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Graphical Abstract.
Keywords: transcriptional reprogramming; oocyte; xenopus; nuclear transfer; resistance; epigenetic; chromatin.
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Figure1. Identification of Genes Resisting Transcriptional Reprogramming by Oocytes(A) Schematic representation shows the nuclear transplantation strategy used to identify genes resistant to reactivation by Xenopus oocytes.(B) MA plot shows log fold change (logFC, y axis) in gene expression between MEF-NT versus ES-NT against the expression in cultured MEFs (x axis, log2 reads per kilobase per million mapped reads [RPKM]; data from Reddington etal., 2013). Red and blue dots, genes differentially expressed (FDR< 0.05, two experiments, n= 32 NT samples per condition).(C) Heatmap representing expression of the differentially expressed genes (rows, Z score of normalized expression level) between MEF-NT and ES-NT in ES and MEF before and 48hr after NT (columns, genes sorted according to fold change between MEF-NT to ES-NT). Expression data for cells before NT are from Reddington etal. (2013) and Bulut-Karslioglu etal. (2014).(D) Boxplot shows the gene expression level before and after NT of genes resistant in MEF-NT (538 genes,top) or resistant in ES-NT (424 genes, bottom). [star] pvalue< 106, Wicoxon rank-sum test.(E and F) The top (E) gene ontology terms and (F)KEGG pathways enriched in the list of differentially expressed (DE) genes restricted in MEF-NT and ES-NT. See also FigureS1 and Tables S1 and S2.
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Figure2. Epigenetic Configuration of MEF Genes Resistant to OOC-Mediated Transcriptional Reprogramming(AE) Shown is the (A) DNase I sensitivity (Yue etal., 2014), (B) H3K4me3 (Kaneda etal., 2011), (C) H3K27me3 (Kaneda etal., 2011), (D) H2Aub (this study), and (E) H3K9me3 (Bulut-Karslioglu etal., 2014) metaplot analysis5 kB around the TSSs, in MEF, of genes with reprogrammed (green), maintained (blue), and resistant (red) expression following nuclear transfer.(F) DNA methylation in the promoter region (3 kb upstream TSS) of reprogrammed, maintained, or resistant genes is shown as the percentage of CpG showing>95% methylation (data from Reddington etal., 2013).(GK) Shown is the (G) DNase I sensitivity (Yue etal., 2014), (H) H3K4me3 (Kaneda etal., 2011), (I) H3K27me3 (Kaneda etal., 2011), (J) H2Aub (this study), and (K) H3K9me3 (Bulut-Karslioglu etal., 2014) metaplot analysis5 kB around the TSSs of genes showing resistance in OOC-NT (red, from this study), mouse egg-NT (orange, from Matoba etal., 2014), transcription factor-induced reprogramming (purple, from Soufi etal., 2012), and cell fusion (gray, from Looney etal., 2014).(L) DNA methylation in the promoter region (3 kb upstream TSS) of genes resistant in OOC-NT, mouse egg-NT, transcription factor-induced reprogramming, and cell fusion is shown as the percentage of CpG showing >95% methylation (data from Reddington etal., 2013). See also FigureS2.
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Figure3. Chromatin Modifier Overexpression Alleviates Resistance to Transcriptional Reprogramming(A) Schematic representation shows the nuclear transplantation strategy used to test the sensitivity of resistant genes to chromatin modifiers.(B) Western blot analysis of modified histones on chromatin recovered 48hr after nuclear transplantation to oocyte expressing single or multiple chromatin modifiers is shown.(C) Summary shows the 12 chromatin configurations tested in the OOC transcriptional reprogramming assay.(D) Boxplot shows the log fold change between resistant gene expression in MEF-NT in various chromatin configurations to that of ES-NT.(E) Number of resistant genes as judged by differential gene expression analysis between RNA-seq data from ES-NT and MEF-NT in various chromatin configurations (FDR< 0.05, two experiments, n= 32 NT samples per conditions). See also FigureS3 and Table S3.
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Figure 4. Multiple Chromatin Modifier Combinations Affect Resistance at a Single-Gene Level
(A) Venn diagram shows the number of genes losing resistance upon four individual chromatin modifier treatments. (B) Example of digital signature of resistance obtained by differential gene expression analysis for a non-resistant (G3pdh) and three resistant (Trap1A, Otx2, and Sox2) genes. The digital signature indicates whether a gene is resistant (shown as 1 when the gene is differentially expressed in MEF-NT compared to ES-NT) or loses resistance (shown as 0, when the gene is not differentially expressed in MEF-NT compared toES-NT) in each of the 12 conditions tested. The bar graphs show qRT-PCR analysis of gene expression (mean SEM of four samples of eight oocytes NT).(C) The numbers of genes for which a synergistic, neutral, adverse, or insensitive effect is observed when combining two chromatin modifier treatments are shown. (D) Venn diagram shows the number of genes sensitive to a histone modifier (Kdm4d, Kdm6b, or USP21) in nuclei with normal (WT) or hypomethylated DNA (Dnmt1N). (E) Loss of resistant gene sensitivity to Kdm6b in DNA-hypomethylated MEF correlates with loss of H3K27me3. The y axis indicates the number of genes that lose H3K27me3 in MEF with hypomethylated DNA compared to WT MEF. Resistant genes are split according to change in sensitivity to Kdm6b after NT when comparing WT MEF to MEF with hypomethylated DNA (red dot). The boxplot shows the background distribution of H3K27me3 change when sampling 10,000 times for a random set of genes of the same size as theresistant gene subset tested. The number in parentheses indicates a p value generated by calculating the proportion of the random background that has a more extreme value than the observed percentages from each of the three groups.(F) Heatmap illustrating the change in gene expression in normal (WT MEF nuclei) or hypomethylated DNA (Dnmt1N MEF nuclei) upon expression of histone modifiers. Ten clusters representing the main trend of gene expression change were selected based on k-means clustering of expression data from RNA-seq analysis (RPKM). The trend of change in gene expression inthese ten clusters is shown in the heatmap as a Z score. See also Table S4.
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Figure5. USP21 Removes Resistance through Deubiquitylation of H2A
(A) Metaplot analysis in MEF of H2AUb ChIP-seq5 kb around the TSSs of genes expressed (green) or not expressed (blue) in MEF is shown. (B) Same as (A) but resistant genes are split according to sensitivity (red line) or insensitivity (red dashed line) to USP21.(C) WB analysis of H2AK119ub1 level in WT or Ring1a&b KO-transplanted nuclei 0 and 48hr after nuclear transfer. Nuclei were transplanted in control oocyte or in oocyte overexpressing USP21 or catalytically inactive XlRing1b (Ring Mut).(D) Boxplot shows expression of the set of genes upregulated in WT MEF transplanted to USP21 compared to WT MEF transplanted to control oocytes (p value< 1011, Welch two-sample t test).(E) Heatmap showing unsupervised hierarchical clustering of genes upregulated in WT MEF transplanted to USP21-expressing oocyte compared to control oocytes in WT MEF transplanted to control oocytes, to USP21-expressing oocytes, to XlRing1b mutant-expressing oocytes, and in Ring1a&Ring1b KO MEF transplanted to control oocytes. Expression value is shown as log2(1+ RPKM).(F) dCas9-USP21 targeted to the Otx2 promoter decreases Otx2 resistance to transcriptional reprogramming. The qRT-PCR analysis of Otx2 and Sox2 gene expression 48hr after MEF transplantation to oocytes injected with water (control), USP21 RNA, or USP21-dCas9 RNA, with or without Otx2 gRNAs mix, is shown ([star]p value< 0.05, Students t test; mean SEM of four experiments). See also FigureS4 and Table S5.
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Figure6. USP21 Improves Transcriptional Reprogramming of Two-Cell-Stage Cloned Mouse Embryos(A) MA plot showing log fold change in gene expression between fertilized embryos and ES clone embryos (logFC, y axis) against the log of gene expression in cultured ES (logCPM, x axis). Differentially expressed genes are shown in red and bluefor genes downregulated and upregulated in clones compared to fertilized embryos, respectively (FDR< 0.05, three experiments).(B) Same as (A) except that USP21 was overexpressed in the cloned embryos.(C and D) Venn diagram indicating the overlap between the downregulated genes (C) and upregulated genes (D) in control and USP21 clones. See also FigureS5 and Tables S6 and S7.
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Supplemental Figure S1 related to Figure 1. Identification of resistant genes by RNA-seq analysis of BrUTP labelled RNA 48h after nuclear transfer. (A) hierarchical clustering of RNA-seq data obtained from samples obtained with (MEF-NT and ES-NT BrUTP)or without BrUTP labelling (MEF-NT No BrUTP) . (B)(C) RT-qPCR analysis of genes identified as resistant in MEF (B) or ES (C). Mean +/- sem from n=3 experiments. (★, P value< 0.05, student t-test).
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Supplemental Figure S2 related to Figure 2. CpG methylation in the gene body of resistant genes
(A) DNA methylation in the gene body of reprogrammed , maintained or
resistant genes shown as percentage of CpG showing >95% methylation (data from (Reddington et al., 2013),). (B) DNA methylation in gene body of genes resistant in OOC-NT, mouse egg-NT, transcription factor induced reprogramming, and cell fusion shown as percentage of CpG showing >95% methylation (data from (Reddington et al., 2013),).
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Supplemental Figure S3 related to Figure 3. Quantitation of histone modification removal from transplanted nuclei upon chromatin modifier expression.
(A) Western blot analysis of histone modifications on the chromatin of
transplanted nuclei collected prior nuclear transfer (0h), or 48h after nuclear transfer to oocytes expressing various combinations of chromatin modifiers. The left and right panels show analysis corresponding to the two experiments used for the RNA-seq analysis. (B) Quantitation of modified histones signal in transplanted nuclei. Intensity of modified histones was averaged from the two experiments shown in B and normalised to average total histone H3 signal intensity obtained by fluorescence measurement (LI-COR detection system). Mean +/- sem from n=2 experiments .
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Supplemental Figure S4 related to Figure 5. USP21-dCas9 mediated H2A deubiquitylation of Otx2.
(A) Titration of USP21-das9 mRNA injection. Oocytes were injected with 0 to 23 ng of USP21-dcas9 mRNA 24h prior to nucler transplantation. 48h after nuclear transfer the transplanted chromatin is recovered and analysed by western blot for H2AK119u1 level. Injection of 0.09 ng of USP21-dcas9 does not globally affect H2AK119u1 level in transplanted chromatin and this amount of mRNA was therefore used for subsequent targeting experiments. (B) Location of guide RNAs targeting the Otx2 promoter (green arrows) as well as the primers used in ChIP-qPCR experiment (red arrows). (C) H2AK119u1 ChIP for Otx2 promoter, Otx2 gene body, and repeat elements of the genome (major satellite and IAP)
following nuclear transfer to control oocytes, USP21 (9ng) injected oocytes, or USP21dcas9 injected (0.09ng) oocytes together with or without co-injection of Otx2 guide RNAs mix. ('star', P value< 0.05, Mann-Whitney test). Mean +/- sem from n=2 experiments
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Supplemental Figure S5 related to Figure 6:USP21 mRNA injection decreases H2AK119u1 level in mouse 2-cell embryos.
(A) H2AK119u1 in the nuclei of control or USP21 injected embryos. Embryos are injected at fertilization with mRNA encoding USP21 and subsequently fixed for immunolabelling at the 2-cell stage. (B) Quantitation of H2AK119u1 signal intensity in control and USP21 injected 2-cell stage embryos. Number of embryos quantitated shown in bracket. ('star', P value< 0.001, Mann-Whitney test).
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