Supplementary Fig. 1 a Kdm3a Kdm4b β-actin Oocyte Testis Oocyte Testis Oocyte Testis b 1.8 Relative expression.6.4.2 Kdm3a Kdm4b RT-PCR analysis of Kdm3a and Kdm4b expression in oocytes and testes. Twenty oocytes were pooled for RT-PCR analysis. The number of PCR cycles was 35. Two independent experiments were conducted. (a) Representative images of electrophoresis. (b) Relative expression levels of Kdm3a and Kdm4b in oocytes compared with those in testes.
Supplementary Fig. 2 a Egfp-zygote Kdm3a-zygote Kdm4b-zygote DAPI Pb H3K9me2 DAPI Pb H3K9me2 DAPI Pb H3K9me2 H3K9me3 Merge H3K9me3 Merge H3K9me3 Merge b H3K9me2 H3K9me3 12 1 Ratio of signal intensity (maternal / paternal) 8 6 4 2 P <.1 P <.1 Egfp (n=14) Kdm3a (n=15) Kdm4b (n=13) Egfp (n=14) Kdm3a (n=15) Kdm4b (n=13) Effects of Kdm3a or Kdm4b expression on H3K9me2/3 states. (a) Representative images of immunofluorescence (IF) analysis in Kdm3a- or Kdm4b-overexpressing fertilised embryos. (b) The box-and-whisker plot shows the ratio of maternal to paternal signal intensities. The P-values were calculated using the Mann-Whitney U-test (U-test). Pb: polar body; n, number of embryos analysed. Scale bars = 2 μm.
Supplementary Fig. 3 Relative expression in 4-cell PEs 3. 2.5 2. 1.5 1..5 Rnf12 Intact (n=5) Egfp (n=1) Kdm3a (n=8) Kdm4b (n=1) Kdm3a/4b (n=7) Intact + TSA (n=9) Egfp + TSA (n=13) Kdm4b + TSA (n=1) IVF (n=5) Analysis of Rnf12 expression using qpcr in parthenogenetic embryos (PEs) harbouring histone modifications and in vitro-fertilised (IVF) embryos at the 4-cell stage. There were no significant differences between groups compared with intact PEs. Error bars indicate the mean ± SEM.
Supplementary Fig. 4 a X-linked genes Plac1 Pgk1 Fmr1nb Mecp2 Uba1 Atrx Rnf12 Symbols Egfp+TSA Kdm4b PEs Kdm4b+TSA Female FEs Tsix is not detectable in all samples. 6. 5. -log1 (P-value) 4. 3. 2. 1.3 1.. -2.5-2. -1.5-1. -.5..5 1. 1.5 2. log2 (Fold change) 2.5 b 6. 5. -log1 (P-value) 4. 3. 2. 1.3 1.. -2.5-2. -1.5-1. -.5..5 1. 1.5 2. 2.5 log2 (Fold change)
Volcano plot analysis of X linked genes based on the average expression of each group in 96h and 12h blastocysts. Values are normalised to the average value of Egfp-PEs. Vertical and horizontal axes indicate P-values and changes in the levels of expression, respectively.
Supplementary Fig. 5 a b Zygote Egfp mrna Kdm4b mrna DAPI DAPI Pb Pb H3K27me3 H3K27me3 Merge Merge Ratio of H3K27me3 signal intensity (maternal / paternal) 4 2 1 Egfp (n=13) Kdm4b (n=14) c d DAPI H3K27me3 Merge Zygote WT TSA DAPI Pb H3K27me3 Merge Ratio of H3K27me3 signal intensity (maternal / paternal) 25 2 15 1 5 WT (n=9) P =.12 TSA (n=9) Effects of H3K27me3 modifications on Kdm4b overexpression and TSA treatment. (a) Oocytes injected with Kdm4b mrna were subjected to ICSI. Samples were fixed 1 11 h after ICSI. (b) After ICSI, oocytes were incubated in KSOM along with TSA for 1 11 h, after which the zygotes were fixed. Representative images are shown on the left. The box-and-whisker plot on the right shows the ratio of maternal to paternal signal intensities of H3K27me3. There were no statistically significant differences between groups. Scale bars = 2 μm.
Supplementary Fig. 6 a Relative expression levels of Rnf12 1.4 1.2 1..8.6.4.2 P <.1 Fresh oocyte (n=3) si-control (n=3) si-rnf12 (n=3) oocytes at 7 hours after sirna injection b c si-control DAPI si-rnf12 DAPI 12 P <.1 RNF12 RNF12 1 Merge Merge Signal intensity 8 6 4 2 si-control si-rnf12 d sirna injection TSA treatment with Egfp mrna and activation (Fig. 3e) 6-7 horus incubation Kdm4b mrna injection and activation (Fig. 3f) Xm- expression analysis at 48 hours after activation
Generation of RNF12-depleted embryos using sirna. (a) Analysis of the expression of maternal Rnf12 in oocytes injected with sirna targeting Rnf12 (si-rnf12) or control sirna compared with that in uninjected fresh oocytes. (b) and (c) Immunofluorescence (IF) analysis of RNF12-depleted parthenogenetic embryos (PEs) at the 4-cell stage. Representative images of si-rnf12- and control sirna (si-control)-injected embryos are shown. Scale bars = 5 μm. The delta symbol shows mitotic blastomeres that were omitted from quantification (b). Signal quantification of sirna-treated embryos. Seven embryos were analysed in both groups (c). (d) Schema of Kdm4b- and Egfp+TSA-PEs derived from maternal/zygotic RNF12-depleted oocytes. The P-values were determined using Student s t-tests. Error bars indicate the mean ± SEM.
Supplemetary Fig. 7 Triton-X Micrococcal nuclease soluble fraction soluble and insoluble fractions Protein kinase K and DNA purification Western blotting H3K9me3 15 bp 5 bp Sample I.D. 1 2 1 2 soluble insoluble 16KDa Scheme of recovery of sperm mononucleosomes. Electrophoresis of mononucleosomal DNA and western blotting analysis from each fraction used for the sperm ChIP-qPCR assay.
Supplementary Fig. 8 dilution preamplification ChIP DNA from > 1E6 cells ChIP DNA from around1e3 cells preamplified DNA ChIP-qPCR Fold changes (preamplified DNA / non preamplified DNA) 1.6 1.4 1.2 1..8.6.4.2 P >.54 P >.94 P >.78 5F XP RA Comparison of ChIP-qPCR results from bulk DNA and pre-amplified DNA. Bulk ChIP DNA from embryonic stem (ES) cells were diluted and pre-amplified. ChIP-qPCR analysis was performed using bulk and pre-amplified DNA with a TaqMan probe targeting three 5 -regions. The P-values were determined using Student s t-tests. Error bars indicate the mean ± SEM.
Supplementary Fig. 9 a H3K9me3 (TT2) Scale chrx: 3 2 kb mm9 1,65, 1,66, 1,67, 1,68, 1,69, WT 1 3 _ Setdb1 KO 1 _ Tsix H3K9me3 rich regions WT 3 Scale chr9: 2 kb mm9 5,365, 5,37, 1 Tex12 b H3K9me3 (E14) Scale chrx: 3 2 kb mm9 1,66, 1,67, 1,68, 1,69, 2i 1 3 Serum 1 Tsix In silico analysis of H3K9me3 at the locus in TT2 and E14 male ES cell lines. ChIP-seq data for male TT2 and E14 ES cell lines acquired by Karimi et al. 3 (a) and Marks et al. 29 (b), respectively, were obtained from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/gds) under accession numbers GSE29413 and GSE23943, respectively. The data were visualised by importing into the Custom track of the University of California, Santa Cruz Genome Browser (http://genome.ucsc.edu/). The Tex12 locus harbours an H3K9me3-rich region similar to the E14 line (data not shown).
Supplementary Fig. 1 a PB 5 repeat CAG Tet3G IRES Egfp PB 5 repeat TRE Kdm4b pa K4B-ES cells (XY) PB 3 repeat PB 3 repeat b Dox KDM4B ACTIN Dox #1 #2 #3 Testis - + - + - + #13 #14 #15 - + - + - + KDM4B ACTIN c #15-4 -2 2 4 correlation coefficient (Pearson's r) :.87-4 -2 2 #1 4 d Number of H3K9me3 positive regions 35 3 25 2 15 1 5 Total Dox - 39416 Dox + 28425 chr1 chr2 chr3 chr4 chr5 chr6 chr7 chr8 chr9 chr1 chr11 chr12 chr13 chr14 chr15 chr16 chr17 chr18 chr19 chrx chry e Dox - Dox + H3K9me3 profiles.4 Nnat Tex12 Spata22 - + - + - +
f dox + K4B-ESCs dox - K4B-ESCs Nuclear transfer KSOM (don +) KSOM (don -) Relative expression levels of K4B-ESCs cloned embryos (4-cell) 1.2 1..8.6.4.2 P =.45 off (n=4) on (n=4) g sirna injection Enucleation NT (ESCs) 5-6 hours incubation 1.5-2.5 hours incubation and activation h Relative expression levels 3. 2.5 2. 1.5 1. * si-control (n=5) si-rnf12 (n=6) * ES clones (4-cell).5 Rnf12
Generation of KDM4B-inducible embryonic stem (ES) cells and RNF12 dependency of ES-cloned embryos. (a) Diagram of the PiggyBac doxycycline (Dox)-inducible expression vector used for introduction of Kdm4b into male ES cells (K4B-ES cells). (b) Western blot analysis of KDM4B expression after dox treatment for 2 days. (c) Scatter plots showing the relationship between two independent Dox-K4B-ES cell lines (#1 and #15). (d) H3K9me3-rich regions in the genomes of K4B-ES cells (#1). H3K9me3-positive regions in doxycycline on/off K4B-ES cell lines were identified using model-based analysis for ChIP-Seq (MACS). The numbers revealed a significantly enriched locus compared with the input data (false discovery rates < 1%, and elevated by a factor of greater than 5-fold). (e) H3K9me3 levels at regions were relatively lower than those at known H3K9me3-rich regions (Nnat, Tex12, and Spata22) 3 in the #1 line. The heat maps show H3K9me3 levels (upper: dox -, lower: dox + ). (f) Effects of H3K9me3 demethylation induced by ectopic expression of KDM4B on expression in the NT embryos from K4B-ES cells (#1). expression was determined using qpcr at the 4-cell stage in a pool of five cloned embryos. (g) Experimental scheme for generating Rnf12-knockdown cloned embryos. (h) qpcr analysis of expression at the 4-cell stage of ES cell-cloned embryos using si-rnf12-treated oocytes as recipients. A pool of five 4-cell embryos represents one biological replicate. Asterisks show P <.4. The P-values were determined using Student s t test. Error bars indicate the mean ± SEM.
Supplementary Fig. 11 a Kdm4b Male blastocysts Egfp AVE. (vs Egfp) b * Pgk1 Plac1 Rnf12 Tsix - 13% 7% 1% - ** * Eif2s3y 15% not detectable <-3.5 1 c Female blastocysts Kdm4b Egfp AVE. (vs Egfp) Pgk1 Plac1 Rnf12 Tsix Eif2s3y 132% 46% 12% 11% - - * * d Kdm4b-FEs E18.5 Egfp-FEs e in vivo development of FEs (E18.5) % 1 8 6 4 2 Fertilized embryos (n) Egfp Kdm4b (32/36) (37/5) Implantations / Transferred embryos Egfp Kdm4b (17/32) (23/37) Viable pups / Implantations
Effects of ectopic expression of Xm- on developmental competency in FEs. (a) Expression of, Pgk1, Plac1, Rnf12, Tsix, and Eif2s3y in individual male 96h blastocysts, as determined using qpcr. The genders of the embryos were determined by the detection of Eif2s3y located on the Y chromosome. The P-values were determined using Student s t-tests. Average expression levels are indicated by Ave. and represent **P <.1 and *P <.1. The coloured bar scale is log 2. (b) FISH analysis of Kdm4b-FEs (female). The asterisks indicate cells with biallelic expression. Scale bars = 5 μm. (c) qpcr analysis of X-linked gene expression in female embryos. The coloured bar scale and asterisks are the same as in (a). (d) Conceptus of Kdm4b- and Egfp-FEs. Representative images of the E18.5 uterus of pseudopregnant mice. Kdm4b-FEs (Xm-XCI) and Egfp-FEs were transferred into the left or right region of the uterus, respectively. (e) Full-term developmental rates. Five independent recipients were analysed, and the numbers of transferred embryos, implantations, and pups are shown in the graph.
Supplementary Fig. 12 a Donor cell No.of ET No.of pups (%) 4-cell 8-cell Morula ICM ES 47 87 9 96 196 15 (32.1) 2 (2.3) 7 (7.3) 14 (7.1) b 9 No. of DRGs (X-linked genes) expresion levels at blastocyst c 6 5 4 3 2 1 7 5 3 1 4CNT 1-2 (male) 4CNT 1-2 (male) SRNT 1-4 (male) SRNT 1-4 (male) DRGs d DRGs (X-linked genes) 29 121 156 4CNT SRNT
Association of developmental competency with XCI in cloned embryos. (a) Development of embryonic cloned embryos 31. (b d) Microarray analysis of 4-cell (4CNT) and Sertoli (SRNT)-cloned blastocysts using the previous reported data (GSE23181) 28. (b) expression states in cloned embryos. (c) X-linked genes (625) expressed in blastocysts were analysed. Genes repressed to less than 3% of the levels in IVF male embryos were identified as downregulated genes (DRGs). Red bars show DRGs. (d) Venn diagram of DRGs in cloned embryos, indicating that 8% of DRGs in 4CNT were the same as in SRNT. These results showed that high developmental competency was retained even when ectopic was expressed and global XCI occurred.
Supplementary Fig. 13 a Kdm4b-PEs (n=3) Egfp-PEs (n=3) IVF (n=2) b (%) 12 Up in Kdm4b-PEs (total 671 transcripts) % of differentially expressed transcripts 1 8 6 4 2 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 X c d % of differentially expressed transcripts (%) 12 1 8 6 4 2 Down in Kdm4b-PEs (total 711 transcripts) 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 X XqA7.3 Xlr3a Xlr3b Xlr4a Xlr4b Xlr5b Xlr5c DXBay18 Gm5639 Slc1a3 DXBay18 Cetn2 Smim9 Brcc3 Mpp1 Irak1 Renbp Pls3 e 1 Xlr-family Magea-family 5c 5b 5a 4b 4a 3b 8 5 3 1 Fold change (vs IVF) -1-2 -3 Egfp Kdm4b PEs -4-5
Transcriptome analysis of Egfp-PEs, Kdm4b-PEs, and IVF embryos at the blastocyst stage (12 h). (a) Unsupervised clustering analysis of gene expression. (b) and (c) Chromosome distribution of significantly differentially expressed transcripts between Egfp- and Kdm4b-PEs (Student s t test: P <.5, and more than 1.5-fold change). Genes upregulated (b) and downregulated (c) in Kdm4b-PEs. (d) Mapping of X-linked downregulated transcripts (DRTs) on the X chromosome. Twenty-one of 75 (28%) X-linked DRTs (17 genes) were located in the XqA7.3 region. (e) Fold-enrichment of Xlr- and Magea-family genes as determined by microarray data in Egfp- and Kdm4b-PEs compared to IVF embryos.
Supplementary Fig. 14 Fertilized embryos Rnf12 Xp Establishment of XCI in TE (imprinted XCI) Parthenogenetic embryos (WT: H3K9me3 +) Rnf12 The majority of embryos loss. Xm Loss of XCI in TE Parthenogenetic embryos (KDM4B overexpression: H3K9me3 -) Rnf12 Xm Establishment of XCI in TE :H3K9me3 XCI: X-chromosome inactivation TE: trophectoderm Summary of regulation during pre-implantation phases and XCI impact on embryo development. Maternal-specific H3K9me3 prevents Xm- activation by RNF12. Erasure of H3K9me3 at the promoter region leads to Xm- activation at the 4-cell stage and markedly improves developmental ability in PEs.
Table 1. FISH analysis at the 4-cell stage. PEs biallelic no signal P-values DAPI cloud pinpoint (n=emrbyos) cells cells (vs Egfp ) Egfp (12) 46 7 4 35 - Kdm4b (16) 62 47 2 21 16 5.6E-11 Egfp +TSA (22) 84 31 16 11 47.5 The P-values were caluculated by Fisher s exact test.
Table 2. in vitro development of PEs. PEs 2-cell 4-cell Morula Blastocyst % (Bl/2C) Egfp 14 138 129 112 8.% Egfp +TSA 52 5 49 45 86.5% Kdm4b 68 67 65 59 86.8% Kdm4b +TSA 85 81 78 73 85.9%
Table 3. Immunofluorescence combined with FISH analysis at the 96 h blastocysts. cloud p - values biallelic H3K27me3+ % Types DAPI ratio cells (vs Egfp) cells (K27me3+ / cloud) Egfp 72 261 36.3% - 16 67.% Egfp +TSA 66 384 63.4% 5.46E-23 36 33.8% PEs kdm4b 69 52 72.8% 6.78E-44 62 41.4% Kdm4b +TSA 973 729 74.9% 4.54E-58 1 22.1% Female FEs 364 333 91.5% 6.66E-75 75.4% The P-values were caluculated by Fisher s exact test.
Table 4. Immunofluorescence combined with FISH at the 12 h blastocysts. Types DAPI cloud ratio p - values (vs Egfp) biallelic cells H3K27me3+ % (K27me3+ / cloud) Egfp 1619 462 28.5% - 88.1% Egfp +TSA 984 539 54.8% 2.93E-4 2 78.7% PEs Kdm4b 1323 838 63.3% 5.98E-81 2 91.8% Kdm4b +TSA 1186 837 7.6% 1.66E-11 23 91.2% Female FEs 491 469 95.5% 4.5E-171 96.8% The P-values were caluculated by Fisher s exact test.
Table 5. Fish analysis in Rnf12 knockdown PEs at the morula stage. Types DAPI cloud pinpoint no signal si-control (n=12) 227 129 52 57 PEs si-rnf12 (n=12) 198 44 55 64
Table 6. in vitro development of Kdm4b mrna injected fertilized embryos. Types 2-cell 4-cell Morula Blastocyst % (Bl/2C) Kdm4b -FEs 64 61 61 56 87.5% Egfp -FEs 26 26 24 22 84.6%
Table 7. Immunofluorescence combined with FISH in the Kdm4b -PEs. ICM TE Types DAPI H3K27me3 DAPI H3K27me3 Egfp (n=13) 128 43 19 158 34 32 PEs Kdm4b (n=16) 156 66 17 1453 754 667 Late stage of blastocysts (12 h) were analyzed.
Table 8. FISH analysis of Rnf12 knockdown Kdm4b -PEs at the blastocyst stage. Types DAPI cloud pinpoint si-control (n=12) 763 396 9 Kdm4b -PEs si-rnf12 (n=1) 552 55 29
Table 9. Primer/Probe sequences. CCTGCAGGTCTAATACGACTCACTATAGGGGCCACCATGG (12 x T)-AAGGTTTGCCCAAACTGGATTCACTG TAATACGACTCACTATAGGGCTCCACTTTGCTGCAACCATGGGGTCC (12 x T)-AGGAGTGGGCAGGATCTAGAAGGGTGCTCC TCTTGGGGATGTGGTGTTTATC TCCTGAGTAAGCCAGAAGCAGT AACCGCAATGGGCTCTACTAT AGTCTCTGCTCGTGATGCTCTCT GGTGGACTTACCTTTCTTTCATTGTTT AAGAATGAAAAGGCAGGTAAGTAT ATGGCTGGAGCAAGCCGTTGCACG TAAAGGTCCAATAAGATGTCAGAA GGAACCAAGGAGCCATTTTGT CTTCTGCATTAGTTGGCGACC ATGAGGAGAGGAAAGGGTAGAAAT TAAAGGTCCAATAAGATGTCAGAA CCCTACCTGAACCACCTCAATAGT AGTTCCCTTTAGGCGTCCCAT AATCAACAAGGTCGGCTTACTCT ATCCGTTTTAGGACTGCGATGTA GCTCCTGAAGATGTAAGCAATAAAG CTTCCTTGCGCCAGTCCCGAG GACGACTTGAAAAATGACGAAATC CATATTCCAGGTCCTTCAGTGTGC AGATGAGGAGAGGAAAGGGTAGAAAT probe FAM-CCTCACAAAATGGC-MGB AGAGAAAGACTAAGATGCCAATGACC GAGCAAGCCGTTGCACG probe FAM-CTTTAACTGATCCGCGGCG-MGB ACCTAAAGGTCCAATAAGATGTCAGAA AAAAAAGAATGAAAAGGCAGGTAAGTAT probe FAM-ACACACAGGTATCCGTGGC-MGB GGTGGACTTACCTTTCTTTCATTGTTT CATCCAGGGACGTGCTGACT probe FAM-CTGCCCTCGTGGACA-MGB TGTGTTCTCCCCTCACTGATCTC Gene I.D. Gapdh Mm99999915_g1 Actb Mm67939_s1 Rnf12 Mm48844_m1 Pgk1 Mm435617_m1 Mecp2 Mm1193537_g1 Plac1 Mm457647_m1 Tsix Mm3455646_m1 Fmr1nb Mm4178826_m1 Atrx Mm494196_m1 Uba1 Mm493988_m1 Mm1232884_m1 Sfmbt2 Mm616783_m1 Gnas Mm1242435_m1 H13 Mm468786_m1 Impact Mm492647_m1 Kdm3a Kdm4b IVT template Kdm3a Kdm4b RT-PCR TaqMan or quantitative probe ChIP-qPCR R2 R3 R4 R5 Gapdh 5'R XP RA H19 IAP Major sattellite R1
targeting exons 6-7 (Figure 1f) probe CCCAAAGCAGCACAGAAAAC FAM-ACCCGAGGATCAACATGCCTGAC-BHQ AGACACACCCACAATACACAC