PRC2-indepdendent actions of H3.3K27M in embryonic stem cell differentiation

The histone H3 variant, H3.3, is localized at specific regions in the genome, especially promoters and active enhancers, and has been shown to play important roles in development. A lysine to methionine substitution in position 27 (H3.3K27M) is a main cause of Diffuse Intrinsic Pontine Glioma, a lethal type of pediatric cancer. H3.3K27M has a dominant-negative effect by inhibiting the Polycomb Repressor Complex 2 (PRC2) activity. Here, we studied the immediate, genome-wide, consequences of the H3.3K27M mutation independent of PRC2 activity. We developed Doxycycline (Dox)-inducible mouse embryonic stem cells (ESCs) carrying a single copy of WT-H3.3, H3.3K27M and H3.3K27L, all fused to HA. We performed RNA-Seq and ChIP-Seq at different timepoints following Dox in undifferentiated and differentiated ESCs. We find increased binding of H3.3 around transcription start sites in cells expressing both H3.3K27M and H3.3K27L compared with WT, but not in cells treated with PRC2 inhibitors. Differentiated cells carrying either H3.3K27M or H3.3K27L retain expression of ESC-active genes, in expense of expression of genes related to neuronal differentiation. Taken together, our data suggest that a modifiable H3.3K27 is required for proper histone incorporation and cellular maturation, independent of PRC2 activity. We generated Doxycycline (Dox)-inducible KH2 FLIP-in mouse ESCs expressing a single copy of WT, K27M or K27L SNAP-HA-H3.3. We performed RNA-seq and ChIP-seq using HA-specific antibodies, H3K27me3-specific antibodies, and H3K27ac-specific antibodies at 4,8,24,72 hrs following Dox addition. We also differentiated our ESCs using retinoic acid and performed RNA-seq and ChIP-seq using HA-specific antibodies at 24 and 72 hrs following Dox.