A Transcriptome (RNA-seq) Transcriptome (RNA-seq) 3. 2.5 2..5..5...5..5 2. 2.5 3. 2.5 2..5..5...5..5 2. 2.5 Cluster Dendrogram RS_ES3.2 RS_ES3. RS_SHS5.2 RS_SHS5. PS_SHS5.2 PS_SHS5. RS_LJ3 PS_LJ3..4 _SHS5.2 _SHS5..4.3.5.2.6.. _LA3.2. _E3d5_LJ3.2 _E3d5_LJ3. _E3d5_LJ3.3 _E2d5_LJ3.3 _E2d5_LJ3.2 _E4d5_LJ3.3 _E4d5_LJ3. _E4d5_LJ3.2 _E2d5_LJ3. _SY2.2 _SY2. HSC. RS_ES3_2 RS_ES3_ RS_SHS5_2 RS_SHS5_ PS_SHS5_2 PS_SHS5_ RS_LJ3 PS_LJ3 _6 _OG _VG _OG2 GS_SHS5_2 GS_SHS5 VG8 _VG5 _Rosa5 _29mix _Rosa3 4 _OG _LA3 _OG2 _OG _E3d5_LJ3_2 _E3d5_LJ3 E3d5_LJ3_3 _E2d5_LJ3_3 _E2d5_LJ3_2 _E4d5_LJ3_3 _E4d5_LJ3 E4d5_LJ3_2 _E2d5_LJ3 SY2_2 _SY2_ HSC_2 HSC_ dist(cor(na.omit(tss.exprs.chip[, c(:, 24, 27, 3, 48:5, hclust 56:4)]))) (*, "complete") Cluster Dendrogram..5..5 2. 2.5 RS_ES3.2 RS_ES3. RS_SHS5.2 RS_SHS5. PS_SHS5.2 PS_SHS5. RS_LJ3 PS_LJ3.2....4.3.5.2.6.4. _E2d5_LJ3.3 _E2d5_LJ3.2 _E3d5_LJ3.2 _E3d5_LJ3. _E3d5_LJ3.3 _E4d5_LJ3.3 _E4d5_LJ3. _E4d5_LJ3.2 _E2d5_LJ3. HSC. RS_ES3_2 RS_ES3_ RS_SHS5_2 RS_SHS5_ PS_SHS5_2 PS_SHS5_ RS_LJ3 PS_LJ3 _OG2 _OG _OG 4 _VG8 _VG5 _Rosa5 _29mix _Rosa3 _VG _OG2 _6 _OG _E2d5_LJ3_3 _E2d5_LJ3_2 _E3d5_LJ3_2 _E3d5_LJ3 E3d5_LJ3_3 _E4d5_LJ3_3 _E4d5_LJ3 E4d5_LJ3_2 _E2d5_LJ3_ HSC_2 HSC_ dist(cor(na.omit(tss.exprs.chip[, c(:, 3, 24:27, 3, 32, hclust 33, 48:5, (*, "complete") 56:4)]))) Epigenome at Promoters (PRIM) Epigenome at Promoters (PRIM).5..5...5..5.4.2..8.6.4...4.6.8..2.4 Cluster Dendrogram P_MCV8_MTS8 P_MCV6_MTS8 _MTS8 _MTS7 _SHS5. _LA3 _WJA2 _MTS7 _MCV8_MTS8.3.2. RS_ES3 PS_LJ3 RS_LJ3 Sperm_ES3 RS_SHS5 PS_SHS5 _HSS4 HSC. LSK_AM ahfsc_lwh qhfsc_lwh HFTAC_LWH _E4d5_LJ3 _E3d5_LJ3 _E2d5_LJ3. P_MCV8_MTS8.prim P_MCV6_MTS8.prim _MTS8.prim _MTS7.prim _H3d3OSKM_D.prim GS_SHS5.prim _OG.prim _VG.prim _6.prim 4_H3d3OSKM.prim _H3d3HA_LA3.prim _WJA2.prim _MTS7.prim _MCV8_MTS8.prim _Rosa.prim _OG_3.prim _OG 2.prim RS_ES3.prim PS_LJ3.prim RS_LJ3.prim Sperm_ES3.prim RS_SHS5.prim PS_SHS5.prim _HSS4.prim HSC_2.prim HSC_.prim MAC.prim LSK_AM.prim ahfsc.prim qhfsc.prim HFTAC.prim _E4d5_LJ3.prim _E3d5_LJ3.prim _E2d5_LJ3.prim _H3d3OSKM.prim dist(cor(na.omit(tss.exprs.chip[, c(:99, 3, 4, 9,, hclust 28, (*, 32, "complete") 4)]))) Cluster Dendrogram..4.6.8..2.4 P_MCV8_MTS8 P_MCV6_MTS8. _LA3 _WJA2 _MTS7.3.2. RS_ES3 PS_LJ3 RS_LJ3 Sperm_ES3 RS_SHS5 PS_SHS5 HSC. LSK_AM ahfsc_lwh qhfsc_lwh HFTAC_LWH _HSS4 _E4d5_LJ3 _E3d5_LJ3 _E2d5_LJ3. P_MCV8_MTS8.prim P_MCV6_MTS8.prim _H3d3OSKM_D.prim _VG.prim _6.prim _OG.prim 4_H3d3OSKM.prim _H3d3HA_LA3.prim _WJA2.prim _MTS7.prim _Rosa.prim _OG_3.prim _OG 2.prim RS_ES3.prim PS_LJ3.prim RS_LJ3.prim Sperm_ES3.prim RS_SHS5.prim PS_SHS5.prim HSC_2.prim HSC_.prim MAC.prim LSK_AM.prim ahfsc.prim qhfsc.prim HFTAC.prim _HSS4.prim _E4d5_LJ3.prim _E3d5_LJ3.prim _E2d5_LJ3.prim _H3d3OSKM.prim dist(cor(na.omit(tss.exprs.chip[, c(:99, 3, 4, 9,, 7:9, hclust 28, (*, "complete") 32, 34, 4)]))) Supplementary Figure. Dendrogram based on (A) expression or () PRIMs of all protein-coding and noncoding genes. Different cell types are distinguished by colors as in Figure. Left, dendrograms include all cell lines. Right, dendrograms without published data on s, Cs, and s. In-house data are highlighed in gray.
A Transcriptome (RNA-seq).4.6.8 PC3 Epigenome at Promoters (PRIM) PC3.4.5.6.7.8.9 -./2/4..2.3/4/5/6..2... _LA3.3 P_MCV6 P_MCV8.2 _MTS7 _WJA2..3. 2 4 6 8 2 4 6 8.4 PC -.3 - -. HSC. PC2 PC2 HSC.. P - HSC Pluripotent P Germ q/a HFSC, Cells HFTAC Somatic Cells / Multipotent Cells HSC, LSK P Pachytene PS Spermatocyte q/a HFSC, q/a-hfsc, HFTAC Round RS Spermatid Spermatozoon HSC Pachytene Spermatocyte q/a HFSC, HFTAC Round Spermatid Sperm Pachytene Spermatocyte 2 4 6 8 Supplementary Figure 2. Round 3D Spermatid PCA plot based on (A) expression or () PRIMs of all Sperm protein-coding and noncoding genes. For s, Cs, and s, only in-house data were analyzed. Different cell types are distinguished by colors as in Figure..4 -. - PC
A. _LA3.2..6.5.4.3.2. _SHS5.2 _SHS5..4..98.98.98.96.98.96.96.96.95.95.94.95.96.94.9.94.95.97.95.94.94.95.97.94.9.93.95.89.86.86.86.86.87.87.86.85.85.87.97.89.86.86.86.86.87.87.86.85.85.87.89.89.87.86.87.87.87.87.87.86.85.86.87.93.9.9.89.88.86.86.86.87.88.86.83.85.86.92.95.88.88.86.86.88.88.88.89.87.88.87.88.88.93.96.94.9.9.88.88.86.86.87.87.88.86.84.86.87. Gene Expression (RNA-seq) Pearson Correlation. 5.5.75..4 _SHS5. _SHS5.2..2.3.95.96.95.96.93.96.96.96.97.95.96.96.98.96.97.95.96.96.98.97.96.98.96.96.96.4.5.6..2 _LA3.98.95.95. PRIM=log2(H3/H3) P_MCV8_MTS8 P_MCV6_MTS8 _MCV8_MTS8. _LA3 _WJA2 _MTS7.3.2. _SHS5..85.76.85.88.86.74.86.86.76.78.79.65.77.76.86.8.65.8.82.74.78.86.8.75.74.8.83.79.67.8.8.78.8.9.84.8.79.73.83.84.8.69.8.83.78.8.93.9.9.84.76.75.82.87.8.68.84.84.78.82. Pearson Correlation. 5.5.75. _SHS5.8.77.76.66.67.79.88.9.77.77.95.78.9.93.8.82.9.88.77.87.89.78.8.77.84.82.67.79.8.7.73.68.85.8.8.73.8.8.73.73..2.3 _MTS7 _WJA2 _LA3.82.8.88.77.8. _MCV8_MTS8 P_MCV6_MTS8 P_MCV8_MTS8 Supplementary Figure 3. Correlation coefficients between in-house and published cell lines. (A) Expression of all protein-coding and noncoding genes. () PRIMs of all protein-coding and noncoding gene promoters with and/or modification. lack boxes denote data produced in-house.
A Pluri-/Multipotent Germline Soma % Total Promoter % 8% 6% 4% 2% Only + Only No Modification % Enrichment 5-5 Depletion (3) (3) (2) (4) E2.5 E3.5 E4.5 PS (2) RS (3) E2.5 E3.5 E4.5 PS RS Sperm LSK HSC qhfsc ahfsc HFTAC Sperm (3) LSK HSC (2) qhfsc ahfsc HFTAC Cell fate commitment, GO:4565 Heart morphogenesis, GO:37 Pancreas GO:36 Eye morphogenesis, GO:48592 Embryonic placenta GO:892 Regulation of organ formation, GO:356 Mesenchymal to epithelial transition, GO:623 Regulation of kidney GO:983 one morphogenesis, GO:6349 Epithelial cell morphogenesis, GO:3382 Development of primary male sexual characteristics, GO:46546 Mammary gland morphogenesis, GO:6443 Gut morphogenesis, GO:48547 Neuron fate specification, GO:48665 Hemopoiesis, GO:397 Cell-cell signaling involved in cell fate commitment, GO:4568 Embryonic skeletal system GO:4876 Ear GO:43583 Lung GO:3324 Anterior/posterior pattern formation, GO:9952 Mesenchyme GO:6485 Vasculature GO:944 Limb GO:673 Supplementary Figure 4. Genes grouped by promoter histone modifications. (A) Using peak detection results at promoters, all 32,58 protein-coding and noncoding genes were assigned to each of four different categories: only (green), only marked by modification; only (red), only marked by modification; + (yellow), marked by both and modifications; and no modification (grey), no significant or modification detected. For each cell type with at least three biological repeats from different cell lines or resources (Supplementary Table S), results are presented as mean values and standard deviations. Number in brackets, sample size. () GO enrichment in bivalent genes identified from different cell types. Red, over representation. lue, under representation.
(522) 5 Depletion Depletion -5 67 396-5 (25) Expression of ivalent Promoters D 8 % Selected Genes p-value =. 6 X7 X6 X5 Expression Change in % 4 Development of primary sexual characteristics, GO:4537 Development of primary sexual characteristics, GO:4537 Heart GO:757 Development of primary sexual characteristics, GO:4537 Heart GO:757 Lung GO:3324 Lung GO:3324 Endocrine system GO:3527 rain GO:742 rain GO:742 Morphogenesis of an epithelium, GO:29 GO:43583 EarEar GO:43583 Gland GO:48732 Gland GO:48732 Gland GO:48732 Ear GO:43583 GO:654 EyeEye GO:654 Lung GO:3324 Urogenital system Urogenital system GO:655 GO:655 Eye GO:654 Kidney GO:822 Kidney GO:822 Urogenital system GO:655 Vasculature GO:944 Vasculature GO:944 Endocrine system GO:3527 Heart GO:757 Endocrine system GO:3527 Skeletal system GO:5 Skeletal system GO:5 rain GO:742 Pattern specification process, GO:7389 process, GO:7389 Pattern specification Vasculature GO:944 Embryonic organ GO:48568 GO:48568 Embryonic organ Kidney GO:822 Morphogenesis of an epithelium, GO:29 Morphogenesis of an epithelium, GO:29 Pattern specification process, GO:7389 Embryonic organ GO:48568 Skeletal system GO:5 2 C (867) X4 X7 5 2958 X3 X5 X2X6 X3 X2 + - + + + - + + - - + + + + + - - - + XX4 Enrichment Enrichment X ivalent Promoter X A X Figure S3 Increase Decrease 8% 6% 4% 2 2% 2 4 6 8 2 % ivalent Genes Univalent & Unmodified Genes Supplementary Figure 5. -like bivalent promoter modification is incompletely established in. (A) Overlap of bivalent genes identified by peak detection in s, s, and cells. () GO enrichment in bivalent genes shared or unique in s, s, and cells. Presence and absence of promoter bivalency in certain cell types is denoted by + and -, respectively. Red, over representation. lue, under representation. (C) Comparison of global gene expression profiles between s and cells. lack dots, bivalent genes identified by peak detection. dashed line, cut-off of twofold (log2) expression differences between s and cells. (D) Percentage of genes with expression increases (black) or decreases (grey) in cells. bivalent genes,,867 genes with both and modifications at promoters. univalent & unmodified genes, 3,74 genes modified with either,, or neither of the two modifications at promoters. p-value =. by Fisher s exact test (one-sided).
.5.4.3...6.4..4..6.4..6.4. 5 5 5 5 5 5 5 5 5 5 K27 7 A % Total Promoter % Total Promoter C % Total Promoter % 8% 6% 4% 2% % % 8% 6% 4% 2% % % 8% 6% 4% 2% % (MTS7) Only + Only No Modification Only + Only No Modification Only + Only No Modification Density Density Density (R) (A) Promoter with Peak Only + Only 2.7 PRIM low PRIM high PRIM (R) (A) 2.2 PRIM low PRIM high PRIM (R) (A) PRIM low 2.2 PRIM PRIM high Gene Expression (log2 FPKM) Gene Expression (log2 FPKM) Gene Expression (log2 FPKM) 2 4 6 8 2 2 4 6 8 2 2 4 6 8 2 Chromatin State Chromatin State Chromatin State Gene Expression (PRIM low ) (PRIM high ) Gene Expression (PRIM low ) (PRIM high ) Gene Expression (PRIM low ) (PRIM high ) D % Total Promoter E % Total Promoter % Only + 8% Only No Modification 6% 4% 2% % (MTS7) % Only + 8% Only No Modification 6% 4% 2% % _MCV8 (MTS8) Density Density (R) (A) PRIM low 2. PRIM (R) (A) PRIM low 2. PRIM PRIM high PRIM high Gene Expression (log2 FPKM) Gene Expression (log2 FPKM) 2 4 6 8 2 2 4 6 8 2 Chromatin State Chromatin State Gene Expression (PRIM low ) (PRIM high ) Gene Expression (PRIM low ) (PRIM high ) Supplementary Figure 6. Distinguishing active and repressive promoter chromatin states by PRIMs in (A) s, () s, (C) s, (D) s, and (E). ased on peak detection results at promoters, all genes were assigned to each of four histone modification categories as in Figure S4A (left). Probability density distribution of PRIMs was made for each of the three categories, only (green Figure line), S4 + (yellow line), and only (red line), and cut-off value between and + or groups was selected by the crossover between and + plot (dashed line). X-axis, PRIMs. Y-axis, probability density. Cut-off value, =2.7, =2.2, =2.2, =2., =2. (middle). Expression of genes in promoter chromatin states delineated by active (above PRIM cutoff) and repressive (at or below PRIM cut-off) marks is shown in boxplot (Right). The bottom and top of the boxes indicate the 25 th and 75 th percentiles, the central bars indicate medians and whiskers indicate non-outlier extremes. Dashed line, median expression value of all genes.
A (PRIM) No 4 2 2 4 6 8 r =.86 No 4 2 2 4 6 8 C Class I Promoters (PRIM) - R N R M A A N Enrichment 5-5 Depletion 2 4 6 8 2 4 () (Modified) (Stable I) A M R N A R N..5 Regulation 2. of cell cycle, 2.5 GO:5726 3. Regulation of fibroblast proliferation, GO:4845 Pattern specification process, GO:7389 Neuron fate commitment, GO:48663 Venous blood vessel morphogenesis, GO:48845 Embryonic organ GO:48568 Cell fate commitment, GO:4565 Vasculature GO:944 Stem cell maintenance, GO:9827 Heart morphogenesis, GO:37 Primary neural tube formation, GO:42 Morphogenesis of an epithelium, GO:29 in utero embryonic GO:7 Placenta GO:89 Lung morphogenesis, GO:6425 Genes 4 7 25 239 9 26 Gene Expression [log2 FPKM] 2 4 6 8 2 4 2 4 6 8 2 4 * Genes Modified Genes Stable I Genes All Class I Genes * *._avg _avg.5 _avg 2._avg _avg 2.5 _avg 3. Type (a) Promoters Type (b) Promoters Type (a) Promoters Genes Type (c) Stable Promoters I Genes Type (b) Modified Promoters Genes All Class I I Genes Promoters Type (c) Promoters All Class I Promoters..5 2. 2.5 3. Figure S7 Supplementary Figure 7. Promoter chromatin states, transcriptional activities, and biological functions of genes activated during reprogramming of s (Class I). (A) Comparison of chromatin states (PRIMs) between s and s for all class I promoters (grey dots). X-axis, PRIMs from s. Y-axis, PRIMs from s. lack dots, class I example genes displayed in Figure 3A. Dashed black line, cut-off between active and repressive chromatin states. Dark grey line, optimal curve fits to all class I promoters. r, correlation coefficient with all class I promoter PRIMs. () Transcriptional activities of selected gene subsets. Genes are grouped by promoter PRIMs as in Figure 4A. The bottom and top of the boxes indicate the 25 th and 75 th percentiles, the central bars indicate medians and whiskers indicate non-outlier extremes. p-values were calculated using Wilcoxon tests., p-value <.. *, p- value <.5. Dashed line, median expression value of all genes. (C) GO enrichment in all Class I genes. Genes are grouped by promoter PRIMs as in Figure 4A. A, chromatin state. R, chromatin state. M, Histone modification ( or ) identifiable. N, Histone modification ( or ) unidentifiable. ottom, number of genes in each group.
A (PRIM) No 4 2 2 4 6 8 r =.9 No 4 2 2 4 6 8 C Class II Promoters (PRIM) 2 4 6 8 2 4 R - A () (Unmodified) (Stable II) N R M A A N M R N A R N..5 2. 2.5 3...5 2. 2.5 3. Regulation of gene expression, epigenetic, GO:429 DNA methylation involved in gamete generation, GO:4346 Spermatogenesis, GO:7283 pirna metabolic process, GO:34587 Meiosis, GO:726 Sexual reproduction, GO:9953 Morphogenesis of an epithelium, GO:29 Pattern specification process, GO:7389 Skeletal system GO:5 Stem cell division, GO:745 Somatic stem cell division, GO:483 Germ cell GO:728 Genes 444 84 82 8 64 3 Gene Expression [log2 FPKM] 2 4 6 8 2 4 2 4 6 8 Repress Genes No Modified Genes Stable II Genes All Class II Genes *._avg _avg.5 _avg 2._avg _avg 2.5 _avg 3. Type (a) Promoters Type (b) Promoters Type (a) Promoters Genes Type (c) Stable Promoters II Genes Type (b) Unmodified Promoters Genes All All Class II II Genes Promoters Type (c) Promoters All Class II Promoters Figure S8 Supplementary Figure 8. Promoter chromatin states, transcriptional activities, and biological functions of gene repressed during reprogramming of s (Class II). (A) Comparison of chromatin states (PRIMs) between s and s for all class II promoters (grey dots). X-axis, PRIMs from s. Y-axis, PRIMs from s. lack dots, Class II example genes displayed in Figure 3A. Dashed black line, cut-off between active and repressive chromatin states. Dark grey line, optimal curve fits to all class II promoters. r, correlation coefficient with all class II promoter PRIMs. () Transcription activities of selected gene subsets. Genes are grouped by promoter PRIMs as in Figure 4C. The bottom and top of the boxes indicate the 25 th and 75 th percentiles, the central bars indicate medians and whiskers indicate non-outlier extremes. p-values were calculated using Wilcoxon tests., p-value <.. *, p- value <.5. Dashed line, median expression value of all genes. (C) GO enrichment in all Class II genes. Genes are grouped by promoter PRIMs as in Figure 4C. A, active chromatin state. R, repressive chromatin state. M, histone modification ( or ) is identifiable. N, histone modification ( or ) is unidentifiable. ottom, number of genes in each group.
A C Suz2 Zfx Nr5a2 Nanog Sox2 Pou5f Klf4 Esrrb Tfcp2l ME ME SME ME ME ME ME SME ME S S S Zic3 Nanog Zbtb6 kb Supplementary Figure 9. K27ac modification in different cell types (blue) and transcription factor enrichment in s (black) at selected genes. (A) Zic3 (Class I, ). () Nanog (Class I, Modified ). (C) Zbtb6 (Class II, ). Core pluripotency regulators (Pou5f, Sox2, Nanog) are highlighted in red. Pink region, Promoter. lue bar at bottom, enhancer. ME, enhancers active in both s and s. SME, enhancers active in, s and s. S, enhancers active only in s. K27ac and transcription factor track range,.
ivalent Promoters (PRIM) in Different Cell Types./2 PC3.4 2.8.6 Epigenome at Promoters - (PRIM).3 PC3 -.... _LA3.3.2. _MTS7 _WJA2 2 4 6 8 2 4 6 8 P_MCV6 P_MCV8 Supplementary Figure Pachytene. Spermatocyte 3D PCA plot based on PRIMs of all promoters with Round Spermatid + bivalent Sperm histone modifications in s. For s, Cs, and s, only in-house data were analyzed. Different cell types are distinguished by colors as in Figure.. PC2.3 PC2 HSC...4 - -. -.3 -.4 PC P HSC Pluripotent P Germ q/a HFSC, Cells HFTAC Somatic Cells / Multipotent Cells HSC, LSK P Pachytene PS Spermatocyte q/a HFSC, q/a-hfsc, HFTAC Round RS Spermatid Spermatozoon HSC Pachytene Spermatocyte q/a HFSC, HFTAC Round Spermatid Sperm 2 4 6 8 PC
A C E2.5 E3.5 E4.5 PS RS Sperm kb Sox2 Cdx2 Gata6 Supplementary Figure. Histone modifications at selected genes in different types of germ cells. Green, modification. Red, modification. track range,. track range,.5.
Specific antibody Control immunoglobulin a6-integrin CD9 Thy- c-kit 83.4% 89.4% 23.7% Count Supplementary Figure 2. Analysis of cell surface marks by flow cytometry.