The Combinatorial Epigenetic Landscape of High Grade Pediatric Gliomas in Single Molecule Resolution

Cell identity in healthy tissues, and during malignant transformation, is governed by the dynamics of chromatin modifications, which expand and diversify phenotypes driven by the genetic code. At the base of chromatin structure are the nucleosomes; histone octamers around which double-stranded DNA is wrapped. Aberrant patterns of DNA and histone modifications are strongly associated with tumorigenesis. Recently, also recurrent mutations in genes encoding the histones themselves were discovered, predominantly within the context of pediatric cancers. Diffuse Midline Pediatric Gliomas are a prominent example of highly aggressive tumors, in which a point mutation in one of the genes encoding H3 results in chromatin dysregulation and alterations in gene expression. Despite the great effort made in recent years to understand the underlying mechanisms by which this mutation drives disease initiation and progression, a comprehensive understanding of the network governing the epigenetic aberrations in these tumors is still lacking.

We applied high resolution, single-molecule imaging on H3 mutant pediatric glioma cells to decode various histone marks on individual nucleosomes, with the aim to decipher the combinatorial epigenome of these tumors. The sensitive and quantitative nature of our method allows us to detect multiple epitopes on individual nucleosomes, and examine the interaction between different histone modifications and the mutant histone itself, in unprecedented resolution and high throughput. Our results point toward specific molecular mechanisms that may drive transcriptional aberrations in these tumors and set the foundation for addressing the ability of different therapeutic agents to modulate the combinatorial epigenetic landscape of these cells.