Hotspots of De Novo Point Mutations in Induced Pluripotent Stem Cells Masahito Yoshihara, Ryoko Araki, Yasuji Kasama, Misato Sunayama, Masumi Abe, Kohji Nishida, Hideya Kawaji, Yoshihide Hayashizaki, Yasuhiro Murakawa  Cell Reports  Volume 21, Issue 2, Pages 308-315 (October 2017) DOI: 10.1016/j.celrep.2017.09.060 Copyright © 2017 The Authors Terms and Conditions

Cell Reports 2017 21, 308-315DOI: (10.1016/j.celrep.2017.09.060) Copyright © 2017 The Authors Terms and Conditions

Figure 1 De Novo Point Mutations in Mouse iPSCs Are Underrepresented in Active Chromatin Regions (A) Study design. iPSC reprogramming per se causes de novo point mutations (star). We integrated the data on these mutations and on human disease-causing single-nucleotide polymorphisms (SNPs) with genomic and epigenomic data and investigated their distribution throughout the genome. RNA Pol II, RNA polymerase II; DHS, DNase I hypersensitive site. (B) Log2-transformed relative enrichment of 2,167 de novo point mutations in mouse iPSCs within genic and intergenic regions. To compute the enrichment, the density of mutations in each region was compared with the average density across the whole genome. (C) Distribution of 2,167 de novo point mutations in mouse iPSCs according to the epigenetic status determined in mouse embryonic fibroblasts (MEFs). The number of mutations, as well as the percentage, in each genomic category is shown. Bar colors represent the p values calculated by Fisher’s exact test (∗p < 0.05). See also Figures S1 and S2 and Tables S1, S2, and S4. Cell Reports 2017 21, 308-315DOI: (10.1016/j.celrep.2017.09.060) Copyright © 2017 The Authors Terms and Conditions

Figure 2 De Novo Point Mutations in Mouse iPSCs Are Overrepresented in LADs (A) Enrichment of 2,167 de novo point mutations in LADs identified in MEFs and mouse embryonic stem cells (mESCs). Bar colors represent the p values calculated by Fisher’s exact test (∗∗∗p ≪ 1.0 × 10−200, ∗∗p = 2.4 × 10−60). (B) Number of de novo point mutations in LADs identified in MEFs and mESCs. Most mutations are commonly observed in LADs identified in MEFs and mESCs. (C) Genome browser view of the distribution of de novo point mutations identified in mouse iPSCs, LADs identified in MEFs and mESCs, and RefSeq genes. See also Figure S2 and Tables S1 and S2. Cell Reports 2017 21, 308-315DOI: (10.1016/j.celrep.2017.09.060) Copyright © 2017 The Authors Terms and Conditions

Figure 3 De Novo Point Mutations in Human iPSCs Are Underrepresented in Active Chromatin Regions and Overrepresented in Inactive Regions, whereas Human Disease-Causing SNPs Show Opposite Trends (A–D) Distribution of 5,448 de novo point mutations within intergenic and genic regions (A). Distribution of 7,747 human disease-causing SNPs within intergenic and genic regions (B). Distribution of de novo mutations according to the epigenetic status determined in normal human dermal fibroblasts (NHDFs) (C). Distribution of human disease-causing SNPs according to the epigenetic status determined in NHDFs (D). The H3K9me3 regions are transcriptionally repressed (C and D). The number of mutations, as well as the percentage, in each genomic category is shown. Bar colors represent the p values calculated by Fisher’s exact test (∗p < 0.05, ∗∗p = 1.3 × 10−88) (A–D). (E–H) A smoothed scatterplot of the number of de novo point mutations against H3K9me3 ChIP-seq intensity (ρ = 0.66, p < 2.2 × 10−16, Spearman’s rank correlation test) (E). A smoothed scatterplot of the number of human disease-causing SNPs against H3K9me3 ChIP-seq intensity (ρ = −0.48, p < 2.2 × 10−16) (F). The x axes represent log2-transformed fold enrichment of H3K9me3 ChIP-seq intensity normalized to input control DNA sequencing data for NHDFs (E and F). A smoothed scatterplot of the number of de novo point mutations against DNase-seq read counts (ρ = −0.53, p < 2.2 × 10−16) (G). A smoothed scatterplot of the number of human disease-causing SNPs against DNase-seq read counts (ρ = 0.49, p < 2.2 × 10−16) (H). The x axes represent the log2-transformed DNase-seq read counts in NHDFs (G and H). The y axes show the number of mutations or SNPs per 10-Mb window of the genome (E–H). See also Figures S3 and S4 and Tables S1, S3, and S4. Cell Reports 2017 21, 308-315DOI: (10.1016/j.celrep.2017.09.060) Copyright © 2017 The Authors Terms and Conditions

Figure 4 De Novo Point Mutations in Human iPSCs Preferentially Occur in LADs (A and B) Enrichment of 5,448 de novo point mutations in LADs identified in IMR90 and TIG3 fibroblasts using ChIP-seq and DamID, respectively (A). Enrichment of 7,747 human disease-causing SNPs in LADs identified in IMR90 and TIG3 fibroblasts (B). The number of mutations, as well as the percentage, in each genomic category is shown. Bar colors represent the p values calculated by Fisher’s exact test (∗p = 9.8 × 10−6, ∗∗∗p ≪ 1.0 × 10−200). (C and D) A smoothed scatterplot of de novo point mutations against LAD ChIP-seq intensity (ρ = 0.38, p = 5.5 × 10−11, Spearman’s rank correlation test) (C). A smoothed scatterplot of human disease-causing SNPs against LAD ChIP-seq intensity (ρ = −0.28, p = 1.8 × 10−6) (D). The x axes represent log2-transformed fold enrichment of LAD ChIP-seq intensity normalized to input control DNA sequencing data in IMR90. The y axes show the number of mutations or SNPs per 10-Mb window of the genome. (E) Left: enrichment of 5,448 de novo point mutations in LADs identified in HT1080 (fibrosarcoma cell line) and human embryonic stem cells (hESCs) by Meuleman et al. (2013) (∗∗∗p ≪ 1.0 × 10−200). Right: number of de novo point mutations in LADs identified in HT1080 and hESCs. Most mutations are commonly observed in LADs identified in HT1080 and hESCs. See also Figure S4 and Tables S1 and S3. Cell Reports 2017 21, 308-315DOI: (10.1016/j.celrep.2017.09.060) Copyright © 2017 The Authors Terms and Conditions

Figure 5 De Novo Point Mutations in LADs of Human iPSCs Occur in a Transversion-Predominant Manner (A) Frequencies of transversions (Tv) and transitions (Ts) in human iPSCs positioned within transcriptionally active DNase I hypersensitive sites (DHSs) identified in hESCs (left) and within LADs identified in IMR90 fibroblasts (center) and TIG3 fibroblasts (right). (B) Genome browser view of the distribution of de novo point mutations identified in human iPSCs, LADs identified in IMR90 and TIG3, and RefSeq genes. In LADs, Tv (orange) are more frequently observed than Ts (blue), whereas mutations are rarely observed in gene-rich regions. See also Figure S5 and Tables S1 and S3. Cell Reports 2017 21, 308-315DOI: (10.1016/j.celrep.2017.09.060) Copyright © 2017 The Authors Terms and Conditions