Volume 26, Issue 2, Pages 302-312.e4 (January 2019) Base-Editing-Mediated R17H Substitution in Histone H3 Reveals Methylation- Dependent Regulation of Yap Signaling and Early Mouse Embryo Development  Guang Yang, Changyang Zhou, Ran Wang, Shisheng Huang, Yu Wei, Xianfa Yang, Yajing Liu, Jianan Li, Zongyang Lu, Wenqin Ying, Xiajun Li, Naihe Jing, Xingxu Huang, Hui Yang, Yunbo Qiao  Cell Reports  Volume 26, Issue 2, Pages 302-312.e4 (January 2019) DOI: 10.1016/j.celrep.2018.12.046 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2019 26, 302-312.e4DOI: (10.1016/j.celrep.2018.12.046) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 BE3-Mediated Histone H3R17H Substitution and Carm1 Truncation Disrupt Pre-implantation Embryo Development (A) Scheme of BE3-mediated mutations in Hist1/2H3. The sgRNA and PAM sequences targeting Hist1H3 and Hist2H3 were shown. Co-expression of BE3 and sgHist1/2H3 induces the substitution mutation from CGC (Arginine, R) to CAC (Histidine, H) at R17 of histone H3. (B) Detection of BE3-mediated H3R17H substitution in 9 variants (A–I) for Hist1H3 and 3 variants (B, C1, and C2) for Hist2H3 in N2A cells by T7EN1 assays. (C) The sgRNA targeting sequence of Carm1 in the catalytic domain of CARM1 (exon 8). A representative Sanger sequencing result was shown, and the red arrows indicate the substituted bases. (D) Schematic diagram of BE3-mediated mutation and phenotype analysis in pre-implantation E4.5 blastocysts and E7.5/E9.5/E12.5/19.5 embryos. (E) The targeting efficiency of BE3-mediated H3R17H substitution (n = 58) and Carm1 truncation (n = 47) in blastocysts was analyzed from Sanger sequencing data by using EditR. 15 representative control (Ctrl) embryos were shown. (F) Morphological analysis of H3R17H and Carm1-stop embryos. The red arrows indicated the abnormal blastocysts. (G) The proportions of 1- or 2- cell embryos (1/2-cell), developmentally delayed embryos (4-32 cell), and normally developed blastocysts (Normal) were calculated for H3R17H substitution and Carm1-stop embryos. Ctrl, sgGFP+BE3. t test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, no significance. Cell Reports 2019 26, 302-312.e4DOI: (10.1016/j.celrep.2018.12.046) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 RNA-Seq Analysis of H3R17-Linked Gene Expression in Early Mouse Embryo (A) Global transcriptome analysis was performed for all detected genes (fragments per kilobase of transcript per million mapped reads [FPKM] > 1 in at least one sample). 3 representative embryos for each group were separately subjected to RNA-seq analysis (developmentally delayed embryos with normal cleavage). The genotypes of the analyzed embryos were shown in Figures 1I–1L. The genes with specific expression patterns in three experimental groups were highlighted with color boxes, and the gene numbers for each cluster were labeled. (B) Heatmapping of gene expression in 6 representative modules from WGCNA. Representative genes, Gene Ontology (GO) terms, and KEGG pathways for each module were also shown. (C) Boxplots showing the distribution of module expression (median FPKM of genes within a given module) for different modules. Targeting groups were compared with the 3 control samples separately for each sample. Two-way repeated measure (RM) ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, no significance. (D) CSI network of all TFs was analyzed and the top hub TFs in the regulatory network are shown. TFs within the same module were grouped together with the same color labeling. Gene expression Pearson correlation coefficient (PCC)-derived CSIs were calculated based on the RNA-seq expression values. The positive correlation between TFs was marked with red lines, and the negative correlation was marked with green lines. (E) The relative expression of pluripotency genes in FPKM was analyzed from RNA-seq data, and the mean values from 3 embryos for each group are shown. False discovery rate (FDR) analysis following t test was performed, and FDR values (q) were displayed. Cell Reports 2019 26, 302-312.e4DOI: (10.1016/j.celrep.2018.12.046) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 Histone H3R17H Substitution and Carm1 Truncation Result in Developmental Defects of Mouse Embryo (A and B) Morphological analysis of E12.5 (A) and E19.5 (B) H3R17H substitution and Carm1-stop embryos. (C) The targeting efficiencies of BE3-mediated H3R17H substitution and Carm1 truncation in E12.5 and E19.5 embryos were analyzed from Sanger sequencing data by using EditR. (D and E) The ratios between the number of embryos (not including empty uteri) and injected zygotes were calculated for three experimental groups at E12.5 (D) or E19.5 (E). (F) The percentages of embryos with developmental defects were calculated in three experimental groups at E12.5. (G) The percentages of dead and development-defective embryos (not including empty uteri) at stage E19.5 were calculated for each group. Same legend bar for (D)–(G). (H) Western blot analysis of H3R17me2a, H3R26me, H3R2me, and CARM1 expression in E12.5 embryos. 4 representative embryos from H3R17H-mutated or Carm1-truncated embryos with high targeting efficiency and a much smaller size were subjected to western blot analysis. (I–K) Representative immunofluorescence images of H3R17me2a (I) and CARM1 (J) expression as well as representative H&E staining images (K) from E12.5 control, H3R17H, and Carm1-stop embryos. Representative embryos from targeting groups with smaller size compared with normal control embryos were subjected to immunostaining and H&E staining assays. t test, ∗p < 0.05, ∗∗p < 0.01; ∗∗∗p < 0.001; ns, no significance. Cell Reports 2019 26, 302-312.e4DOI: (10.1016/j.celrep.2018.12.046) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 CARM1-Mediated H3R17 Methylation Regulates Embryo Development through Yap Signaling Pathway (A) Analysis of signaling pathways enriched in each module in Figure 2B. Red and green colors indicate activated and inhibitory enrichment, respectively (I, inhibit; A, activate). The value is calculated from the log10(FDR), and the red/green borders indicate significant enrichments (FDR < 0.01). (B and C) GSEA showing enrichment of the YAP (B) and cell cycle (C) gene signatures in the control embryo relative to H3R17H mutation or Carm1 truncation embryos. (D) The relative expression of Yap1 target genes in FPKM was analyzed from RNA-seq data. The mean values from 3 embryos for each group are shown. (E–H) Yap1 overexpression rescues the H3R17H mutation- and Carm1 truncation-elicited phenotypes in E4.5 blastocysts (E and F) and E12.5 embryos (G and H). Yap1 mRNA was co-injected with BE3 and sgRNAs targeting Hist1/2H3 or Carm1. The morphology of embryos was captured in (E) and (G). The proportion of normal E4.5 blastocysts was shown in (F), and the percentage of E12.5 embryos with developmental defects was show in (H). sgRNAs targeting Yap1 and SpCas9 were also co-injected and E12.5 embryos were collected for morphological analysis in (G) and (H). (I) Enrichment of H3R17me2a around the TSSs of Yap1, Ccne2, Vgll3, and Itga5 by analyzing the ChIP-seq data from GEO: GSM1874213. (J) ChIP-qPCR analysis of H3R17me2a enrichment around the TSSs of the indicated genes. Three replicates were subjected to ChIP assays. The black lines under the peak in (I) indicate the region of PCR products. IgG and a pair of primers inside the gene body (about 1 kb proximal to the enrichment region) served as negative control. t test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ns, no significance. Cell Reports 2019 26, 302-312.e4DOI: (10.1016/j.celrep.2018.12.046) Copyright © 2018 The Author(s) Terms and Conditions