ATRX is a member of the SWI/SNF family of helicase/ATPases. Mutations in ATRX result in developmental abnormalities including a-thalassaemia severe mental retardation, facial dysmorphism and urogenital abnormalities. A number of studies have shown that ATRX acts together with Death-domain associated protein (DAXX) as a histone chaperone complex for the H3 variant H3.3. The ATRX/DAXX/H3.3 complex is important for heterochromatin assembly at repetitive sequences, such as ribosomal DNA (rDNA), retro-transposons, and pericentric and telomeric repeats.

In ATRX patient cells that the loss of ATRX function gives rise to DNA hypomethylation at rDNA repeats. Here we investigate the role of ATRX in directing H3.3 incorporation and heterochromatin assembly at rDNA repeats in mouse embryonic stem cells (ESCs). Gene knockout (KO) of ATRX results in a significant loss of H3K9me3 and H4K20me3 at rDNA repeats in ESCs. This loss of heterochromatin maintenance at rDNA is accompanied with changes in the binding of UBF (rDNA transcription factor) and ribosomal RNA transcription. Furthermore, we detect a reduction of rDNA copy number in ATRX KO cells. We also investigate the impact of the loss of ATRX on rDNA maintenance in human cancer cells that use ALT (Alternative Lengthening of Telomeres) for telomere DNA elongation.  This study demonstrates the importance of ATRX/H3.3 in controlling heterochromatin formation and genomic stability at rDNA repeats, providing insights into epigenetic defects associated with ATRX mutation and development of ALT cancers.