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. Author manuscript; available in PMC: 2020 Mar 23.
Published in final edited form as: Nat Struct Mol Biol. 2019 Sep 23;26(10):899–909. doi: 10.1038/s41594-019-0293-z

Fig. 4. Chromatin-associated G-tract RNA removes PRC2 from specific genes in cells.

Fig. 4

(a) Hypothesis: G-tract RNA, tethered to chromatin with dCas9, should compete with CGI chromatin for PRC2, reducing H3K27me3. The same length RNA that is equally G-rich but lacking G-tracts or RNA in which the G-tracts are replaced with A-tracts, both of which bind PRC2 only weakly, should both have no effect.

(b) Top: Position of the Fgf11 sgRNA and primer pairs A and B. Bottom: Change in HA-dCas9, SUZ12, H3K27me3 and total H3 occupancy at Fgf11 and Pax7 measured by ChIP-qPCR after dox-mediated induction of HA-dCas9 expression in cells containing the Fgf11 sgRNA, to which G-tract, G-rich or A-tract RNA is appended (mean and S.D., n=3 independent dox inductions. P-values: Fgf11-A G-tract RNA SUZ12=0.0018, H3K27me3=0.14. Fgf11-B G-tract RNA SUZ12=0.0052, H3K27me3=0.03. Fgf11-B G-rich RNA SUZ12=0.03, Welch’s one-tailed t-test).

(c) Change in H2AK119ub, H3K27ac and total H3 at Fgf11 and Pax7 before and after incubation with dox (mean, S.D., n=3 independent dox inductions, no significant changes, Welch’s one-tailed t-test).

(d) Change in HA-dCas9, SUZ12, H3K27me3 and total H3 occupancy at Fgf11 and Pax7 before and after dox treatment (day 6) and after subsequent dox washout (day 12) (mean and S.D., n=3 independent dox inductions. P-values: Dox induction Fgf11-A G-tract RNA: SUZ12=0.02, H3K27me3=0.0066. Fgf11-B G-tract RNA SUZ12=0.046, H3K27me3=0.49. Dox washout Fgf11-A G-tract RNA: SUZ12=0.041, H3K27me3=0.0094. Fgf11-B G-tract RNA SUZ12=0.052, H3K27me3=0.091, Welch’s one-tailed t-test).

(e) Top: Fgf11 RNA sequence spanning the first exon-intron junction was appended to Fgf11 sgRNA. Bottom: As (b), except using Fgf11 sgRNA to which the Fgf11 RNA sequence has been appended (mean and S.D., n=3 independent dox inductions. P-values: Fgf11 A SUZ12=2.9x10-4, H3K27me3=0.0019. Fgf11 B SUZ12=0.045, H3K27me3=0.026, Welch’s one-tailed t-test).