Abstract
While comprehensive molecular profiling of histone H3.3 mutant pediatric high-grade glioma has revealed extensive dysregulation of the chromatin landscape, the exact mechanisms driving tumor formation remain poorly understood. Since H3.3 mutant gliomas also exhibit high levels of copy number alterations, we set out to address if the H3.3K27M oncohistone leads to destabilization of the genome. Hereto, we established a cell culture model allowing inducible H3.3K27M expression and observed an increase in mitotic abnormalities. We also found enhanced interaction of DNA replication factors with H3.3K27M during mitosis, indicating replication defects. Further functional analyses revealed increased genomic instability upon replication stress, as represented by mitotic bulky and ultrafine DNA bridges. This co-occurred with suboptimal 53BP1 nuclear body formation after mitosis in vitro, and in human glioma. Finally, we observed a decrease in ultrafine DNA bridges following deletion of the K27M mutant H3F3A allele in primary high-grade glioma cells. Together, our data uncover a role for H3.3 in DNA replication under stress conditions that is altered by the K27M mutation, promoting genomic instability and potentially glioma development.
Highlights
Pediatric high-grade glioma (HGG) is a common childhood brain malignancy for which no adequate treatment exists [1]
We discovered that the H3.3K27M oncohistone increases sensitivity to replication stress during S-phase, leading to mitotic aberrancies
We found that histone H3.3 mutant gliomas have a greater degree of copy number variations (CNVs) compared to histone H3.1 mutant gliomas and H3 wild type gliomas (Fig 1A and S1 Table), with H3.3G34R/V HGG being more aneuploid than H3.3K27M HGG
Summary
Pediatric high-grade glioma (HGG) is a common childhood brain malignancy for which no adequate treatment exists [1]. Several functional studies have confirmed the tumor-driving capacity of the oncohistones and revealed their impact on chromatin modifying proteins, chromatin composition and gene expression [7,8,9,10,11,12,13,14]. This is in line with the apparent selective pressure to mutate residues that either are, or flank epigenetic modification sites involved in gene regulation
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