H3.3G34W promotes growth and impedes differentiation of osteoblast-like mesenchymal progenitors in Giant Cell Tumour of Bone [ChIP-Seq]

Glycine 34 to tryptophan (G34W) substitutions in H3.3 arise in ~90% of giant cell tumour of bone (GCT). Here, we show H3.3G34W is necessary for tumour formation. Profiling the epigenome, transcriptome and secreted proteome of patient samples and tumour-derived cells CRISPR/Cas9-edited for H3.3G34W shows that H3.3K36me3 loss on mutant H3.3 induces a shift of the repressive H3K27me3 mark from intergenic to genic regions, beyond areas of H3.3 deposition. This promotes the redistribution of antagonistic chromatin marks and aberrant downregulation of contractile myofibroblast-associated genes altering cell fate in mesenchymal progenitors. Single-cell transcriptomics reveals that H3.3G34W stromal cells recapitulate a neoplastic trajectory from an SPP1+ osteoblast progenitor-like population towards an ACTA2+ myofibroblast population, which secretes extracellular matrix ligands predicted to recruit and activate osteoclasts. Our findings suggest that H3.3G34W leads to GCT by sustaining a transformed state in osteoblast-like progenitors which promotes neoplastic growth, pathological recruitment of giant osteoclasts, and bone destruction. We performed ChIP sequencing of isogenic unedited H3.3G34W and isogenic clones where the mutant allele was KO or repaired to WT by CRISPR/Cas9 from two patient-derived cell lines. We performed ChIP-seq of H3.3, H3.3G34W, H3K27ac, H3K27me3, H3K36me2, H3K36me3, H3K9me3, SUZ12.