(A) Targeting p300 to the WT1 promoter results in an increase in H3K27ac and a reduction of H3K27me3. Top: a schematic strategy for targeting dCas9-HA-p300-HA fusion proteins to the promoter of WT1. ChIP-PCR analysis of dCas9-HA and dCas9-HA-p300-HA fusion proteins (first panel), H3K27me3 (second panel), Ezh2 (third panel), and H3K27ac (last panel) at the promoter of WT1 was performed using SF8628 cells transfected with indicated combinations of dCas9-HA, dCas9-HA-p300-HA and two different sgRNAs targeting to the WT1 promoter. Data are mean ± SD (N = 3 independent replicates, **p<0.01). (B) The expression of WT1 increases after targeting dCas9-HA-p300-HA to the WT1 promoter. The expression of WT1 was analyzed by quantitative RT-PCR. Data are mean ± SD (N = 3 independent replicates, **p<0.01). (C) SF8628 cell proliferation is inhibited by targeting dCas9-HA-p300-HA to the WT1 promoter. Data are mean ± SD (N = 3 independent replicates, **p<0.01). (D) A model for reduction and retention of H3K27me3 in cells with H3.3K27M mutation. In wild type cells, the PRC2 complex co-localizes with H3K27me3 peaks at the major PRC2 sites. Since these genes are silenced, the levels of H3.3 are low. Poised enhancers contain low levels of H3K27me3, PRC2 complex and H3.3. In H3.3K27M mutant cells, the PRC2 complexes, once recruited to the poised enhancers, are trapped there, likely due to higher concentration of H3.3K27M mutant proteins at poised enhancers. This will lead to a reduction of the PRC2 complex at strong PRC2 sites and a global reduction of H3K27me3. However, the amount of H3.3K27M mutant proteins at major PRC2 sites are low and thus having limited effect on the ability of the PRC2 complex, which is at reduced levels compared to wild type cells, to methylate H3K27.