TMOD-15. Native Histone H3.3K27M Sustains Progenitor Identity and Impairs Differentiation to Rewire Neural Development

Abstract The H3.3K27M oncohistone defines a majority of diffuse midline gliomas (DMGs), including diffuse intrinsic pontine gliomas (DIPGs), yet how this mutation reprograms neurodevelopment to create a DMG-susceptible cellular ecosystem remains unresolved. Here, we develop a Cre-inducible knock-in mouse model expressing untagged H3.3K27M to interrogate its developmental impact across the cortex, cerebellum, and pons. To model the effects of H3.3K27M, we generated a knock-in mouse expressing untagged H3.3K27M. In contrast to tagged H3.3K27M system, our model preserves native histone structure. We find that H3.3K27M expression in neural progenitor cells elicits widespread brain malformations, including cortical dyslamination, cerebellar hypoplasia, and motor deficits. H3.3K27M promotes expansion of progenitor populations, impairs neuronal and glial differentiation, and disrupts regional programs governing pontine patterning. These developmental defects are accompanied by global loss of H3K27me3, highlighting a failure of epigenetic repression in neural progenitors. Our findings position H3.3K27M as both an epigenetic disruptor and developmental reprogrammer, establishing a proliferative and stem-like state for DMG initiation. This work uncovers critical windows of vulnerability in brain development that may be leveraged for early therapeutic intervention in pediatric high-grade gliomas.