Fig. 5: Identification of an Ikaros motif in the MYC BENC enhancer region required for β-catenin-mediated repression of MYC.
a–e, BCR–ABL1 transformed β-catenin(S33;S45)+/fl B-ALL cells were edited with gIkzf1/3 or gNT. Clonal cell lines that are WT or knockout for Ikzf1/3 were generated and transduced with 4-OHT-inducible Cre-ERT2 or ERT2 vectors. Transcriptional targets of Ikzf1, Ikzf3 and β-catenin and changes in histone mark H3K27ac were studied by ChIP-seq in Ikzf1/3 WT (dark-green box) or Ikzf1/3 knockout (white box) cells in the presence (red box) or absence (light-green box) of β-catenin stabilization (GSE196745). a, Venn diagram showing the number of regions bound by β-catenin only (4,356), Ikaros factors only (4,596) or both (11,354). Of 15,710 β-catenin peaks, 11,354 (72.2%) were also bound by Ikaros factors. b, ChIP-seq analysis of histone mark H3K27ac is shown as a heat map for the Myc locus, including upstream Myc promoter regions and long-range transcriptional enhancers of Myc in B-ALL cells from β-catenin(S33;S45)+/fl mice. H3K27ac distribution marking active enhancer regions shows that most of the H3K27ac enhancer activity is concentrated in BENC regions in B-ALL cells. In humans, single-nucleotide polymorphisms in this region are associated with increased risk for B-ALL (rs4617118, rs75777619 and rs28665337). c, Enrichment of β-catenin, Ikzf1 and Ikzf3 binding to BENC elements C and D and changes in H3K27ac upon induction of β-catenin (red box) in B-ALL cells with (white box) or without (dark-green box) deletion of Ikaros factors compared to baseline conditions (light-green box). Identification of Ikaros-binding motifs in the BENC-C (m1 and m2) and BENC-D (m3) elements. d, Quantification of H3K27ac signals (mean value of n = 2 cell lines, technical replicates) by ChIP-seq at BENC enhancer regions, other regions with binding of both Ikaros factors and β-catenin (cobound) and all other regions. H3K27ac ChIP was performed with (red box) and without (light-green box) β-catenin accumulation and in the presence (white box) or absence (dark-green box) of Ikaros factor deletion. Box plots display boxes at the first and third quartiles with a line at the median and whiskers from the minimum to maximum data points within 1.5× the interquartile range (BENC, n = 2; cobound, n = 1,043; other, n = 42 peaks). e, ChIP–qPCR analysis for recruitment of NuRD complex components (MTA2 and CHD4) to the BENC-C enhancer region in β-catenin(S33;S45)+/fl B-ALL cells that are WT (dark-green box) or knockout (white box) for Ikzf1/3 under baseline β-catenin (light-green box), following β-catenin stabilization (red box) or deletion of β-catenin (white box). Data represent a pool of six independent experiments. Mean values from independent experiments (n = 3 technical replicates) were used to compare changes in MTA and CDH4 binding upon β-catenin stabilization compared to baseline and β-catenin knockout condition compared to baseline (two-sided unpaired t-test). f, HDR-mediated editing of the BENC-C m1 motif to abrogate binding of Ikzf1 and Ikzf3 by mutating Ikaros core motif GGGAA and generation of an EcoR1 recognition sequence (GAATTC). Following EcoR1 digestion to confirm mutation of the BENC-C m1 motif, single-cell-derived clones were generated and clones carrying the BENC-C m1 motif mutation were identified by Sanger sequencing. g,h, Single-cell-derived clones from β-catenin(S33;S45)+/fl B-ALL cells with WT or mutated BENC-C Ikaros m1 motifs were transduced with vectors expressing Cre-ERT2 or ERT2 along with GFP. g, GFP+ cells were sorted by FACS and western blot analysis was performed to characterize β-catenin and Myc protein levels 1 day after 4-OHT treatment in B-ALL cells carrying intact or mutated BENC-C Ikaros m1 motifs in the presence (Cre-ERT2) or absence (ERT2) of β-catenin stabilization. h, GFP+ cells with intact (WT 1G11) or mutated BENC-C Ikaros m1 motifs (mut 1C12) were mixed with cells with an intact BENC-C Ikaros m1 motif and the effects of β-catenin stabilization were measured in competitive cell culture experiments by flow cytometry. Data depict the means ± s.d. calculated from three independent experiments (n = 3 technical replicates).
