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. 2019 Sep 26;47(19):10115–10133. doi: 10.1093/nar/gkz801

Figure 4.

Figure 4.

Ash2l/Oct4/Sox2/Nanog complex locates on the super-enhancers to regulate enhancer activation. (A) A Venn diagram shows the corresponding numbers of Ash2l-bound genes at Distal II elements (4817 genes, obtained from ChIP-seq data), genes that were affected by Ash2l knockdown (9393 genes, obtained from microarray data; 7597 genes, obtained from RNA-seq data). Ash2l-bound genes at Distal II elements were detected by ChIP-seq, and the genes that were affected by Ash2l knockdown were analyzed by microarray and RNA-seq. A total of 805 genes in the intersection were identified as Ash2l-affected genes at Distal II element, including 107 upregulated genes and 698 downregulated genes. (B) A box plot showing the 805 super-enhancer-driven genes that are affected by Ash2l knockdown at Distal II elements. (C) Categorization of the 698 genes that bound by Ash2l at Distal II elements and down-regulated upon Ash2l knockdown into the binding targets of Ash2l-Oct4 (AO; 608 genes), Ash2l-Wdr5 (AW; 64 genes) and Ash2l-Oct4-Wdr5 (AOW; 46 genes). (D) A Venn diagram shows the numbers of binding loci for the Ash2l-Oct4 overlap, Sox2, Nanog at Distal II elements. (E) Gene Ontology analysis shows the downregulated genes in Ash2l knockdown ESCs and co-bound by Ash2l and Oct4 (AO), Ash2l and Wdr5 (AW), or Ash2l, Oct4 and Wdr5 (AOW) at Distal II elements. (F) Gene set enrichment analysis of the 608 AO co-bound genes at Distal II elements and down-regulated upon Ash2l knockdown. These genes include upregulated genes in ESCs (the first on the left) and downstream genes targeted by Oct4, Sox2 and Nanog, respectively (the second/third/fourth on the left). (G) Genome browser tracks showing the ChIP-seq results of Med1, H3K27ac, Oct4, Sox2, Nanog, Ash2l and Wdr5 at Jarid2, Oct4, Sox2 and Nanog enhancers from the existing ChIP-seq database (Upper) RNA-seq results showing the production of enhancer RNA (eRNA) at Ash2l-bound super-enhancers (lower). (H) qPCR of the eRNA production levels at super-enhancers of Oct4, Jarid2 and Nanog in ESCs with or without Ash2l-knockdown. The eRNA expression at each super-enhancer in Ash2l-knockdown ESCs was shown as relative levels to that in ESCs with shCtrl. (I) ChIP-qPCR shows the enrichment of RNA polymerase II and H3K27ac at the super-enhancers of Oct4, Jarid2 and Nanog in ESCs with or without Ash2l knockdown. Data were normalized with IgG control and shown as relative enrichment to that in ESCs with shCtrl. (J) Re-ChIP-qPCR analysis revealed the binding relationship of Ash2l, Oct4, Sox2 and Nanog at Jarid2, Oct4, Sox2 and Nanog super-enhancer in ESCs with or without Ash2l knockdown. The first ChIP-qPCR with anti-Ash2l antibody revealed the direct binding of Ash2l at the super-enhancer of Oct4. The second ChIP-qPCR with indicated antibodies showed that Ash2l, Oct4, Sox2 and Nanog virtually co-bound to the gene loci at the super-enhancer of Jarid2, Oct4, Sox2 and Nanog. Data were shown as relative fold change to the IgG control. (K) Western blot reveals that Ash2l knockdown suppressed the protein levels of Oct4, Sox2 and Nanog, but not p300, Med1 and H3K27ac in ESCs with shAsh2l. Data are presented as mean ± SD; *P< 0.01.