Fig 2: NOTCH1 signalling represses SAHF formation partially by repressing HMGA proteins. a, b qRT-PCR (n = 6) (a) and immunoblotting (b) for the indicated mRNA and proteins in IMR90 ER:HRASG12V cells stably infected with control vector or N1ICD-FLAG ± 100 nM 4OHT for 6 days. c, d qRT-PCR (n = 4) (c) and immunoblotting (d) of IMR90 cells expressing doxycycline (DOX)-inducible N1ICD-FLAG (iN1ICD) and infected with a mVenus control vector or dnMAML1-mVenus ± 1000 ng/mL DOX for 3 days. e Quantification of SAHFs in IMR90 cells expressing ER:HRASG12V, iN1ICD and EGFP or an EGFP-HMGA1 fusion ± 100 nM 4OHT for 6 days and ±1000 ng/mL DOX for 3 days. f qRT-PCR for the indicated mRNA in IMR90 cells expressing rThy1-mRFP and co-cultured with RPE1 cells expressing mVenus or JAG1-mVenus, isolated using MACS (n = 3). Statistical significance calculated using one-way ANOVA with Tukey’s correction for multiple comparisons (a, c) or two-sample t-test (e, f). *p = 0.05, **p = 0.01, ***p = 0.001

Fig 3: Tumour cells can repress SAHFs and chromatin opening in adjacent RIS fibroblasts. a Normalised mRNA expression values from the Cancer Cell Line Encyclopedia (CCLE) and immunoblotting of JAG1 in the tumour cell lines indicated. b Quantification of SAHFs in IMR90 ER:HRASG12V cells expressing mRFP co-cultured with the tumour cell lines indicated for 6 days +100 nM 4OHT ± 10 µM DAPT (left) and representative images (right) (scale bar = 25 µm). n = 3 biological replicates except for Hep3B cultures where n = 6. c SAHF quantification in IMR90 ER:HRASG12V cells expressing mRFP+100 nM 4OHT±10 µM DAPT. d Schematic showing experimental set-up. IMR90 ER:HRASG12V cells expressing mRFP were cultured with tumour cell lines +100 nM 4OHT before flow sorting to isolate red cells. e qRT-PCR of mRNA isolated from the cells described in c. n = 3 biological replicates. f Immunoblotting of JAG1 in the cells indicated. g Quantification of SAHFs in IMR90 ER:HRASG12V expressing mRFP co-cultured with the tumour cells indicated for 6 days +100 nM 4OHT. n = 3 biological replicates. h Volcano plots showing regions of altered accessibility in RIS cells co-cultured with MCF7, A549 and Hep3B cells (as in c) relative to RIS cells cultured alone. Regions that also become more accessible in RIS (red) and NIS (purple) vs. growing are indicated where numbers indicate the total number of significant alterations (log2 fold change <-0.58 or >0.58 and FDR < 0.01) and <-1 indicates the number with a log2 fold change of <-1. i Number of more accessible regions in RIS (identified in Fig. 4c) that are repressed by co-culture with MCF7, A549 and Hep3B. b, c, e, g Statistical significance calculated using one-way ANOVA with Tukey’s correction for multiple comparisons; *p = 0.05, **p = 0.01, ***p = 0.001, NS = not significant

Fig 4: NOTCH1 signalling mediates non-cell autonomous regulation of chromatin structure at the microscopic and nucleosome scale. Lateral induction of NOTCH1 activity in a signal-receiving cell by JAG1 on the surface of an adjacent cell (including cancer cells) can drive NIS. NIS cells form unique chromatin-accessible regions and microscopically ‘smoothened’ chromatin. In the context of RIS, non-cell autonomous activation of NOTCH1 signalling can repress the formation of AT-rich RAS-driven accessible regions at the nucleosome level and SAHF formation at the microscopic level. Mechanistically, N1ICD represses HMGA1, which is responsible for SAHF formation and at least partially for the formation of ectopic-accessible chromatin in RIS cells