400 ensures the proper execution of the developmental program, emphasizing its critical role in maternal‐to‐embryonic transition.


Supplemental Figures
(c) Heatmap of differentially expressed genes in GV oocytes between control and cKO group.(d) Relative mRNA level of "P53 signaling" genes in GV oocyte from control and cKO mice by qRT-PCR.Data are presented as means ± SD (n=3).mRNA levels of genes were normalized to Actb.Two-tailed student's t-test was used to calculate p values.ns, not significant, * p < 0.05, * * p < 0.01.(e) TOM20 staining in control and Ep400-depleted GV oocytes to show mitochondrial distribution.Data are presented as means ± SD (n=15).Two-tailed student's t-test was used to calculate p values.Scale bar, 25μm.* * * p < 0.001.(f) JC1 staining in control and Ep400-depleted GV oocytes to detect MMP.MMP level is indicated by red/green ratio.Data are presented as means ± SD (n=14).Two-tailed student's t-test was used to calculate p values.Scale bar, 25μm.ns, not significant.
(g) GM130 staining in control and Ep400-depleted GV oocytes to show distribution of Golgi apparatus.Data are presented as means ± SD (n=10).Two-tailed student's t-test was used to calculate p values.Scale bar, 25μm.ns, not significant.(h) GV oocytes were stained with Lyso-Tracker Red to show lysosome distribution.Data are presented as means ± SD (n=12).Two-tailed student's t-test was used to calculate p values.Scale bar, 25μm.ns, not significant.

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Figure S1.Generation of maternal Ep400 deficient mice.(a) Representative genotyping results of Zp3 Cre , and Fl, Wt, Deleted (Del) alleles.(b) Western blotting of EP400 using protein extracts from mouse ESC with indicated cell numbers.(c) Dot blot of EP400 using protein extracts from mouse ESC with indicated cell numbers for imaging assessment.(d) Dot blot of EP400 using protein extracts from mouse GV oocytes with indicated cell numbers for imaging assessment with short (left) and long (right) exposure.

Figure S2 .
Figure S2.Organelle aggregation of oocytes from cKO mice.(a) TUNEL staining of ovarian sections from adult control and cKO mice.Data are presented as means ± SD (n=8).Two-tailed student's t-test was used to calculate p values.Scale bar, 100μm.* * * p < 0.001.(b) γH2A.X signal of NSN and SN GV oocytes from control and cKO mice.(c) Heatmap of differentially expressed genes in GV oocytes between control and cKO group.(d) Relative mRNA level of "P53 signaling" genes in GV oocyte from control and cKO mice by qRT-PCR.Data are presented as means ± SD (n=3).mRNA levels of genes were normalized to Actb.Two-tailed student's t-test was used to calculate p values.ns, not significant, * p < 0.05, * * p < 0.01.(e) TOM20 staining in control and Ep400-depleted GV oocytes to show mitochondrial distribution.Data are presented as means ± SD (n=15).Two-tailed student's t-test was used to calculate p values.Scale bar, 25μm.* * * p < 0.001.(f) JC1 staining in control and Ep400-depleted GV oocytes to detect MMP.MMP level is indicated by red/green ratio.Data are presented as means ± SD (n=14).Two-tailed student's t-test was used to calculate p values.Scale bar, 25μm.ns, not significant.

Figure S3 .
Figure S3.Maturation failure and quality decline of Ep400-depleted oocytes.(a) MI oocytes (8h following IVM) were stained with Mito-Tracker Red to show mitochondrial distribution.Scale bar, 25μm.(b) Images of fertilized and unfertilized oocytes from control and cKO mice.Scale bar, 50μm.(c) TOM20 staining in control and Ep400-depleted MII oocytes to show mitochondrial distribution.Data are presented as means ± SD (n=15).Two-tailed student's t-test was used to calculate p values.Scale bar, 25μm.* * p < 0.01.(d) JC1 staining in control and Ep400-depleted MII oocytes to detect MMP.MMP level is indicated by red/green ratio.Data are presented as means ± SD (n=15).Two-tailed student's t-test

Figure S4 .
Figure S4.Effect of Ep400 depletion on transcriptome of MII oocyte.(a) Volcano plot comparing transcripts of MII oocytes between control and cKO group.Gene expression with log 2 (fold change) increased or decreased more than 1 in cKO group is highlighted with red and blue respectively.(b) Heatmap of differently expressed genes in MII oocytes between control and cKO group.(c) Gene ontology analysis of upregulated and downregulated genes in MII oocytes by Metascape.

Figure S5 .
Figure S5.Effect of Ep400 depletion on early embryos.(a) Comparison of γH2A.X signal of early 2-cell embryos between control and cKO group.Data are presented as means ± SD (n=16).Scale bar, 25μm.Two-tailed student's t-test was used to calculate p values.ns, not significant.(b) Representative images of embryos obtained from natural cycle ovulation of control and cKO female mated with WT male mice and cultured in KSOM medium.Scale bar, 100μm.

Figure S6 .
Figure S6.Effect of Ep400 depletion on transcriptome of early 2-cell embryos.(a) Volcano plot comparing transcripts of early 2-cell embryos between control and cKO group.Gene expression with log 2 (fold change) increased or decreased more than 1 in cKO group is highlighted with red and blue respectively.E2C, early 2-cell embryos.(b) Heatmap of differently expressed genes in early 2-cell embryos between control and cKO group.(c) Heatmap of expression (log 2 FPKM) of representative ZGA genes in early 2-cell embryos.(d) Gene ontology analysis of downregulated genes in early 2-cell embryos by Metascape.(e) Overlapping of dysregulated genes at early and late 2-cell stages.

Figure S7 .
Figure S7.Preimplantation embryo development arrest of maternal Ep400-depleted embryos.(a) JC1 staining in control and Ep400-depleted 2-cell embryos to detect MMP.MMP level is indicated by red/green ratio.Data are presented as means ± SD (n=12).Two-tailed student's t-test was used to calculate p values.Scale bar, 25μm.ns, not significant.(b) GM130 staining in control and Ep400-depleted 2-cell embryos to show Golgi apparatus.Data are presented as means ± SD (n=8).Two-tailed student's t-test was used to calculate p values.Scale bar, 25μm.ns, not significant.(c) Lysosome distribution in control and Ep400-depleted 2-cell embryos.Data are presented as means ± SD (n=11).Two-tailed student's t-test was used to calculate p values.Scale bar, 25μm.ns, not significant.(d) Nuclear morphology of Ep400 depleted 2-cell embryos.Red arrowheads indicate abnormal nuclei.Scale bar, 25μm.

Figure S8 .
Figure S8.Impact of maternal Ep400 on epigenetic modifications at late 2-cell stage.(a) Cumulative curve illustrates increased PI value in cKO group relative to control group.(b) Density plot of H3.3, Pol II, and H3K27ac around enhancer (H3K27ac-marked regions) and 2 kb upstream/downstream of enhancer in control and maternal Ep400-depleted late 2-cell embryos (upper panel), and corresponding heatmap (lower panel).

Figure S9 .
Figure S9.Ep400 deficiency impacted expression of minor and major ZGA genes.(a) Relative mRNA level of minor ZGA genes in late 2-cell embryos from control and cKO mice by qRT-PCR.Data are presented as means ± SD (n=3).mRNA levels of genes were normalized to Actb.Two-tailed student's t-test was used to calculate p values.* * * p < 0.001.

Figure S10 .
Figure S10.Major ZGA regulates mitochondrial functions.(a) Gene ontology analysis of major ZGA genes.(b) JC1 staining in late 2-cell embryos treated with or without DRB to detect MMP.MMP level is indicated by red/green ratio.Data are presented as means ± SD (n=7).Two-tailed student's t-test was used to calculate p values.Scale bar, 10μm.* * * p < 0.001.

Figure S11 .
Figure S11.Knockdown of Ep400 in early mouse embryos led to reduced expression of Major ZGA genes.(a) Mouse GV oocytes were injected with siRNAs against Ep400, followed by oocyte collection at 48h post culturing in IVM medium with Milrinone for knockdown efficiency analysis by qRT-PCR.Data are presented as means ± SD (n=3).mRNA levels of genes were normalized to Actb.Two-tailed student's t-test was used to calculate p values.* * p < 0.01, * * * p < 0.001.

Figure S14 .
Figure S14.Ep400 deficiency led to reduced enrichment of H3.3 and Pol II at Idh3b and Psat1 promoters in mouse ESC.(a) qRT-PCR was performed to verify knockdown efficiency at 48h post transfection of siRNA-1 against Ep400 in mouse ESC.Data are presented as means ± SD (n=3).mRNA level of Ep400 was normalized to Actb.Two-tailed student's t-test was used to calculate p values.* * * p < 0.001.(b) ChIP-qPCR was performed to examine H3.3 and Pol II enrichment at Idh3b and Psat1 promoter regions in control and Ep400-knockdown ESC.qPCR signals from ChIP samples were normalized to that of respective Input samples.Data are presented as means ± SD (n=3).
). Two-tailed student's t-test was used to calculate p values.Scale bar, 25μm.* * * p < 0.001.(e) MII oocytes were stained with Lyso-Tracker Red to show lysosome in MII oocytes.Data are presented as means ± SD (n=21).Two-tailed student's t-test was used to calculate p values.Scale bar, 25μm.* * * p < 0.001.(f) Images of sperm binding to the zona pellucida of control and Ep400-depleted 2-cell embryos.LCA-FITC staining in control and Ep400-depleted GV oocytes show CGs.Data are presented as means ± SD (n=10).Two-tailed student's t-test was used to calculate p values.Scale bar, 25μm.* * * p < 0.001.(i) Ovastacin staining in control and Ep400-depleted GV oocytes.Data are presented as means ± SD (n=10).Two-tailed student's t-test was used to calculate p values.Scale bar, 25μm.
b) Relative mRNA level of major ZGA genes in late 2-cell embryos from control and cKO mice by qRT-PCR.Data are presented as means ± SD (n=3).mRNA levels of genes were normalized to Actb.Two-tailed student's t-test was used to calculate p values.* * * p < 0.001.Comparison of expression changes of Maternal RNA in early/late 2-cell embryos from control/cKO group by RNA-seq data.Mann-Whitney U test was used to calculate p values.(k) Major ZGA genes were evenly divided into two groups based on EP400 enrichment at gene promoters.Mann-Whitney U test was used to calculate p values.(l) Expression of Major ZGA genes with strong EP400 association by RNA-seq data.Note that these genes had significantly reduced gene expression at late 2-cell stage in absence of maternal Ep400.Mann-Whitney U test was used to calculate p values.
). mRNA levels of genes were normalized to Actb.Two-tailed student's t-test was used to calculate p values.* * * p < 0.001.((m)Expression of Major ZGA genes with weak EP400 association by RNA-seq data.Note that these genes had no evident changes of gene expression at late 2-cell stage in absence of maternal Ep400.Mann-Whitney U test was used to calculate p values.