Fig. 2

NB-specific genes in CRC potentially induce high expression of SMAD9. A, B SEdb analysis showing the highest number of SE counts in NB (A) and other cancer types described above in terms of rank (B). C Gene track showing NB-specific binding signals in the SMAD9 SEs region based on SEdb. D Gene track showing high binding signals for TFs in CRC as well as H3K27ac in the SMAD9 enhancer region. Sg-1#, 2# and 3# indicate the sgRNA primers based on the locations for subsequent CRISPRi experiments. E Matrix showing the gene-gene correlation value among MYCN, GATA3, PHOX2B, HAND2 and SMAD9 expression in NB cells in the DepMap portal. F, G Q-RT-PCR analyses of TF genes in CRC and SMAD9 after knockdown of one of these TFs in BE(2)-C (F) and SK-N-BE2 (G) cells. H Q-RT-PCR analyses of SMAD9 with disrupted binding of the CRC enhancer region to SMAD9 using the CRISPRi system in MYCN-amp dCas9 STCs. I, J Cell viability on day 6 (I), colony formation (J; left panel) and quantification (J; right panel) of MYCN-amp dCas9 STCs with disrupted binding of the CRC enhancer region to SMAD9. CRC: core regulatory circuit; dCas9: dead Cas9; GBM: glioblastoma multiforme; MYCN-amp: MYCN amplification; NB: neuroblastoma; SEs: super-enhancers; SEdb: super-enhancers database; shSCR: shRNA scrambled control; sgSCR: sgRNA scrambled control; STCs: stably transfected cells. *P < 0.05, **P < 0.01 and *** P < 0.001