Investigating Epigenomic Features of Paediatric Glioblastoma Dependent on the Histone 3 K27M Mutation
1. Department of Human Genetics, McGill University, Montreal, Quebec, Canada; 2. Research Institute of McGill University Health Centre, Montreal, Quebec, Canada; 3. Department of Pediatrics, McGill University, Montreal, Quebec, Canada; 4. Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada
Paediatric glioblastoma is a malignant brain tumour that remains incurable. A subgroup of this disease is defined by the mutation of lysine 27 to methionine (K27M) in one of several histone 3 genes. As an important site of acetylation and methylation for epigenetic writer and reader proteins involved in control of gene expression, the K27M mutation is found in tumours with specific epigenomic features that distinguish them from histone-wild-type tumours. Most strikingly, these tumours exhibit massive loss of histone 3 lysine 27 trimethylation (H3K27me3) via a dominant-negative effect on polycomb repressive complex 2 that is responsible for the deposition of this mark.
With the CRISPR-Cas9 genome editing technique, we have introduced the K27M mutation into the endogenous locus of several histone 3 genes in the cell line HEK-293T. We have also introduced deletion mutations into the endogenous K27M allele of primary patient-derived brain tumour cells, rendering it functionally null. Through immunoblot and chromatin immunoprecipitation with next generation sequencing (ChIP-seq), we see the global loss of H3K27me3 accompanied by experimental introduction of K27M in both histone-WT glioblastoma and the unrelated lineage, HEK-293T. The removal of endogenous K27M from the relevant tumour cell context reciprocally results in genome-wide gain in H3K27me3. The resulting changes in gene expression upon loss of K27M inform of the influence of H3K27me3 on transcriptional regulation. These results demonstrate the dependence of H3K27me3 loss on the presence of the K27M mutation in this disease, and not other contributors such as a tumour’s cell-of-origin.