Fig 2: CTBP1 directly interacts with EZH2 and EED in granulosa cells. (A) The efficiency of RNA pulldown was detected by reverse transcription-quantitative PCR. Data are presented as the mean ± SEM. **P<0.01 vs. control. (B) Samples from RNA pulldown assays were collected for SDS-PAGE and Coomassie brilliant blue staining. Arrows indicated the proteins identified by mass spectrometry. (C) Interaction between CTBP1-AS and EZH2 or EED were identified in granulosa cells and KGN cells by RNA pulldown and immunoblotting analysis. CTBP1-AS, C-terminal-binding protein 1 antisense; EED, embryonic ectoderm development protein; EZH2, enhancer of zeste homolog 2; PARP, poly-ADP-ribose polymerase; RBM12B, RNA binding motif protein 12B; RBM3, RNA binding motif protein 3.

Fig 3: Schematic illustration of the dynamics of Xist expression and X-chromosome inactivation (XCI). In WT embryos, Xist on the maternal X chromosome (Xm) is silenced by oocyte-derived maternal H3K27me3, allowing Xist expression only from the paternal X chromosome (Xp), which becomes inactive X (Xi). In Eed matKO embryos, Xist on Xm is derepressed at preimplantation, resulting in transient Xm inactivation. Nevertheless, this aberrant XCI state is resolved at the late blastocyst stage, which leads to a proper XCI state in postimplantation embryos. In Eed/Xist matDKO embryos, it is expected that ectopic expression of Xist from Xm is prevented.

Fig 4: Maternal XCI is prevented in Eed/Xist maternal doubleKO (matDKO) embryos. (A) Representative images of Xist RNA-FISH in morula embryos. The sex of each embryo was assessed by simultaneous DNA-FISH to detect the Atrx locus on the X chromosome (green spots). Blue indicates DNA stained with DAPI. The numbers of embryos examined (n) are indicated. Scale bars, 10 µm. (B) The ratios of blastomeres showing the indicated numbers of Xist RNA clouds. Each bar represents an individual embryo. (C) Box plot showing the relative expression (matKO/WT) of genes on the maternal autosomes and the maternal X chromosome, respectively, in morula embryos. Genes with ≥20 SNP-containing reads in all replicates were analyzed. The middle lines in the boxes represent the medians. Box edges and whiskers indicate the 25th/75th and ±1.5 × interquartile range, respectively. P, Mann–Whitney–Wilcoxon test. (D) The relative expression (matKO/WT) of individual maternal X-linked genes with ≥20 SNP-containing reads in all replicates. The total number of genes is 216. The numbers of down-regulated and up-regulated genes with a cutoff of fold change 2 are indicated.

Fig 5: Eed deletion impairs OPC proliferation and differentiation.(A and B) In situ hybridization for Pdgfra (A) and quantification of Pdgfra+ OPCs (B) in different brain regions from control and Eed cKO mice at P7 (n = 4 animals per genotype). (C and D) Immunolabeled for Ki67 and PDGFRα (C) and quantification of Ki67+ PDGFRα+ cells (D) in the striatum and superficial layers from P7 control and Eed cKO brains (n = 4 animals per genotype). (E) P14 control and Eed cKO brains labeled for cleaved caspase 3 (CC3) and DAPI. (F) Immunostaining for H3K27me3 and OLIG2 in OPCs isolated from control and Eed cKO cortices. (G) Control and Eed cKO OPCs treated with T3 for 3 days and immunostained for OLIG2, MBP (left). The percentages of MBP+ cells relative to OLIG2+ cells (right, n = 4 independent experiments). (H) Diagram depicting tamoxifen administration strategy on control and OPC-Eed iKO mice. (I) Control and OPC-Eed iKO brains at P14 stained for H3K27me3 and CC1. (J) Immunolabeling for CC1 (left) and quantification of CC1+GFP+ cells (right) in control and OPC-Eed iKO brains at P14 (n = 7 animals per genotype). (K) The white matter of control and OPC-Eed iKO mice at P7 stained for MBP. Scale bars, 50 μm (A), 60 μm (C, I, and K), 100 μm (E, G, and J), and 20 μm (F). Data are means ± SEM. **P < 0.01, ***P < 0.001. Photo credit: Jiajia Wang, CCHMC.