Extended Data Fig. 13: NR3C1 binding is abrogated by specific BCL2-SE mutations in the LY10 cell line.
From: Super-enhancer hypermutation alters oncogene expression in B cell lymphoma
a. NR3C1 ChIP–qPCR in wild-type (TMD8, top) and mutant (LY10, bottom) DLBCL cell lines, using primers encompassing the BCL2-SE hotspot, the known NR3C1 target NFKBIA, and a control non-target region (mean +/− SD; n = 3 technical replicates from one representative experiment out of 2 that gave analogous results; one-way ANOVA with Bonferroni correction). Data are expressed as % of input normalized on control IgG IP. b. Schematic of the BCL2 locus, with the hypermutated SE shown in red, and the primers used in the NR3C1-ChIP–PCR approximately positioned below the map. c. Gel electrophoresis of NR3C1 ChIP–PCR amplicons from the LY10 cell line, as compared to input and control IgG ChIP (data shown are from one representative experiment out of 2 independent experiments that gave analogous results). Band quantification was obtained by densitometry and the relative values are provided below the image, with input set as 1. d. Sequencing analysis of the PCR products shown in c. On the top panel, the reference BCL2 genomic sequence (NM_000624) and the sequence of the three BCL2 alleles (LY10 carries a trisomy 18) are aligned to the predicted NR3C1 binding motif. Sequencing traces of the ChIP–PCR amplicons document that only the wild-type “G” mutant allele is efficiently immunoprecipitated, as compared to the input, documenting abrogation of NR3C1 binding by the two mutations (Sanger sequencing performed with the reverse primer). e. Relative BCL2 expression changes in isogenic clones from the indicated cell lines, colour coded as in Fig 5f (n = 4 each except for LY10, where only 2 corrected clones were recovered, and control neutral clones, were 8 were used to exclude biological variability). For each cell line, the mean value of the unmanipulated clones is arbitrarily set as 1 (two-tailed unpaired t-test). f. Absolute BCL2 mRNA levels in the 3 cell lines used in CRISPR-Cas9 experiments, measured by RNA-seq (LY10, mutated; BJAB and SUDHL5, unmutated). g. Overlap between cases of DLBCL with mutations in the BCL2-NR3C1-BS, BCL2 translocations (Tx), and/or coding mutations in the NR3C1 gene. Data are from 328 cases analysed by WGS or Sanger sequencing. h. BCL2 expression levels in cases of primary DLBCL, stratified based on the presence of the indicated genetic lesions (n = 181 cases with available WGS and RNA-seq data). Data are expressed as TPM, and statistically significant differences were calculated by one-way ANOVA with Bonferroni correction. i. Relative distribution of cases harbouring the indicated genetic lesions in various DLBCL COO subtypes. P-values were calculated by two-tailed Fisher’s exact test to determine specific enrichment of a genetic lesion in each COO group versus the other groups combined (UNC < unclassified; ND, not determined). The total number of cases analysed within each subtype is provided in brackets, and the number of mutated cases is shown on the top. j. Relative distribution of cases harbouring the indicated genetic lesions in LymphGen genetic classes. A two-tailed Fisher’s exact test was used to calculate the enrichment of each genetic lesion in each LymphGen class versus the other classes combined. The total number of cases analysed within each class is provided in brackets, and the number of mutated cases is shown on the top.
