Pasque V , Gillich A , Garrett N , Gurdon JB .

081277 Wellcome Trust , RG44593 Wellcome Trust , RG54943 Wellcome Trust , WT081277 Wellcome Trust

	Figure 1. The inactive X chromosome of differentiated somatic cells is remarkably resistant to reprogramming by Xenopus oocytes. (A) Nuclear transfer experimental scheme. Female MEFs with an X-linked CMV-GFP transgene on the active (Xa) or on the inactive (Xi) X chromosome were sorted, permeabilized with Streptolysin O (SLO) and the resulting nuclei transplanted into the germinal vesicles (GVs) of stage V Xenopus oocytes. Transplanted oocytes were incubated at 18°C and samples were collected at several time points for transcriptional analysis. Transcriptional reactivation of X-GFP was assayed by qRT–PCR. (B) The Xi of MEFs is resistant to transcriptional reprogramming by oocytes. qRT–PCR analysis of GFP (black), Oct4 (white) and Sox2 (grey) expression in transplanted nuclei immediately and 3 days after nuclear transfer. The arrow highlights maintenance of Xi-GFP repression. P<0.05, n=3, error bars are mean±s.d. The table shows transcript levels mean±s.d. a.u. represents arbitrary unit. (C) The imprinted Xi of trophoblast stem (TS) cells is resistant to transcriptional reprogramming by oocytes. Quantitative analysis of GFP (black) and Sox2 (grey) expression in transplanted Xi-GFP TS and Xa-GFP MEFs nuclei. Arrows highlight maintenance of imprinted Xi-GFP silencing. P<0.05 for GFP, except samples marked *P<0.06. For Sox2, P<0.05, except samples marked *P<0.1, n=3, error bars are mean±s.d. The table shows transcript levels mean±s.d. a.u. represents arbitrary unit.
	Figure 2. The Xi of EpiSCs can be reactivated by nuclear transfer to Xenopus oocytes. (A) Schematic representation of Xi-GFP EpiSCs nuclear transfer experiments. Undifferentiated female EpiSCs cultured on feeders were sorted from differentiating cells by flow cytometry of SSEA1-positive, GFP-negative EpiSCs. After SLO permeabilization, Xi-GFP EpiSC nuclei were transplanted to oocyte GV, and the resulting oocytes were cultured for 3 days. (B) Xi-GFP of EpiSC nuclei can be reactivated after nuclear transfer. Quantitative RT–PCR of X-GFP expression after nuclear transfer. Time points and types of transplanted nuclei are indicated. Transcript levels are shown in table±s.e.m. P<0.05, except samples marked *P<0.2, n=3, error bars show s.e.m. a.u. represents arbitrary unit. (C) Rlim allele-specific RT–PCR. Validation of allele-specific Rlim RT–PCR on cells derived from embryos resulting from a cross between X-GFP Musculus and Castaneus mice (maternal genotype denoted first). MEFs and EpiSCs were derived from embryos genotyped for sex (Ube1 expression) and X-GFP transgene expression and sorted by flow cytometry based on GFP expression. (D) Rlim can be reactivated after nuclear transfer. Allele-specific Rlim RT–PCR of Xi-GFP mus/cast MEFs and EpiSCs, immediately after (day 0) or on day 3 after nuclear transfer.
	Figure 3. DNA methylation does not correlate with reversibility of the Xi before nuclear transfer. Bisulphite analysis of G6pdx, X-GFP and Hprt1 promoter and coding regions in female MEFs, EpiSCs and TS cells. All regulatory regions tested are fully methylated on the Xi of all cell types (black circles), and unmethylated on the Xa allele (open circles). The proportion of methylated CG residues is indicated. No circles represent mutated or missing CpGs.
	Figure 4. H3K27me3 does not correlate with reversibility of the Xi before and after nuclear transfer. (A) Immunofluorescence of X-GFP female EpiSCs, MEFs and TS cells against H3K27me3. Confocal images of H3K27me3 immunostainings (green) counterstained with DAPI (magenta) show that H3K27me3 is enriched on the Xi of female EpiSCs grown on feeders (93%, n=84), the Xi of MEFs (98%, n=123) and the Xi of TS cells (100%, n=32). Scale bars=10 μm. Images are projected Z-sections. (B) H3K27me3 is maintained on the Xi after nuclear transfer. Immunofluorescence of transplanted female MEFs nuclei against H3K27me3 (green). A high proportion (>48%) of female nuclei retain an H3K27me3-labelled Xi up to 72 h after nuclear transfer (arrowheads). The proportion of nuclei carrying an H3K27me3-labelled Xi is shown. n=number of nuclei. DAPI is shown in magenta. Scale bars=2 μm. Images are single Z-sections.
	Figure 5. The long noncoding RNA Xist dissociates from chromatin of the Xi after nuclear transfer. (A) RNA FISH for Xist RNA (green) on transplanted female MEF nuclei. Oocyte GVs containing transplanted nuclei were dissected, fixed and subjected to RNA FISH against Xist RNA. Confocal images reveal that the Xist RNA cloud of female MEFs (0 h) is lost from the Xi 18 h after nuclear transfer. Note the presence of punctate Xist RNA FISH signal dispersed throughout the nucleus of some of the 18 h transplanted nuclei. DAPI is shown in magenta. Low (scale bars=25 μm) and high (scale bars=5 μm) magnification pictures are shown. P denotes permeabilized nuclei. Images are projected Z-sections. (B, C) Xist RNA is lost from the Xi after nuclear transfer of female MEFs (B) and female EpiSCs (C). Xist RNA FISH of nuclear transfer female MEFs and EpiSCs. Samples were collected and fixed at indicated time points. The Xist RNA cloud characteristic of the Xi is maintained up to 3 h after nuclear transfer, then decreases to give a pinpoint signal at 12 and 16 h, and is completely lost from transplanted nuclei by 24–48 h after nuclear transfer. The proportion of nuclei with a Xist RNA cloud is indicated. DAPI is shown in magenta. n=number of nuclei. Scale bars=5 μm in (B) and 2 μm in (C). Images are projected Z-sections. (D) Xist expression levels in transplanted female MEF and EpiSC nuclei. qRT–PCR analysis of Xist (dark grey) and Sox2 (light grey) expression in transplanted nuclei. Xist transcript levels increase after nuclear transfer. Error bars are s.e.m. a.u. represents arbitrary unit.
	Figure 6. Nuclear transfer reverses epigenetically stable, Xist-induced and Xist-independent gene repression. Reversibility of PGK-puro silencing following nuclear transfer of clone 36 cells. To obtain the Xist-dependent (XD) PGK-puro repressed state, clone 36 ES cells were induced to express Xist for 4 days. To obtain the Xist-independent (XI), stable PGK-puro repressed state, clone 36 ES cells were induced to differentiate with RA for 4 days while being induced with Xist at the same time. The nuclei of XD and XI PGK-puro repressed cells were transplanted to oocytes. Biological triplicates were collected immediately or 2 days after nuclear transfer. Nuclei induced to ectopically express Xist after nuclear transfer, within the GV is indicated (+). Transcriptional analysis of puro (dark grey) and Xist (light grey) expression by qRT–PCR of oocytes transplanted with nuclei obtained as described in Supplementary Figure S6B is shown. P<0.05, n=3. Error bars are s.e.m. a.u. represents arbitrary unit.
	Figure 7. mH2A correlates with stable Xi and is maintained after nuclear transfer to Xenopus oocytes. (A) Immunostaining of female EpiSCs, MEFs and TS cells against mH2A1 (green), ubH2A (red) and SSEA1 (red). Undifferentiated female EpiSCs do not exhibit accumulation of mH2A1 on the ubH2A-labelled Xi. mH2A1 is induced in differentiated EpiSCs, marked by loss of the pluripotency marker SSEA1 (right panel, left column). mH2A1 is incorporated in chromatin of the Xi in differentiated EpiSCs, as shown by co-localization with Xi marker ubH2A (right panel, right column). Note that the Xi of undifferentiated EpiSCs is stained with ubH2A only. mH2A1 was found accumulated in 95% of female MEFs, 85% of female TS cells and 0% of female EpiSCs. DAPI is shown in blue. Scale bars=10 μm. Images are projected Z-sections. (B, C) mH2A1-GFP remains associated with heterochromatic regions in transplanted nuclei and reveals chromatin reorganization. Projections of confocal images of mH2A1-GFP MEF (B) and sable mH2A1-GFP C2C12 (C) nuclei transplanted into oocytes preloaded with Bmi1-cherry by mRNA injection. Note the persistence of mH2A1-GFP on the Xi (arrowheads), bound by Bmi1-cherry imported from the oocyte, and the appearance of mH2A1-GFP-labelled pericentric heterochromatin foci. Scale bars=10 μm. Images are projected Z-sections.
	Figure 8. mH2A depletion improves reprogramming by nuclear transfer. (A) qRT–PCR analysis of mH2A1 and mH2A2 expression following shRNA-mediated mH2A RNAi. (B) Western analysis of mH2A1 in shRNA expressing Xi-GFP MEFs. (C, D) qPCR analysis of GFP (black), Sox2 (grey) and Oct4 (white) expression in transplanted Xi-GFP MEFs nuclei subjected to mH2A RNAi and/or TSA treatment. P<0.05 except samples marked *P<0.06 in (C), or *P<0.08 in (D), n=3. Error bars are s.e.m. Note the differences in y axis.

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