Molecular and Cellular Biology: A SNAI2/CTCF Interaction is Required for NOTCH1 Expression in Rhabdomyosarcoma (Ignatius Lab)
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
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Since 2004, UT Health San Antonio, Greehey Children’s Cancer Research Institute’s (Greehey CCRI) mission has been to advance scientific knowledge relevant to childhood cancer, contribute to understanding its causes, and accelerate the translation of knowledge into novel therapies. Greehey CCRI strives to have a national and global impact on childhood cancer by discovering, developing, and disseminating new scientific knowledge. Our mission consists of three key areas — research, clinical, and education.
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Since 2004, UT Health San Antonio, Greehey Children’s Cancer Research Institute’s (Greehey CCRI) mission has been to advance scientific knowledge relevant to childhood cancer, contribute to understanding its causes, and accelerate the translation of knowledge into novel therapies. Greehey CCRI strives to have a national and global impact on childhood cancer by discovering, developing, and disseminating new scientific knowledge. Our mission consists of three key areas — research, clinical, and education.
Stay connected with the Greehey CCRI on Facebook, Twitter, LinkedIn, and Instagram.