Figure 4.
Noncoding WT1 variants lead to allele-specific downregulation of WT1, and biallelic WT1 inactivation is recurrently found in APL patients. (A) ChIP-seq tracks of H3K4me1, H3K4me3, H3K27me3, and H3K27ac occupancy in the WT1 locus in NB4 cells without noncoding WT1 variants. The site for noncoding WT1 variants is marked with a red line. (B) Impairment of the enhancer activity by noncoding WT1 variants. Wild-type or mutated intronic sequences were cloned into the pGL3-promoter plasmid, and the luciferase reporter assay was performed in NB4 cells (without noncoding WT1 variants). Data are plotted as means plus or minus standard deviation (n = 3). ***P < .001. (C) Decreased H3K27ac signals on the intronic region of WT1 in noncoding WT1 variants containing APL patients compared with patients without such variants. The P value was calculated by the Wilcoxon test. (D) Significant downregulation of WT1 expression in APL patients with noncoding WT1 mutations/variants compared with patients without these alterations. Triangle represents the case with the somatic mutation. Quadrilateral represents the case with the germline variant. Star represents the case harboring both somatic mutation and germline variant. Circular represents the case without the noncoding mutation/variant. (E) Allelic imbalance of H3K27ac ChIP-seq signals and WT1 expression in noncoding WT1 variants for APL patients. Upper panel: diagram of the WT1 locus. Left panel: the mutation status in each allele of WT1 in APL samples with noncoding WT1 variants. Middle panel: allelic distribution of H3K27ac ChIP-seq reads with and without the indicated noncoding variant. Right panel: allele distribution of WT1 expression based on RNA-seq reads mapped to the indicated heterozygous exonic SNPs or exonic mutations. The red and blue colors represent different alleles, and gray represents the indistinguishable alleles. One-tailed binomial test (expected probability 0.5) was performed: *P < .05; **P < .01; ***P < .001; ****P < .0001. LOH, loss of heterozygosity; NA, not available; NC, noncoding variant; No hete SNP, no heterozygous exonic SNP.

Noncoding WT1 variants lead to allele-specific downregulation of WT1, and biallelic WT1 inactivation is recurrently found in APL patients. (A) ChIP-seq tracks of H3K4me1, H3K4me3, H3K27me3, and H3K27ac occupancy in the WT1 locus in NB4 cells without noncoding WT1 variants. The site for noncoding WT1 variants is marked with a red line. (B) Impairment of the enhancer activity by noncoding WT1 variants. Wild-type or mutated intronic sequences were cloned into the pGL3-promoter plasmid, and the luciferase reporter assay was performed in NB4 cells (without noncoding WT1 variants). Data are plotted as means plus or minus standard deviation (n = 3). ***P < .001. (C) Decreased H3K27ac signals on the intronic region of WT1 in noncoding WT1 variants containing APL patients compared with patients without such variants. The P value was calculated by the Wilcoxon test. (D) Significant downregulation of WT1 expression in APL patients with noncoding WT1 mutations/variants compared with patients without these alterations. Triangle represents the case with the somatic mutation. Quadrilateral represents the case with the germline variant. Star represents the case harboring both somatic mutation and germline variant. Circular represents the case without the noncoding mutation/variant. (E) Allelic imbalance of H3K27ac ChIP-seq signals and WT1 expression in noncoding WT1 variants for APL patients. Upper panel: diagram of the WT1 locus. Left panel: the mutation status in each allele of WT1 in APL samples with noncoding WT1 variants. Middle panel: allelic distribution of H3K27ac ChIP-seq reads with and without the indicated noncoding variant. Right panel: allele distribution of WT1 expression based on RNA-seq reads mapped to the indicated heterozygous exonic SNPs or exonic mutations. The red and blue colors represent different alleles, and gray represents the indistinguishable alleles. One-tailed binomial test (expected probability 0.5) was performed: *P < .05; **P < .01; ***P < .001; ****P < .0001. LOH, loss of heterozygosity; NA, not available; NC, noncoding variant; No hete SNP, no heterozygous exonic SNP.

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