Histone H3.3 mutations are a hallmark of pediatric gliomas, but their core 
oncogenic mechanisms are not well-defined. To identify major effectors, we used 
CRISPR-Cas9 to introduce H3.3K27M and G34R mutations into previously 
H3.3-wildtype brain cells, while in parallel reverting the mutations in glioma 
cells back to wildtype. ChIP-seq analysis broadly linked K27M to altered 
H3K27me3 activity including within super-enhancers, which exhibited perturbed 
transcriptional function. This was largely independent of H3.3 DNA binding. The 
K27M and G34R mutations induced several of the same pathways suggesting key 
shared oncogenic mechanisms including activation of neurogenesis and NOTCH 
pathway genes. H3.3 mutant gliomas are also particularly sensitive to NOTCH 
pathway gene knockdown and drug inhibition, reducing their viability in culture. 
Reciprocal editing of cells generally produced reciprocal effects on 
tumorgenicity in xenograft assays. Overall, our findings define common and 
distinct K27M and G34R oncogenic mechanisms, including potentially targetable 
pathways.