Fig 1: RBBP4, but not RBBP7, is indispensable for maintaining the identity of mESCs, and RBBP4 degradation activates 2C gene expression. (A) Heatmap of ChIP-seq signals for RBBP4 and RBBP7. (B) Pearson correlation of ChIP-seq signals between RBBP4 and RBBP7. (C) Schematic diagram of the AID system to degrade RBBP4 or RBBP7. The endogenous Rbbp4 gene or Rbbp7 gene was tagged with AID–mCherry in OsTir1 knockin parental mESCs. (D) Western blots showing protein levels of RBBP4 and RBBP7 in cells treated with IAA at different time points. (E) MA plot showing the expression changes of genes and TEs after degradation of RBBP4 or RBBP7. The horizontal red and blue lines define log2FC >1 and < −1, respectively. (F) Gene set enrichment analysis (GSEA) of early-2C genes in RBBP4- or RBBP7-depleted RNA-seq data. (G) RT–qPCR to detect expression of 2C genes and MERVL elements following degradation of RBBP4 or RBBP7 (n = 3). (H) Comparison of our scRNA-seq data with previously published scRNA-seq data of mouse pre-implantation embryos from zygote to E4.5, naïve mESCs and EpiSCs.
Fig 2: (A) Kaplan–Meier curves for survival time according to protein expression of RBBP7. Blue curve = negative expression; red curve = low/medium expression; green curve = high expression. (B,C) Images showing histopathological expression of RBBP7 in patients diagnosed with pancreatic cancer. High expression of the protein corresponds to an IRS score of 3, whereas low/medium expression corresponds to an IRS score of 1/2.
Fig 3: Identification of a super-enhancer that drives RBBP7 expressions in BCa. (A) The putative enhancers of the super-enhancer (E1-E3) were identified based on markers of H3K27ac, P300, and DNase I in HeLa, HCT-116 and MCF-7 cells. (B) ChIP-qPCR analysis showing the H3K27ac enrichment on RBBP7-SEs in MCF-7, T-47D, and ZR-75-1 cells. (C) Illustration of genetic deletion of RBBP7-SE regions via CRISPR sgRNA technology. (D) ChIP-qPCR analysis showing the p300 enrichment on RBBP7-SE in Ctrl and p300-KD MCF-7 or T-47D cells. (E) ChIP-qPCR analysis of RBBP7 mRNA expression in 4 WT cell clones and 4 clones with CRISPR-deleted RBBP7-SE (n = 5). (F) Western blot and RT-qPCR analysis comparing the expression levels of RBBP7 in control and p300-KD cells (MCF-7, T-47D). (G) Western blot showing the RBBP7 expressions in response to increasing doses of JQ1 treatment. (H) CCK-8 analysis showing the cell proliferation rates of BCa cells (MCF-7, T-47D) with or without RBBP7-SE depletion. (I) Colony formation assays showing the growth capacities of BCa cells (MCF-7, T-47D) with or without RBBP7-SE depletion. (J) Western blotting assays showing the RBBP7 levels in MCF-7, and T-47D cells. The overexpression vector GV230-RBBP7 was constructed to express RBBP7-EGFP fusion protein, and then transfected into the RBBP7-SE homozygous deletion SE−/− cell lines. (K) Proliferation and migration abilities were measured by colony formation and transwell assays. Data represent the Mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using a un-paired Student’s t-test (B, D, E, F, K) and the 2-way ANOVA followed by Tukey’s multiple comparisons test (H)
Fig 4: (A) RBBP7 mRNA expression levels in women with CVD compared to HC group (** p < 0.01). (B) IRS scores assessing RBBP7 expression in placental tissue of women with CVD and without this condition (* p < 0.05). (C,D) Representative immunostaining images displaying RBBP7 expression in placental villi from CVD and HC samples.
Fig 5: RBBP7 recruits demethylase LSD1 to epigenetically regulate SOX9/SOX2/OCT4/CCND1 levels. (A) Co-IP and western blot assays showing the potential interactions between RBBP7 and a series of NuRD complex components. (B) Representative graphs showing the in vitro sphere-formation assay that were conducted in control and siRNA-treated MCF-7 cells. (C) Western blot assays showing the knocking down RBBP7 expressions in cells treated with or without siRNA. (D) ChIP-qPCR analysis of Pol II-S2P (left) and S5P (right) in the promoter regions of the indicated targets in WT and LSD1-KD MCF7 cells with or without LSD1 restoration. (E) ChIP-qPCR analysis of H3K9me3 enrichment on the promoters of genes in WT and LSD1-KD MCF7 cells with or without LSD1 restoration. (F) ChIP-qPCR analysis of RBBP7 enrichment on the promoters of genes in WT and RBBP7-KD MCF7 cells with or without RBBP7 restoration. (G) ChIP-qPCR analysis of Pol II-S2P (left) and S5P (right) in the promoter regions of the indicated targets in control and RBBP7-KD MCF7 cells with or without LSD1 restoration. (H) RT-qPCR analysis revealing the mRNA levels of SOX9/SOX2/OCT4/CCND1 in RBBP7-overexpressing MDA-MB-231 or MDA-MB-453 cells with or without LSD1 knockdown. (I) Graphs and tumor volumes of indicated MDA-MB-231-cell-derived xenograft tumors (Ctrl-shCtrl group, RBBP7-shCtrl group, and RBBP7-shLSD1 group). (J) Quantification of tumor weight in tumors derived from indicated groups in (I). (K) IHC graphs of Ki-67, and CCND1 markers in tumors derived from indicated groups in (I). Data represent the Mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using a un-paired Student’s t-test (B, D-H, J), and the 2-way ANOVA followed by Tukey’s multiple comparisons test (I)
Supplier Page from Abcam for Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade