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

Abstract

Rhabdomyosarcoma (RMS) is a pediatric malignancy of the muscle with characteristics of cells blocked in differentiation. NOTCH1 is an oncogene that promotes self-renewal and blocks differentiation in the fusion negative-RMS sub-type. However, how NOTCH1 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 NOTCH1 locus marking a sub-topologically associating domain (TAD) boundary. Deletion of the SNAI2-CTCF peak showed that it is essential for NOTCH1 expression and viability of FN-RMS cells. Reintroducing constitutively activated NOTCH1 -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 NOTCH1 . Cells that re-establish NOTCH1 expression after SNAI2 ablation are unable to differentiate robustly as SNAI2 shRNA knockdown cells; yet, SNAI2 -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 NOTCH1 expression. Further, we demonstrate that SNAI2 suppression of apoptosis post-radiation is independent of SNAI2 / NOTCH1 effects on self-renewal and differentiation.