The transcriptional co-repressor Runx1t1 is essential for N-Myc-driven neuroblastoma tumorigenesis [ATAC-Seq]
Changes in epigenetic regulation are believed to be a major contributing factor to neuroblastoma development. Using a large-scale in vivo mutagenesis screen in Th-MYCN transgenic mice, we identified a single point mutation in the transcriptional corepressor Runx1t1, that can block N-Myc-driven neuroblastoma tumorigenesis. The loss of function mutation disrupts a highly conserved zinc finger domain (NHR4) within Runx1t1. Crossing an independent Runx1t1 knockout model with Th-MYCN mice, demonstrated that Runx1t1 haploinsufficiency is enough to prevent neuroblastoma development and reverse ganglia hyperplasia. Silencing RUNX1T1 in human neuroblastoma cells resulted in decreased colony formation in vitro, and significant inhibition of tumor growth in vivo. Our results show that RUNX1T1 forms part of a transcriptional LSD1-CoREST3-HDAC repressive complex that regulates the epigenomic landscape and chromatin accessibility, to control neuron-specific pathway genes and maintain an undifferentiated state. Runx1t1 thus represents an entirely novel and highly promising target not previously described in neuroblastoma. Overall design: KELLY cells containing shRNA to Runx1t1 were treated with doxycycline, or control, to induce knock-down. After 72 hours, 50,000 cells were collected in duplicate per condition to perform ATAC-sequencing based on methods previously described (Buenrostro, Wu et al. 2015). Briefly, cells were lysed to prepare the nuclei pellet, followed by Tn5 tagmantation using the Nextra kit (Illumina), according to the manufacturer's instructions. Transposed reaction was cleaned with a DNA Clean & Concentrator-5 kit (Zymo, cat# D4014). The transposed DNA was amplified using NEBNext High-Fidelity 2x PCR Master Mix (NEB), with SYBR Green I dye (Invitrogen) and Nextera primers Ad1 and Ad2 at 25 µM. The number of PCR cycles were determined and optimised for 8 cycles. Finally, amplified libraries were purified using the DNA Clean & Concentrator-5 kit  (Zymo), according to manufacturer's instructions. An additional cleaning step was performed using Ampure XP beads, to remove primer dimers and large fragments >1,000 bp. The quality of purified libraries was assessed using the 2200 TapeStation (Agilent). The final concentration of the libraries was 2nM and the sequencing was performed with Illumina NovaSeq 6000 S1 pair-end 100bp reads to obtain average depth of 100 million reads per sample.