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

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© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 5. — Ash2l binding motif is crucial for the recruitment of OSN to Jarid2, Oct4 and Nanog super-enhancers. (A) Schematic illustration of the design of sequence-specific sgRNA for blocking of Jarid2, Nanog or Oct4 super-enhancers using monoallelic silencing (labeled as sgRNA_AB) or biallelic silencing (labeled as sgRNA_AC, sgRNA_DE, sgRNA_DF, sgRNA_GH and sgRNA_GI). The dCas9 nuclease is targeted to super-enhancer of Jarid2, Nanog or Oct4 by either set of sgRNA. Three amplicons for each enhancer (Jarid2: R1: enhancer; R2: promoter, R3: desert; Nanog: R4: enhancer; R5: promoter, R6: desert; Oct4: R7: enhancer; R8: promoter, R9: desert) were designed to evaluate protein enrichment in ChIP-qPCR assay. (B) ChIP-qPCR analysis showing the enrichment of indicated protein at Jarid2, Nanog and Oct4 super-enhancers in ESCs with CRISPRi/dCas9-mediated interference of Ash2l binding. ChIP-qPCR analysis with indicated antibodies was conducted in CRISPRi/dCas9-modified ESCs, and the enrichment of each protein at R1–3 regions, R4–6 regions or R7-R9 regions was evaluated with specific primers. Data showed that CRISPRi/dCas9-mediated interference of Ash2l binding at Jarid2 enhancer (upper), Nanog enhancer (middle) and Oct4 enhancer (lower), specifically increased dCas9 binding and decreased the binding of Ash2l, Oct4, Sox2, Nanog and H3K27ac, to R1, R4 and R7, but not R2, R3, R5, R6, R8 and R9. (C) qPCR results showing the effect of CRISPRi/dCas9-mediated interference of Ash2l binding on mRNA expression of Jarid2, Nanog and Oct4. (D) Western blot showing the effect of CRISPRi/dCas9-mediated interference of Ash2l binding on the protein content of Jarid2, Nanog and Oct4. (E) qPCR shows CRISPRi/dCas9-mediated interference of Ash2l binding at indicated enhancers abrogate the upregulation of Jarid2, Nanog, Oct4 transcripts induced by Ash2l overexpression. (F) Reporter assay shows that Flag-Ash2l and Myc-Oct4 induce a synergistic effect on enhancer activity in Jarid2, Nanog and Oct4 super-enhancers. The Ash2l-mediated enhancement of Jarid2, Nanog and Oct4 enhancer activation can be abrogated by specific sgRNA for indicated enhancer. Data are presented as mean ± SD; *, P< 0.01.