Fig 1: E2F6 is dispensable for the maintenance of epigenetic silencing.a Experimental design. We either derived MEFs from WT and E2f6−/− embryos or generated E2f6-KO clones and WT control clones by CRISPR-Cas9. b Western blotting of E2F6 in MEFs derived from WT and E2f6−/− embryos and MEFs CRISPR-Cas9 clones. β-ACTIN was used as a loading control. c RT-qPCR analysis of the expression of E2F6 target genes in embryo-derived WT and E2f6−/− MEFs (top), and MEF CRIPSR-Cas9 clones (bottom) (mean fold change compared to WT controls, n = 2 independent cell lines or clones per condition, expression normalized to Gusb, Rpl13a, and Mrpl32). d Heatmap representing the percentage of CpG methylation profiled by RRBS in the promoters of E2F6 target genes in embryo-derived WT and E2f6−/− MEFs (left), and MEF CRISPR-Cas9 clones (right). e Western blotting of E2F6 in WT and E2f6−/− MEFs, and E2f6−/− MEFs transduced with lentiviruses expressing E2F6 or E2F6 E68 fused to 3×-HA. α-TUBULIN was used as a loading control. f RT-qPCR analysis of the expression of E2F6 target genes in WT and E2f6−/− MEFs, and E2f6−/− MEFs rescued with E2F6. The values are represented as the fold change compared to WT (mean ± SEM, n = 3 independent experiments, expression normalized to Gusb, Rpl13a, and Mrpl32). *p < 0.05, **p < 0.01, ns: not significant (two-tailed t-test compared to E2f6−/− cells). g Methylation analysis by COBRA of the Tuba3a and Gpat2 promoters in WT and E2f6−/− MEFs, and E2f6−/− MEFs rescued with E2F6. h Model: E2F6 binds to and represses promoters of germline genes in preimplantation stages. At implantation, E2F6 recruits DNA methylation and initiates long-term epigenetic silencing of its target germline genes and subsequently becomes dispensable for the maintenance of the epigenetic repression in differentiated cells. Source data are provided as a Source Data file.
Fig 2: E2F6 binding at target genes shown by ChIP-qPCRChIP-qPCR showing enrichment of E2F6 at target genes Zcwpw2 and Rab8a as shown by fold enrichment over the negative region. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data are represented as mean ± SEM.
Fig 3: MGA is essential for PRC1.6 genomic binding in tumor cells.(A) Venn diagram showing overlap between MAX, MGA, E2F6, and L3MBTL2 bound genes in KP cells. (B) Heatmaps showing genome-wide promoter proximal (±2 kb) binding by the indicated transcription factors in control and sgMga KP cells. (C) Meta-plots of occupancy by the indicated transcription factors in Empty and sgMga KP cells. (D) Volcano plot of differentially expressed genes that are directly bound by MGA (red and blue dots indicate bound genes, up and down as indicated; green dots with labels indicate genes functionally implicated in MGA activity). (E, F) representative tracks for MGA, L3MBTL2, E2F6, MAX, and MYC binding at the (E) Stag3/Gpc2 promoter and (F) Podxl2 loci in KP cells and sgMGA KP cells. (G–I) Meta-plots of RNA pol2 enrichment at the TSS ±2 kb in sgMGA KP cells (red lines) vs KP cells with wild-type MGA (black lines) at (G) MGA-bound genes, (H) genes upregulated, and (I) genes downregulated in sgMGA KP cells. (J–L) Box plots of RNA pol2 peak areas at TSS ±2 kb in KP vs sgMGA KP cells for (J) loci upregulated (p-value=2.1e-07), (K) loci downregulated (p=1.6e-06). The p-values in (J–L) calculated by Welch two sample t-test. (L) Box plots of RNA pol2 peak areas at MYC-bound loci in KP and sgMGA-KP cells (p=8.7e-05). (M) Venn diagram depicting the numbers of genes bound by RNA polymerase II, MYC, and MGA and their extent of overlap in KP cells.
Fig 4: ChIP-seq analysis and interactome of E2F6 in ES cells.a Genome browser tracks showing E2F6 ChIP-seq and input signals in E2F6+/+ and E2f6−/− ESCs over the Tuba3a gene. UCSC CGI and RefSeq gene annotations are shown below the tracks. b Density histogram representing the distance of E2F6 peak summits to their closest TSS (n = 2533). c Percentage of E2F6 peaks overlapping with annotated UCSC CpG islands (CGIs). d The E2F6 and MAX E-box CACGTG motifs are the most enriched sequence motifs identified in E2F6 peaks (p-value: hypergeometric test). e Metaplot representing the position of E2F6 and MAX E-box motifs relative to the summits of E2F6 peaks (n = 2533). f Genome browser tracks of ChIP-seq signals in mouse ESCs showing that E2F6 peaks frequently colocalize with peaks of other PRC1.6 subunits. UCSC CGI and RefSeq gene annotations are shown below the tracks. g Heatmaps of E2F6, MGA, L3MBTL2, PCGF6, RYBP, and RING1B ChIP-seq read densities ±2.5 kb around E2F6 peak summits (n = 2533), ordered by E2F6 read densities. h Western blotting with an anti-E2F6 antibody showing the expression of the tagged E2F6 in ESCs transfected with 3×HA-E2F6. α-TUBULIN was used as a loading control. i. Volcano plot of E2F6 interaction partners in ESCs. Anti-HA immunoprecipitation was performed on nuclear extracts from control ESCs and ESCs expressing 3×HA-E2F6 (n = 5 independent experiments). Significantly enriched proteins are indicated in red. j Co-immunoprecipitation of E2F6 with PRC1.6 in mouse ESCs. Western blotting was performed on the input and anti-HA immunoprecipitate from control ESCs and ESCs expressing 3×HA-E2F6 using the indicated antibodies. GLP was used as a control nuclear protein that does not interact with E2F6. Source data are provided as a Source Data file.
Fig 5: The effect of E2F6 knockdown on the immunosuppressive effect of tacrolimus. Real-time PCR analysis showed E2F6 mRNA expression (A) and Western blots verified E2F6 knockdown (B). Real-time PCR analysis of IL-2 mRNA expression (C,D) and GM-CSF mRNA expression (E,F) with ELISA analysis of IL-2 and GM-CSF. (n = 3). ***, p < 0.001; *, p < 0.05. p-values were calculated using Student’s t-test.
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