Fig 1: Transcriptomic correlation analysis reveals a functional link of Rif1 to RNF2 and Pcgf6. A Known regulators controlling the pluripotency-to-totipotency transition of mESCs. B STRING visualization of the protein-protein interaction network of the previously reported 21 repressors of totipotency. C Principal component analysis (PCA) of gene expressions of the mESCs with depletion of the indicated genes. D PCA based on the expressions of repetitive elements. E Correlation matrix showing the unbiased and pairwise comparisons of the global changes in gene expression upon depletion of the indicated genes in mESCs. F Correlation matrix generated with the transcriptional changes of repetitive elements upon depletion of the indicated genes in mESCs. Color bars represent Pearson correlation coefficient. G Venn diagram illustrating the differentially expressed genes (adjusted p-value < 0.05 and |Log2 Fold Changes (FC)| > 1) in the mESCs depleted of Rif1, Pcgf6, or RNF2. H Venn diagram of the differentially expressed repetitive elements (adjusted p-value < 0.05 and |Log2 FC| > 1)
Fig 2: The AKT-mTOR pathway was activated by circACTG1/miR-940/RIF1 axis. (a and b) Expression of RIF1 after transfecting miR-940 mimic. (c and d) Expression of RIF1 after transfecting shcircACTG1. (e and f) Expression of RIF1 with circACTG1 knockdown or combined with miR-940 inhibitor. (g) Overall survival of RIF1 expression. (h–j) AKT-mTOR pathway molecule expression after transfecting miR940 mimic (h), shcircACTG1 (i), and shcircACTG1 and miR-940 inhibitor (j).
Fig 3: mLncZFHX2 regulates RIF1 expression via recruiting KLF4 onto RIF1 promoter. (A) Venn diagram of overlaps between ChIRP-seq (probe mLnc/input) and RNA-seq (Ad mLnc/Ad NC). (B) Heatmap of 10 genes in MCs treated with Ad mLnc or Ad NC. (C) RT-qPCR of mRNA levels of 10 genes in MCs treated with Ad mLnc or Ad NC (n = 6, mean ± SD). (D) MCs cultured in hypoxia for 48 h and subjected to ChIRP assay using biotin-labeled probes against mLncZFHX2. Precipitated DNA fragments were extracted for RT-qPCR to determine mLncZFHX2 enrichment on the RIF1 promoter (n = 6, mean ± SD). (E) Complementarity between the RIF1 promoter (−1323 to −1313) with the mLncZFHX2 sequence (82 to −92). (F) Venn diagram of overlap between probe mLnc specifically conjugated proteins and mouse transcription factors from CIS-BP. (G) RNA pulldown assay on biotin-labeled fragments of mLncZFHX2 from MC lysates. (H) Computed electrostatic potentials of mLncZFHX2 and KLF4 from molecular docking experiments. (I) A RIP assay of KLF4 and its mutants interacting with mLncZFHX2 was conducted using anti-Flag (n = 6, mean ± SD). (J) EMSA using biotin-labeled mLncZFHX2 and recombinant KLF4 with or without anti-KLF4. (K) Schematic of putative KLF4-binding sites within the RIF1 promoter region (2000 bp). KLF4 ChIP assay of the RIF1 promoter. (L) Luciferase reporter assay using RIF1 promoters with mLncZFHX2 or KLF4 overexpression in HEK-293 cells (n = 6, mean ± SD). (M) Luciferase assay of RIF1 wild-type and mutant promoter activity under mLncZFHX2 and KLF4 co-overexpression in HEK293 cells. (N) MCs were transfected with Ad NC or Ad mLnc and then subjected to KLF4 ChIP assay targeting the RIF1 promoter. (O) Schematic of mLncZFHX2 functional mechanisms. Graphs show P values from unpaired two-tailed t-test (C, D), one-way ANOVA (I, L, M) and two-way ANOVA (N).
Fig 4: Similar transcriptomic changes caused by depletion of Rif1 or Pcgf6. A Schematic illustration of the compositions of the canonical PRC1 (cPRC1) and non-canonical PRC1 (ncPRC1) complexes. B-C Correlation analysis of the differentially expressed genes (B) or repetitive elements (C) in the mESCs depleted of the indicated Pcgf proteins or Rif1. Color bar represents Pearson correlation coefficient. D Gene Ontology (GO) analysis of differentially expressed genes (|FC| > 1.5, p < 0.05) among the mESCs depleted of Rif1 or the indicated Pcgf proteins. Bubble color indicates the adjusted p value, and bubble size represents the number of genes in each category
Fig 5: The interaction between Rif1 and PRC1.6 is mediated by Pcgf6. A The LacO-LacI induced colocalization experiments reveal that knockdown of different components of the PRC1.6 complex has no effect on the interaction between Rif1 and Pcgf6. GFP-Rif1 is shown in green, LacI-DsRed fused with Pcgf6 is shown in red, and DNA is stained with DAPI (blue). Bar: 10 μm. B RT-qPCR analysis of the indicated transcripts after the transfection of the corresponding shRNAs in the U2OS-LacO cell line. **** p < 0.0001 by t-test, Error bars represent SD, n = 3. C The relative enrichment of GFP-Rif1 in the indicated experimental groups. ns: not significant by t-test, Error bars represent SD, n = 10–23 cells per group. D The LacO-LacI induced colocalization experiments reveal that the interaction between Rif1 and RNF2 is weakened after the knockdown of Pcgf6. GFP-Rif1 is shown in green, LacI-DsRed fused with RNF2 is shown in red, and DNA is stained with DAPI (blue). Bar: 10 μm. E RT-qPCR analysis of Pcgf6 after the transfection of shRNAs targeting NT or Pcgf6 in the U2OS-LacO cell line. **** p < 0.0001 by t-test, Error bars represent SD, n = 3. F The relative enrichment of GFP-Rif1 in the indicated experimental groups. **** p < 0.0001 by t-test, Error bars represent SD, n = 29 in shNT group, n = 14 in shPcgf6 group. G Western blot showing the protein level of the indicated members of the PRC1.6 complex in the presence or absence of Rif1. H Coimmunoprecipitation of RNF2 and RYBP with endogenously tagged HA-Pcgf6 in the mESCs treated with shRNA targeting NT or Rif1. I Gel filtration of nuclear extracts from the Rif1 WT or Rif1 CKO mESCs. The corresponding elution volumes for each analyzed fraction are labeled. The arrow indicates the approximate elution volume of a 440 kDa protein complex
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