Extended Data Fig. 4: Allelic imbalance in FGF3 and FGF4 activation.

a, Two heterozygous SNPs in FGF4 (both 3′ UTR) enabled us to evaluate allelic expression in three SDH-deficient GISTs (tumours S6, S1 and S4). Both alleles for each SNP were detected in DNA sequencing data for these tumours, but only one allele was detected in RNA-seq data of tumours S1 and S6, indicative of mono-allelic FGF4 expression. Both alleles are detected in tumour S4, indicating bi-allelic expression of FGF4. b, Heterozygous SNPs in FGF3 exons (both synonymous base substitutions) enabled us to evaluate allelic expression in the SDH-deficient GISTs (tumours S2 and S5). In both cases, DNA sequencing confirmed heterozygosity at the genome level (C/A and T/C, respectively), but RNA-seq data demonstrated mono-allelic FGF3 expression. c, Both alleles of heterozygous SNPs in ANO1 exons were found in the RNA-seq data derived from SDH-deficient GIST samples, confirming bi-allelic expression of ANO1. Similarly, both alleles of heterozygous SNPs were found in the histone H3K27ac ChIP–seq data, confirming the bi-allelic nature of the super-enhancer (not shown). d, One SDH-deficient GIST sample was heterozygous for a SNP (rs386829467) located about 50 bp from the CTCF motif of Peak 2 in the FGF insulator. Allele-agnostic methylation data confirmed 43% methylation of the CTCF peak in this tumour, while essentially no methylation was detected in the SDH-intact tumours (left). Separation of the two alleles using the heterozygous SNP revealed strong allelic bias in the SDH-deficient tumour: one allele was largely unmethylated (~3% methylation), while the other was highly methylated (~75% methylation), consistent with mono-allelic methylation of the CTCF site (right). e, Schematic depicts 4C-seq experimental protocol and primer design for detecting SNPs. DNA elements in close physical proximity are crosslinked and restricted with an enzyme that leaves nucleotide overhangs. These overhangs are then proximity ligated to crosslinked fragments. A second restriction enzyme (with different restriction sites) is then used to circularize the ligated fragments, allowing for inverse PCR. Here we selected restriction enzymes and designed a custom read 2 primer to capture a heterozygous SNP within the super-enhancer. This second read is normally non-informative as contact frequencies are determined through the viewpoint primer (read 1), but in this case enabled us to detect the SNP and assign each ligated fragment to a specific allele. f, The left trace (grey) depicts standard 4C-seq data (allele agnostic), which demonstrates strong interaction between super-enhancer viewpoint and FGF4. However, the SNP covered in the non-viewpoint read enabled us to distinguish interactions involving the minor (top right) or major (bottom right) allele. This revealed that the major allele (purple) is responsible for ~97% of super-enhancer–FGF4 interactions.