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

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

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)547-565
Number of pages19
JournalMolecular and cellular biology
Volume43
Issue number11
DOIs
StatePublished - 2023

Keywords

  • CRISPR/Cas9
  • CTCF
  • ChIP-seq
  • Chromatin
  • HiC
  • NOTCH1
  • Notch
  • Rhabdomyosarcoma
  • SNAI2
  • gene regulation

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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