Histone variant H3.3 is incorporated into DNA by ATRX to facilitate the assembly of DNA repeats, including telomeres and ribosomal repeat DNA (rDNA) into heterochromatin domains. One striking finding in cancer epigenetics has been the identification of H3.3 mutations typically observed in paediatric gliomas: H3.3K27M and H3.3G34R. Importantly, H3.3G34R frequently co-occurs with ATRX mutations, and the loss of ATRX is strongly associated with the activation of the ALT telomere maintenance pathway to enable cell immortality. In gliomas, H3.3G34R disrupts chromatin assembly and promotes ALT by inhibiting the function of H3K9/K36 KDM4B demethylase.
The nucleolus is a subnuclear domain that harbours ribosomal DNA repeats. The rDNA repeats are transcribed by RNA Polymerase I to produce rRNA, a crucial component of the ribosome. Like the telomere, ATRX-mediated H3.3 deposition is required for maintaining DNA stability at rDNA repeats, and the inactivation of ATRX led to rDNA repeat loss and instability causing reduced ribosomal RNA transcript output and sensitivity to RNA Polymerase I inhibition.
Here, we examine how and if co-driver mutations that co-occur with ATRX mutations in gliomas (H3.3G34R/K27M) could also affect the formation of rDNA chromatin, and organization of its “phase separated” nucleolar domain. We show that, like ATRX loss, H3.3 mutations also affect rDNA copy number, rRNA transcription, along with a failure to form a proper nucleolar domain. In addition, H3.3 mutations limit the capacity of rRNA synthesis by RNA Polymerase I, and as a result, these cells show an increased sensitivity to RNA Polymerase I inhibition. Combined, this study indicates that ATRX/H3.3 dependent chromatin assembly pathway is required for a functional nucleolar subnuclear domain and the inactivation of this pathway compromises rRNA transcription output. As a result, RNA polymerase I inhibitors can be used as a potential therapeutic treatment towards H3.3 and ATRX-mutated gliomas.