Fig 1: PCGF6 positively regulates pluripotency genes via super-enhancers. (A) Distribution of PCGF6 binding sites at promoter (−3 kb to +3 kb), gene body, and intergenic regions. (B) Average ChIP-Seq density of H3K4me1 and H3K27ac near the PCGF6 peak center. (C) Average ChIP-Seq density of MED1 near the PCGF6 peak center. (D) The average ChIP-Seq density of PCGF6 and MED1 in typical enhancers (TEs) and super enhancers (SEs). (E) Box plot of MED1 (left) and PCGF6 (right) ChIP-Seq density (reads per million reads per base) at the TE and SE regions. (F) Relative expression of SE-associated genes in Pcgf6-depleted group compared with empty vector group. The genes that closest to SEs were selected. (G) The enrichment of PCGF6, MED1, and H3K4me1 at the SE region of Ccnd3 and Agtrap. (H–K) Promoter-associated interactions of Ccnd3 (H) and Agtrap (I) derived from promoter capture Hi-C reads. Relative expression of Ccnd3 (J) and Agtrap (K) after Pcgf6 knockdown were presented. The regions and genes above are closest to SE and PCGF6 binding site. *P < 0.05 compared with control cells. (L) eRNA expression of Oct4, Sox2 and Nanog SEs after 5 days of Pcgf6 knockdown. Data are presented as mean ± SEM (n = 3). *P < 0.05 compared with control cells. **P < 0.01 compared with control cells
Fig 2: Role of OCT4 and PCGF6 in Super-enhancer Regions. (A) Average ChIP-Seq density of OCT4, SOX2 and NANOG near the PCGF6 peak center. (B) Heatmaps of PCGF6 binding loci that are sorted by H3K4me3 and H3K27me3, and the distribution of OCT4, SOX2 and NANOG at PCGF6 binding sites. (C and D) The relative expression pattern of PCGF6 and OCT4 during somatic cell reprogramming (C) and EB differentiation (D). (E) Distribution of PCGF6/OCT4 co-binding sites at promoter (−3 kb to +3 kb), intergenic, and other regions. (F) Relative expression of PCGF6/OCT4 co-binding genes in Pcgf6-depleted group compared with empty vector group. (G) Validation of physical associations of PCGF6 and OCT4 in 293T cells by co-immunoprecipitation. (H) Protein levels after 23 h of dox treatment in ZHBTc4 mESCs. (I–J) The enrichment of PCGF6, MED1 and OCT4 at the SE region of Ccnd3 and Polr3gl. Dox treatment decreased the enrichment of PCGF6 at the SEs of Ccnd3 and Polr3gl. Data are presented as mean ± SD from three independent replicates. **P < 0.01 compared with control cells
Fig 3: The functional model of PCGF6 and OCT4. In this model, PCGF6 function as a transcription activator for pluripotency in mESCs. In detail, PCGF6 activates cell cycle gene including Ccnd3 and Polr3gl via the super-enhancer dependent chromatin Interactions to promote mESCs proliferation, meanwhile, OCT4 is required for the binding of PCGF6 at these transcription activation regions
Fig 4: Transcription activation function of PCGF6. (A) Heatmaps of PCGF6 binding loci are sorted by the active marker H3K4me3 and the repress marker H3K27me3. H3K27ac is an active marker. DNase I is an open chromatin markers. (B and C) Percentage of PCGF6 targeted peaks (B) and genes (C) in active (K4me3) regions, repressive (bivalent) regions, and repressive (K27me3) regions. (D) Box plot of gene expression of PCGF6 targeted genes in mESCs. **P < 0.01 compared with active (K4me3) group. (E) Relative expression of PCGF6 targeted genes in Pcgf6-depleted group compared with empty vector group. Data are presented in two independent replicates. (F) Heatmaps showing the dynamic expression of PCGF6 targeted genes during embryonic development and somatic cell reprogramming. (G) Heatmaps shows the dynamic expression of PCGF6/H3K4me3 co-binding genes during embryonic development and somatic cell reprogramming. (H) Relative expression of ESC-associated genes from the RNA-Seq data. The gene list generated from ESC-enriched genes (Ben-Porath et al., 2008) and SE associated genes (Whyte et al., 2013). (I–L) The enrichment of PCGF6, H3K27ac, H3K4me3 and H3K27me3 at the locus of Mfge8 (I) and Ifitm1 (K). Relative expression of Mfge8 (J) and Ifitm1 (L) after Pcgf6 knockdown were presented. **P < 0.01 compared with control cells. *P < 0.05 compared with control cells
Fig 5: Effects of PCGF6 on the maintenance and establishment of pluripotency in mESCs. (A) The expression of PCGF6 in different tissues. (B) Knockdown efficiency of Pcgf6 in mESCs was validated by real-time quantitative PCR (qPCR). Data are presented as mean ± SD from three independent replicates. **P < 0.01 compared with control cells. (C) Knockdown efficiency of Pcgf6 in mESCs was validated by Western blot. (D) Morphology of Pcgf6 knockdown mESCs with AP staining in the day 5, respectively. Scale bar represents 50 μm. (E) Volcano map of the RNA-Seq expression data from empty vector and Pcgf6 knockdown mESCs. 1.3 fold change and P < 0.05 was significantly. (F) The dynamic expression of genes Pcgf6, Oct4, Sox2 and Nanog during somatic cell reprogramming. The data was analyzed using the microarray data for gene expression from GSE19023 (Heng et al., 2010). (G) Schematic overview of the reprogramming process using stable pre-iPS cell lines and medium switch from serum/LIF to N2B27/2i/LIF. (H) The knockdown of Pcgf6 significantly reduces the reprogramming efficiency. Bright field (left), GFP field (middle) and AP staining (right) in day 10 after 2i + LIF medium switch. Scale bar represents 500 μm. (I) Quantification of the numbers of Oct4-GFP colonies at day 10 of N2B27/2i/LIF treatment. Data are presented as mean ± SD from three independent replicates. **P < 0.01 compared with control cells. (J) Quantification of the numbers of colony formation at day 10 of N2B27/2i/LIF treatment. Data are presented as mean ± SD from three independent replicates. **P < 0.01 compared with control cells. (K) Total expression of Oct4, Sox2, Nanog, Klf4 and Pcgf6 in day 0 of serum/LIF and day 10 of N2B27/2i/LIF treatment. Data are presented as mean ± SD from three independent replicates
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