Fig 2: The core regulatory circuitry of MES cells includes transcription and activation of the NOTCH pathway. a ChIP-sequencing profiles of H3K27ac mark in neural crest cells (NCC, in green), MES neuroblastoma cells (691-MES, SH-EP2, 700-MES, 717-MES, in orange) or ADRN neuroblastoma cells (691-ADRN, SH-SY5Y, 700-ADRN, 711-ADRN, 753-ADRN, in blue). The number of reads per 20 million mapped reads is shown on the y-axis. Chromosomal gene position is shown on the x-axis. Super-enhancers (SE) are indicated by horizontal bars. Gene color depicts orientation on sense (green) and anti-sense (red) DNA strands. b Box plots of mRNA expression for the four MES cell lines and five ADRN cell lines shown in panel a. Whiskers denote the interval within 1.5 times the interquartile range (box edges) of the median (center line). Expression values (2log-transformed) for each cell line are indicated by open circles. Affymetrix mRNA probesets are indicated for each gene. c Western blot analysis of NOTCH1, NOTCH2, NOTCH3, the NOTCH co-factor MAML2 and the NOTCH target gene HES1 in membrane (M) or nuclear (N) lysates. Protein lysates were from MES- and ADRN cell line pairs of isogenic origin, depicted in orange and blue, respectively. IC intracellular domain, FL full-length, TM transmembrane. PARP and α-tubulin were used as loading controls for the nuclear- and membrane fractions, respectively. Source data are provided as a Source Data file

Fig 3: NOTCH-IC paralogues induce a reciprocal feed-forward signaling cascade. a SH-SY5Y cells with inducible overexpression of NOTCH3-IC were profiled for gene expression up to 21 days. Z-scores of mRNA expression values are shown for JAG1, NOTCH1, NOTCH2, NOTCH3, MAML2, and HES1. The transcription of NOTCH receptors from endogenous loci is measured using probesets located in the 3′UTRs of NOTCH1 (probeset 218902_at), NOTCH2 (probeset 202443_x_at) and NOTCH3 (probeset 203238_s_at). Notably, the 3′UTR is absent in the NOTCH3-IC transgene. b. Western blot analysis of NOTCH1, NOTCH2, NOTCH3, MAML2, and JAGGED1 proteins in SH-SY5Y with NOTCH1-IC, NOTCH2-ICFLAG or NOTCH3-IC inducible overexpression. β-actin was used as loading control. Time of induction was 96 h. Source data are provided as a Source Data file. c Western blot analysis of SH-SY5Y cells with (+dox) or without (−dox) NOTCH3-IC expression that were cultured in the presence (+) or the absence (−) of the gamma-secretase inhibitor RO4929097. Protein lysates were analyzed for NOTCH pathway genes (NOTCH1, MAML2, HES1), MES markers (FN1, YAP1, SNAI2) and ADRN markers (PHOX2A, PHOX2B, GATA2, TFAP2B). β-actin was used as loading control. dox, doxycycline. Source data are provided as a Source Data file. d Analysis of MES and ADRN mRNA signature scores in SH-SY5Y cells with NOTCH3-IC induction that were treated with the gamma-secretase inhibitor RO4929097. Control cells (-dox) treated with DMSO or RO4929097 are shown in dark- and light blue, respectively. NOTCH3-IC expressing cells with DMSO or RO4929097 are shown in red and purple, respectively. Cells were induced with doxycycline and treated with RO4929097 for 14 days. Dashed circles indicate groups of MES- or ADRN-type cell lines as previously determined1

Fig 4: NOTCH3-IC induces epigenetic remodeling of super-enhancer landscapes. a Analysis of H3K27ac marked super-enhancer regions (n = 1662) that were previously shown to be associated with MES or ADRN cells in neuroblastoma1. Shown are changes of H3K27ac signal in SH-SY5Y cells with induced expression of NOTCH3-IC (T = 7 days of induction). Red (increased) or blue (decreased) H3K27ac signal of the super-enhancers is depicted by horizontal bars on the genomic regions. Expanded views show the super-enhancer (SE) regions that distinguish MES and ADRN lines (indicated by orange and blue vertical bars, respectively) (right). b, c H3K27ac profiles in SH-EP2, SH-SY5Y and in SH-SY5Y cells with (NOTCH3-IC on) or without (NOTCH3-IC off) 7 days of doxycycline-induced expression of NOTCH3-IC. Shown are examples of b ADRN-specific SE-associated TF genes (ASCL1, PHOX2A) and ADRN lineage genes (DBH, DDC). In c, examples of MES-specific SE-associated TF genes (SMAD3, ELK4, NOTCH2, MAML2) are shown. SEs associated with ADRN- and MES cells are indicated by blue and orange horizontal bars, respectively. Gene color depicts orientation on sense (green) and anti-sense (red) DNA strands. d. Motif analysis on regions with increased H3K27ac signal. Statistical significance of enrichment was calculated using Fisher’s exact test with Bonferroni correction

Fig 5: NOTCH3-IC is a master regulator of ADRN-to-MES reprogramming a. Analysis of mRNA signature scores for MES and ADRN cells in a panel of neuroblastoma cell lines and a time series of NOTCH3-IC induction. Control (-dox) SH-SY5Y-NOTCH3-IC cells are depicted in blue; SH-SY5Y cells with induction of NOTCH3-IC (+dox) are shown in red. Time of NOTCH3-IC induction is indicated in days. Dashed circles group cell lines of MES- or ADRN-type as previously determined1. b Z-score mRNA expression of core super-enhancer associated transcription factors specific for ADRN or MES cells. Time of NOTCH3-IC induction (in days) is indicated above. dox, doxycycline. c Western blot analysis showing proteins of ADRN-markers (PHOX2A, PHOX2B, DBH, and TFAP2B), MES-markers (FN1, SNAI2, YAP1, and SOX9) and members of the NOTCH pathway (JAG1, NOTCH1, NOTCH2, NOTCH3, MAML2, and HES1) in SH-SY5Y with inducible overexpression of NOTCH3-IC. Time of NOTCH3-IC induction is indicated above the western blots. Protein lysates from 691-MES and 691-ADRN cells are loaded on the same blot as a reference for MES and ADRN states, respectively. β-actin was used as loading control. dox, doxycycline. Source data are provided as a Source Data file