American Society of Hematology

Figure 2

$Characterizing the effects of pathogenic WAS missense mutations on WASp:SWI/SNF associations in vivo. (A) Multidomain structure of WASp is shown along with the indicated pathogenic mutations within its different domains. Recurring “hot spot” mutations are indicated in red along with their reported clinical severity grades (stable XLT vs XLT>WAS 5A/5M progressive disease; 5A, grade 5 with autoimmunity; 5M, grade 5 with malignancy). See supplemental Figure 3A for a detailed description of the mutations and their corresponding disease severity grades. (B) RT-qPCR quantitation of candidate TH1 or TH2 genes in WASnull T-cell line (HTLV-1–immortalized) reconstituted with transfected FL-WASp or the indicated mutants after CD3/28 activation under TH1 or TH2 skewing or TH0 nonskewing conditions. Normal CD4 TH cell line (HTLV-1 immortalized) is the control. UT, untranfected WASnull T cells. The mRNA copy numbers derived from the control TH0 cells are not shown but were subtracted from the displayed final mRNA values of the TH1- or TH2-skewed cells. Absolute copy numbers adjusted to GAPDH are displayed as fold change (up or down) in TH1 or TH2 cells compared with their TH0 controls. Data represent the average from at least 3 biological replicates, with bars indicating SEM. Wilcoxon nonparametric test using GraphPad InStat software determined the P values comparing the data between FL and mutants (red asterisk, P < .01; black asterisk, P > .01 but ≤ .05). In data where the differences did not reach statistical significance (ie, P > .05), an asterisk is not shown. (C). MNase ChIP-qPCR assays were performed for the indicated proteins as described in the legend to Figure 1C. The genomic location of PCR primer/probes is indicated by a red asterisk in the gene diagram shown at the top. For TBX21, the 5′ UTR primers were designed within the genomic region that also contains a GAS (γ-activated sequence) site (5′-TTCAGGCAA-3′ at about −770 bp from first coding ATG). For IFNG, the primers are located between −200 to −250 bp from first coding ATG, a region known to contain functional promoter elements. The intergenic region between COL8A2 and TRAPPC3 genes on Chr.1, which does not contain known protein-coding genes, served as a negative control. See supplemental Table 1 for primer/probe details. (D). Nuclear fraction of Jurkat (TH1-skewed, TCR-activated) cells stably expressing Flag/Myc dual-tagged FL-WASp or its indicated WASp mutants were sequentially IPed in the indicated combination (eg, Flag>Myc denotes first IP: Flag, second IP: Myc) and analyzed by sequential western blotting of the same gel with the indicated antibodies.$

Characterizing the effects of pathogenic WAS missense mutations on WASp:SWI/SNF associations in vivo. (A) Multidomain structure of WASp is shown along with the indicated pathogenic mutations within its different domains. Recurring “hot spot” mutations are indicated in red along with their reported clinical severity grades (stable XLT vs XLT>WAS 5A/5M progressive disease; 5A, grade 5 with autoimmunity; 5M, grade 5 with malignancy). See supplemental Figure 3A for a detailed description of the mutations and their corresponding disease severity grades. (B) RT-qPCR quantitation of candidate T_H1 or T_H2 genes in WAS^null T-cell line (HTLV-1–immortalized) reconstituted with transfected FL-WASp or the indicated mutants after CD3/28 activation under T_H1 or T_H2 skewing or T_H0 nonskewing conditions. Normal CD4 T_H cell line (HTLV-1 immortalized) is the control. UT, untranfected WAS^null T cells. The mRNA copy numbers derived from the control T_H0 cells are not shown but were subtracted from the displayed final mRNA values of the T_H1- or T_H2-skewed cells. Absolute copy numbers adjusted to GAPDH are displayed as fold change (up or down) in T_H1 or T_H2 cells compared with their T_H0 controls. Data represent the average from at least 3 biological replicates, with bars indicating SEM. Wilcoxon nonparametric test using GraphPad InStat software determined the P values comparing the data between FL and mutants (red asterisk, P < .01; black asterisk, P > .01 but ≤ .05). In data where the differences did not reach statistical significance (ie, P > .05), an asterisk is not shown. (C). MNase ChIP-qPCR assays were performed for the indicated proteins as described in the legend to Figure 1C. The genomic location of PCR primer/probes is indicated by a red asterisk in the gene diagram shown at the top. For TBX21, the 5′ UTR primers were designed within the genomic region that also contains a GAS (γ-activated sequence) site (5′-TTCAGGCAA-3′ at about −770 bp from first coding ATG). For IFNG, the primers are located between −200 to −250 bp from first coding ATG, a region known to contain functional promoter elements. The intergenic region between COL8A2 and TRAPPC3 genes on Chr.1, which does not contain known protein-coding genes, served as a negative control. See supplemental Table 1 for primer/probe details. (D). Nuclear fraction of Jurkat (T_H1-skewed, TCR-activated) cells stably expressing Flag/Myc dual-tagged FL-WASp or its indicated WASp mutants were sequentially IPed in the indicated combination (eg, Flag>Myc denotes first IP: Flag, second IP: Myc) and analyzed by sequential western blotting of the same gel with the indicated antibodies.

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