Fig. 4

A recurrent non-coding somatic mutation at the ZNF143 locus affects TF binding, 3D chromatin architecture and expression of multiple distal genes. a Genomic region of the recurrent mutation at the ZNF143 promoter and the neighboring genes is shown. Inset shows the number of BRCA-EU patients with mutations around the ZNF143 promoter. The sequence flanking the C to T mutation and the relative position of the ER peak (gray shadow) to the mutations are shown. Relevant ENCODE sequencing tracks are also displayed. b Motif score ratios were calculated between genomic sequences with and without the mutation. Blue bars indicate higher motif scores with the mutation, thus motif created; red bars represent higher motif scores without the mutation, thus motif disrupted. Downward black arrows indicate the mutation position within each motif. c EMSA results demonstrate protein binding affinity for WT and mutant (with the C>T mutation; Mut) oligonucleotides. The three lanes for each case are biotin-labeled oligos only, biotin-labeled oligos plus nuclear extract, and biotin-labeled oligos plus nuclear extract and competitor probes from left to right. Competitor probes are unlabeled oligos to examine DNA-protein binding specificity. Non-specific interactions are labeled as “n.s.”. d Schematic representation of the CRISPR base editor approach to introduce the C to T mutation into MCF-7 cells. qPCR was utilized to screen genomes of more than 400 single cell colonies to detect the specific mutation. e Sanger sequencing results show the genomic sequences at and around the mutation site in WT and two mutant (Mut) MCF-7 clones. f ChIP-qPCR analysis shows ZBTB7A enrichment at the mutation site in MCF-7 WT cells and a mutant clone. Error bars represent standard errors of four independent data points (biological replicates). The P value was calculated using one-sided Student’s t test. g qRT-PCR results show relative mRNA levels of genes that are topologically or spatially associated with the mutant site in WT and mutant MCF-7 clones. Error bars represent standard deviations from 11 biological replicates. h Bar graphs show contact frequency between the mutated site and the four other proximal sites in WT and mutant MCF-7 cells as measured by the Chromatin Conformation Capture (3C) assay. Interacting sites from the MCF-7 Pol2 ChIA-PET data are colored in magenta. Hypothetical interaction with the control site is indicated with a gray dash line. The blue boxes at the end of the interaction curves indicate the primer positions used in the 3C assay. Error bars represent standard deviations (2 biological replicates). i Crystal violet colony formation assay measures the relative size and viability of colonies for WT and mutant MCF-7 cells in response to control, estradiol (E2) and tamoxifen (Tam.) treatment. Images and corresponding quantifications are shown. Error bars represent standard deviations from 12 biological replicates. All the P values were calculated with two-sided Student’s t test unless indicated otherwise. ***P < 0.001, **P < 0.01, *P < 0.05