Figure 1: Alterations in histone modifications derepress Xm-Xist expression.

(a–d) Oocytes injected with Kdm3a (a,b), Kdm4b (c,d) or Egfp mRNAs were subjected to ICSI. After 7–8 h, embryos were fixed and analysed for H3K9me2 (a) and H3K9me3 (c) using IF. Nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI) are shown in blue. Representative images are presented on the left. The box-and-whisker plot shows the ratio of maternal to paternal signal intensities. The horizontal line indicates the median. The P-values were calculated using the Mann–Whitney U-test (U-test). Pb, polar body; n, number of embryos analysed (b,d). (e) Schema of the generation of PEs with altered histone modifications. To examine the effects of histone demethylation on Xm-Xist expression, either H3K9me2 demethylase (Kdm3a) or H3K9me3 demethylase (Kdm4b) mRNAs were injected into MII oocytes that were then activated. To assess the effects of inhibition of histone deacetylation on Xm-Xist expression, oocytes were activated and incubated in the presence of TSA for 24 h. After 48 h, ten four-cell PEs were pooled and analysed as one biological replicate using qPCR. (f) Analysis of Xm-Xist expression at the four-cell stage. The expression level of Xm-Xist in female embryos derived from IVF was defined as 1. One or two asterisks indicate Xm-Xist expression in one or two replicates, respectively. The P-values were determined using Student’s t-tests. Error bars indicate the mean±s.e.m. (g–i) Xist FISH analysis of Kdm4b- and Egfp+TSA-PEs at the four-cell stage. (g) Representative images of FISH results. (h) Ratio of cells with Xist signal to the total number of cells. n, number of interphase cells analysed. (i) Ratio of cells with biallelic expression to total cells. The detailed FISH results are shown in Supplementary Table 1. Scale bars, 20 μm.