Genetics and Epigenetics combine to deadly effect in Pediatric Glioblastoma.


As I’ve mentioned several times before, cancer involves a combination of genetic and epigenetic changes that result in alterations of cell signalling and gene expression patterns that go on to establish the hallmarks of cancer.

The recent availability of a wealth of mutational data has led to the identification of recurrent mutations in multiple genes that read, write and modify chromatin marks, serving to highlight a direct link between cancer genetics and epigenetics [1] . In most of these cases though, we’ve observed changes in the enzymes that mediate epigenetic processes, ranging from mutation to amplification/overexpression/silencing.

Enzymes always have substrates, and perhaps not altogether surprisingly, a very recent discovery found mutations in the primary substrate of EZH2 [2], which trimethylates lysine 27 of the histone tail of histone H3, which represses gene expression, in paediatric glioma.

Histone 3 is one of the four core histones that comprise the nucleosomes round which DNA is wrapped, and in humans there are two genes that produce a variant of Histone 3, called Histone 3.3, and the authors of this paper found mutations that converted lysine 27 in one of those genes to a methionine. The mutations were heterozygous (the other H3k27 was intact) and so they consequently went on to investigate what the mutation did to H3k27 epigenetic modifications in neurospheres they established from patient tissue, compared to adult glioma and a normal neural cell line of the same differentiation status.

They found global reductions in dimethylated and trimethylated H3k27, and subsequent evidence that this was not attributable to changes in the levels of Ezh2 and Suz12, which are components of the PRC2 complex that mediates H3k27 methylation and silencing. Much to their surprise, they note other H3 marks, including H3k27 acetylation, didn’t differ significantly, which is in direct opposition to findings in [3], even if that was based on mutant Ezh2 as opposed to a mutant histone.

They then had to confirm the reduction was actually due to the mutated histone variant, and to do this they expressed the mutant gene in 293T cells at very low levels, and also established cell lines that contained another previously known histone 3 mutation, albeit not at lysine 27 (K27), and also a version of H3.1 that had a K27 mutation. They again found dramatic reductions in H3k27me3 levels, and were able to validate these results in human astrocyte cultures and in murine embryonic fibroblasts, suggesting a tissue independent mechanism of reduction of H3k27me3 levels was at work.

The introduction of H3.1 and H3.3 K27 mutants is associated with global reductions in H3k27me3 and me2 levels by western blotting and fluorescent microscopy. Transfection experiments into MEF’s reveals reductions associated solely with k27 mutants and also that this is gradual (bar graph and fluorescent micrographs at the bottom) .

They then carried out ChIP-seq and gene expression experiments to understand what altered levels of H3k27me3 did to the gene expression profiles of the mutant cell lines they had developed, and by doing ChIP-seq on both Ezh2 and H3k27me3, they observed that there were no significant reductions in Ezh2 peaks, but there were regions, compared to normal neural stem cells, where the local levels of H3k27me3 were higher than in neural stem cells.  They confirmed this using ChIP-qPCR on two other patient samples and also found that the peaks (indicating maximum binding/concentration of H3k27me3 and/or Ezh2 based on what they’d done ChIP on) identified a strong overlap in the mutant cells but not in normal neural stem cells, suggesting enrichment in those areas might be down to the mutant histones trapping Ezh2.

In order to confirm Ezh2 was actually co-localising more with the mutant histone than the wild-type histone, they pulled all three down with specific antibodies to check if more Ezh2 was pulled down with the mutant histone, and indeed, this is exactly what they found.

Genes that had gained H3k27me3 specifically in mutant glioma cell lines were found to be associated with H3k4me3 as well, marking what are commonly known as “bivalent” genes, that have both activating and repressive chromatin marks and are poised to swing either way, and are responsible for driving tissues to mature and differentiate  [4]. They found using RNA-seq that the expression of these genes was far lower than normal stem cells. They do admit that adult neuronal stem cells are far from the ideal controls to use, and that transfecting proper neurons with mutant histones would help consolidate findings further.

Finally, they conclude that the lack of histone mutations in adult glioma might have to do with the context in which paediatric and adult gliomas develop, with the former being in context of a developing brain very early in life.

So, yeah, I think that is a cool paper.



Ankur Chakravarthy.


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