Prethish Sreenivas,Long Wang,Meng Wang,Anil Challa,Paulomi Modi,Nicole Rae Hensch,Berkley Gryder,Hsien-Chao Chou,Xiang R. Zhao,Benjamin Sunkel,Rodrigo Moreno-Campos,Javed Khan,Benjamin Z. Stanton & Myron S. Ignatius
Abstract
Rhabdomyosarcoma (RMS) is a pediatric malignancy of the muscle with characteristics of cells blocked in differentiation. NOTCH1 is an oncogene promoting self-renewal and blocking 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 the 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 continue 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.
Keywords: Rhabdomyosarcoma, SNAI2, CTCF, NOTCH1, Notch, Chromatin, HiC, ChIP-seq, CRISPR/Cas9, gene regulation