A SNAI2/CTCF Interaction is Required for NOTCH1 Expression in Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a pediatric malignancy of the muscle with characteristics of cells blocked in differentiation. is an oncogene that promotes self-renewal and blocks differentiation in the fusion negative-RMS sub-type. However, how expression is transcriptionally maintained in tumors is unknown. Analyses of SNAI2 and CTCF chromatin binding and HiC analyses revealed a conserved SNAI2/CTCF overlapping peak downstream of the locus marking a sub-topologically associating domain (TAD) boundary. Deletion of the SNAI2-CTCF peak showed that it is essential for expression and viability of FN-RMS cells. Reintroducing constitutively activated -ΔE in cells with the SNAI2-CTCF peak deleted restored cell-viability. Ablation of SNAI2 using CRISPR/Cas9 reagents resulted in the loss of majority of RD and SMS-CTR FN-RMS cells. However, the few surviving clones that repopulate cultures have recovered . Cells that re-establish expression after SNAI2 ablation are unable to differentiate robustly as SNAI2 shRNA knockdown cells; yet, -ablated cells continued to be exquisitely sensitive to ionizing radiation. Thus, we have uncovered a novel mechanism by which SNAI2 and CTCF maintenance of a sub-TAD boundary promotes rather than represses expression. Further, we demonstrate that SNAI2 suppression of apoptosis post-radiation is independent of / effects on self-renewal and differentiation.