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Fig. 3 | Genome Biology

Fig. 3

From: Recurrent mutations at estrogen receptor binding sites alter chromatin topology and distal gene expression in breast cancer

Fig. 3

A recurrent intergenic somatic mutation disrupts TF binding and decreases expression of distal genes. a Genomic region of two recurrent somatic mutations and their neighboring genes is shown. Inset shows the number of BRCA-EU patients with mutations in the intergenic locus between the LRRC3C and GSDMA genes. Nucleotide changes for the two recurrent mutations and the relative position of the ER peak (gray shadow) are shown. Relevant tracks (ENCODE) and positions of the sgRNAs used in panel d are also displayed. b Motif scores were calculated with and without each mutation using the PWMEnrich package [57], which performs DNA motif enrichment analysis against databases such as MotifDb. Motif score ratios were displayed as blue and red bars representing higher motif scores with and without the mutation, respectively. Downward black arrows indicate the mutation position within each motif. c EMSA results demonstrate protein binding affinity for WT and mutant oligonucleotides (oligos) with either double or single mutations. 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 Neighboring gene expression levels were assessed by qRT-PCR in MCF-7 cells with CRISPR-dCas9-based interference of control and the mutation sites. All the P values were calculated with two-sided Student’s t test. ***P < 0.001. Error bars represent standard deviations from six biological replicates

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