Supplementary Figure 6: G-tract RNA tethering mimics dynamic changes in PRC2 gene occupancy.
From: G-tract RNA removes Polycomb repressive complex 2 from genes
(a) Immunoblotting for HRasV12 in parental NIH/3T3 cells and in cells stably expressing HRasV12. (b) Change in NIH/3T3 cell morphology upon expression of HRasV12. Scale bars indicate 400 μm. (c) Change in expression of Adcy7, Sorcs2 and Smad6 nascent unspliced RNA (top) and mature spliced mRNA (bottom) upon expression of HRasV12 relative to nascent or mature Actb mRNA, respectively. Nascent RNA: Adcy7 P=0.01, Sorcs2 P=0.005, Smad6 P=0.04. mRNA: Adcy7 P=0.005, Sorcs2 P=0.0003, Smad6 P=0.002). (d) HA-dCas9, SUZ12, H3K27me3 and total H3 occupancy (as % input, with non-specific IgG control) at Adcy7, Sorcs2 and Actb before and after induction of dCas9 in cells expressing sgRNA targeted to Adcy7 and appended with either G-tract or A-tract RNA. Representative data from a single set of ChIP experiments are shown (mean and S.D. from 3 technical replicates). Data from 3 independent experiments are shown in Fig. 5d. (e) As D, except in cells expressing sgRNA to Sorcs2. Data from 3 independent experiments are shown in Fig. 5e. (f) As D, except in cells expressing HRasV12 and sgRNA targeted to Smad6. Data from 3 independent experiments are shown in Fig. 6c. (g) Spliced (mRNA) and unspliced (pre-mRNA) Smad6 RNA, relative to spliced or unspliced Actb RNA, respectively, in HRasV12 cells expressing sgRNAs specific Smad6 before and after induction of dCas9 expression (mean and S.D., n=3).
