ATRX (Alpha thalassemia/mental retardation X-linked) complexes with DAXX to deposit histone variant H3.3 into repetitive heterochromatin. A number of studies have shown that specific point mutations in H3.3, ATRX and DAXX are initiating events in a range of cancers including brain cancers, pancreatic neuroendocrine tumours, chondroblastomas and osteosarcomas. In these cancers, ATRX mutations are linked to an Alternative Lengthening of Telomeres (ALT) phenotype. ALT is an aberrant DNA recombination mechanism that drives telomere DNA elongation independent of telomerase activity and is found in 15% of all cancers. Here we investigate chromatin defects associated with H3.3 mutation (a G34R substitution) and ATRX knockout in embryonic stem cells (ESCs).
We introduced a single-copy H3.3 G34R mutation in ESCs. The expression of H3.3 G34R mutant was found to lead to increases in H3K36me3 and H3K9me3 profiles across the genome which were comparable to a KDM4A/B/C triple-knockout. Our study illustrates that histone point mutations can exert a whole-genome effect to promote cancer formation. To study the role of ATRX in tumourigenesis, we knocked out the Atrx gene in ESCs. We showed that depletion of ATRX in mouse embryonic stem cells led to repeat instability and copy loss in ribosomal RNA gene (rDNA). Supporting this, ALT positive human sarcoma tumour samples show a substantially lower rDNA copy than ALT negative samples. Further investigation shows that the rDNA instability is caused by a disruption in H3.3 deposition and thus, a failure in heterochromatin formation in the absence of ATRX. In addition, ATRX-depleted cells are reduced in rRNA transcription output, and show increased sensitivity to RNA polymerase I (Pol I) transcription inhibitor CX5461. Our study provides insights into the contribution of ATRX loss of function to tumourigenesis through the loss of rDNA stability, and suggests the therapeutic potential of targeting Pol I transcription in ALT cancers.