Figure 5.
Noncoding variants in the intronic enhancer of WT1 disrupt MYB binding and MYB-mediated transcriptional activation of WT1. (A) Noncoding WT1 variants disrupted the binding site of MYB. The binding motif of MYB was determined by the MYB-specific ChIP-seq data for NB4 cells (detailed in supplemental Methods). The lower panel shows patient-derived mutation sequences with mutated nucleotides labeled in red. (B) ChIP-seq tracks showing MYB binding to the WT1 intronic enhancer in NB4 cells (without noncoding WT1 variants). (C) DNA pulldown assays with anti-MYB antibodies and cell lysates from HEK293T cells stably expressing MYB. (D-E) The dual-luciferase assays on the enhancer activity of nonmutated and mutated constructs with or without MYB expression in 293T cells (D) and NB4 cells (E). The firefly luciferase activity was normalized to the renilla luciferase and presented as the ratio relative to the pGL3-SV40 promoter vector. (F) The knockout efficiency of sg-MYB was tested by western blot. (G) MYB knockout significantly reduced the H3K27ac signals of the WT1 enhancer and expression of WT1. MYB binding signals (left), H3K27ac signals (middle), and relative expression of WT1 (right) were determined. (H) The schematic diagram of the single guide RNA (sgRNA) target sites in the H3K27ac ChIP-seq track for NB4 cells. The red box indicates the sgRNA targeting the MYB motif, and the blue boxes indicate control sgRNAs targeting regions surrounding the MYB motif within intron 3. The protospacer adjacent motif (PAM) sequence is shown in the purple frame. The red arrowhead indicates the expected cleavage site. (I) Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated targeted mutagenesis of the MYB motif within the third intronic region of WT1 inhibited WT1 expression. The left panel shows the relative WT1 expression in mutated clones targeted by the indicated sgRNAs and the unedited clones. CRISPR/Cas9-edited sequences were validated by Sanger sequencing. Data are normalized against the mean expression level of the unedited clones and are plotted as means plus or minus SD (n = 4). The middle panel shows the edited genomic sequences from CRISPR/Cas9-edited single-cell clones. The right panel shows the mRNA levels of WT1 expression in parental and CRISPR/Cas9-edited clones. Data are represented as the fold change relative to the expression of the parental cells and are plotted as means plus or minus SD (n = 3). (J) Disruption of the MYB motif resulted in the loss of MYB binding at the WT1 enhancer region. ChIP–quantitative polymerase chain reaction (qPCR) assays were performed to detect MYB binding using an antibody specific for MYB in the representative MYB motif-mutated clones and the parental cells. Data are calculated as the percentage of input and are plotted as means plus or minus SD (n = 3). The statistical significance was determined using a 2-tailed Student t test. *P < .05; **P < .01; ***P < .001. IgG, immunoglobulin G; ns, not significant; si-NC, negative control small interfering RNA (siRNA); si-MYB, siRNA targeting MYB; pklv-vector, sgRNA empty vector control; sg-MYB, sgRNA targeting MYB; SD, standard deviation.

Noncoding variants in the intronic enhancer of WT1 disrupt MYB binding and MYB-mediated transcriptional activation of WT1. (A) Noncoding WT1 variants disrupted the binding site of MYB. The binding motif of MYB was determined by the MYB-specific ChIP-seq data for NB4 cells (detailed in supplemental Methods). The lower panel shows patient-derived mutation sequences with mutated nucleotides labeled in red. (B) ChIP-seq tracks showing MYB binding to the WT1 intronic enhancer in NB4 cells (without noncoding WT1 variants). (C) DNA pulldown assays with anti-MYB antibodies and cell lysates from HEK293T cells stably expressing MYB. (D-E) The dual-luciferase assays on the enhancer activity of nonmutated and mutated constructs with or without MYB expression in 293T cells (D) and NB4 cells (E). The firefly luciferase activity was normalized to the renilla luciferase and presented as the ratio relative to the pGL3-SV40 promoter vector. (F) The knockout efficiency of sg-MYB was tested by western blot. (G) MYB knockout significantly reduced the H3K27ac signals of the WT1 enhancer and expression of WT1. MYB binding signals (left), H3K27ac signals (middle), and relative expression of WT1 (right) were determined. (H) The schematic diagram of the single guide RNA (sgRNA) target sites in the H3K27ac ChIP-seq track for NB4 cells. The red box indicates the sgRNA targeting the MYB motif, and the blue boxes indicate control sgRNAs targeting regions surrounding the MYB motif within intron 3. The protospacer adjacent motif (PAM) sequence is shown in the purple frame. The red arrowhead indicates the expected cleavage site. (I) Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated targeted mutagenesis of the MYB motif within the third intronic region of WT1 inhibited WT1 expression. The left panel shows the relative WT1 expression in mutated clones targeted by the indicated sgRNAs and the unedited clones. CRISPR/Cas9-edited sequences were validated by Sanger sequencing. Data are normalized against the mean expression level of the unedited clones and are plotted as means plus or minus SD (n = 4). The middle panel shows the edited genomic sequences from CRISPR/Cas9-edited single-cell clones. The right panel shows the mRNA levels of WT1 expression in parental and CRISPR/Cas9-edited clones. Data are represented as the fold change relative to the expression of the parental cells and are plotted as means plus or minus SD (n = 3). (J) Disruption of the MYB motif resulted in the loss of MYB binding at the WT1 enhancer region. ChIP–quantitative polymerase chain reaction (qPCR) assays were performed to detect MYB binding using an antibody specific for MYB in the representative MYB motif-mutated clones and the parental cells. Data are calculated as the percentage of input and are plotted as means plus or minus SD (n = 3). The statistical significance was determined using a 2-tailed Student t test. *P < .05; **P < .01; ***P < .001. IgG, immunoglobulin G; ns, not significant; si-NC, negative control small interfering RNA (siRNA); si-MYB, siRNA targeting MYB; pklv-vector, sgRNA empty vector control; sg-MYB, sgRNA targeting MYB; SD, standard deviation.

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