The histone variant H3.3 G34W substitution in giant cell tumor of the bone link chromatin and RNA processing

While transcription as regulated by histones and their post-translational modifications have been well described, the connection between histone variants and changes to transcription remains poorly characterized. Potentially important insight into this process is provided by the frequently occurring mutations of H3.3, leading to G34 substitutions in childhood glioblastoma and giant cell tumor of the bone (GCTB). In this study, we have established primary cell lines from GCTB patients and used them to uncover the influence on cellular growth behavior, gene expression, and chromatin compaction. The primary cells with G34W show increased colony formation, infiltration and proliferation, hallmarks in aggressive tumor development. Isogenic cell lines with G34W recapitulated the increased proliferation observed in primary cells. Transcriptomic analysis of primary cells and biopsies suggest that most genes are downregulated, which is also reflected in increased chromatin compaction. We identified components of the spliceosome complex specifically interacting with G34W in established isogenic cell lines with a GFP-tagged H3.3. RNA-sequencing analysis and hybridization-based validations further enforced splicing aberrations. Our data uncover a role in RNA processing and chromatin modulation that is blocked by G34W, potentially driving the tumorigenic process in GCTB. We performed gene expression microarray on 29 samples, of which 27 were GCTB and 2 were control cell lines (mesenchymal stem cell line KM1234 and the hFOB1.19 osteoblast cell line). Collectively, 19 samples were H3.3G34W and 10 were H3.3WT. The samples were run on a HumanHT-12 v4 Expression BeadChip (Illumina).