Nancy S. Lee, Sarah F. Clatterbuck Soper & Paul Meltzer

• Submitter: Nancy Lee (Student or postbac)
• email: leenas@nih.gov

The alternative lengthening of telomeres (ALT) is an aberrant telomere maintenance mechanism which occurs in the absence of telomerase. ALT is believed to be a DNA template-based repair mechanism characterized by long telomeres of heterogenous lengths, accumulation of extrachromosomal telomeric repeats (ECTRs), and formation of ALT-associated promyelocytic leukemia (PML) bodies. Loss of function of the ATRX-DAXX histone chaperone complex is strongly associated with the ALT phenotype in osteosarcoma and other cancers of mesenchymal origin. However, the exact mechanism in which these mutations lead to ALT activation remains unknown. Here, we establish a model system to observe the early events of ALT acquisition by stably expressing wild-type ATRX fused to a mutant FKBP domain (dTag) in an ALT-positive, ATRX-negative osteosarcoma cell line (U2OS). Using the dTag protein degron system, we achieve rapid, targeted degradation of exogenous ATRX, which reversibly modulates the ALT phenotype. The ablation of ATRX activity on a proteomic level allows to us to transition from the non-ALT to ALT state in a rapid, synchronized manner, which provides a distinct advantage in capturing early molecular drivers of ALT over methods that rely on transcriptional regulation. Characterizing the transcriptional and epigenetic events that initiate ALT is a crucial step in developing informed treatments against ALT-positive osteosarcomas.