Figure 1.
The transcription factor TFAP4 is induced by MYC in developing B cells and GCB cells and is mutated in lymphoid cancers. (A) Z score heat map showing expression of genes that are differentially expressed at least threefold by pro/pre-B cells from Eμ-Myc−Tfap4+/+ (non-Tg; n = 3) and Eμ-Myc Tfap4+/+ (n = 5) mice, as determined by RNA sequencing. (B) Z score heat map showing expression of genes that are differentially expressed at least threefold by c-MYC+ vs c-MYC− mouse splenic GCB cells, as determined by microarray (GSE38304). (C) Overlap of DEGs in (A) and (B). (D) Overlapping genes from panel C involved in gene regulation with direct c-MYC binding to each genomic region and mutation frequencies in hematopoietic tumors in COSMIC. The list is sorted by the numbers of point mutations in hematopoietic tumors after prioritization of c-MYC–bound genes. (E) Quantitative reverse transcription polymerase chain reaction (left panel) and immunoblot analysis (right panel) of Tfap4 mRNA and TFAP4 protein in BM B220+ IgM− cells of Eμ-Myc−Tfap4+/+ (non-Tg) and Eμ-Myc+ Tfap4+/+ and Tfap4+/− mice. Tfap4 mRNA expression was normalized to spike-in control RNA, ERCC-00108. Histone H3 serves as a loading control in immunoblot. (F) Frequency of coding TFAP4 mutations registered across BL subtypes.26 (G) Mapping of recurrent (≥4 independent) somatic missense mutations of TFAP4 identified in primary human tumors from TCGA, PeCan, and COSMIC databases. Each mutation is shown by an asterisk and is mapped to its position in the TFAP4 protein; PeCan lymphoid tumors are shown in blue, and Duke BL cases are shown in red. The basic region (b) functioning as a DNA binding domain and the helix-loop-helix (HLH) domain are shown. A caret (^) indicates that the R129W mutation is found outside of this region. (H) List of recurrent somatic mutations in the DNA binding region and their cancer types in panel G with hematopoietic malignancies shown in bold. (I) Assay to determine the function of somatic TFAP4 variants in upregulation of CD25, which is a direct TFAP4 target (supplemental Figure 1C-E), in Tfap4−/− CD8 T cells (left panel). Expression of CD25 in Tfap4−/− CD8 T cells retrovirally expressing each TFAP4 somatic variant (right panel). Data are representative of 3 independent experiments . *P < .05 by 1-way ANOVA. Ctrl, control; MFI, median fluorescence intensity; rIL-2, recombinant interleukin-2; RV; retrovirus; α, anti-.

The transcription factor TFAP4 is induced by MYC in developing B cells and GCB cells and is mutated in lymphoid cancers. (A) Z score heat map showing expression of genes that are differentially expressed at least threefold by pro/pre-B cells from Eμ-Myc−Tfap4+/+ (non-Tg; n = 3) and Eμ-Myc Tfap4+/+ (n = 5) mice, as determined by RNA sequencing. (B) Z score heat map showing expression of genes that are differentially expressed at least threefold by c-MYC+ vs c-MYC− mouse splenic GCB cells, as determined by microarray (GSE38304). (C) Overlap of DEGs in (A) and (B). (D) Overlapping genes from panel C involved in gene regulation with direct c-MYC binding to each genomic region and mutation frequencies in hematopoietic tumors in COSMIC. The list is sorted by the numbers of point mutations in hematopoietic tumors after prioritization of c-MYC–bound genes. (E) Quantitative reverse transcription polymerase chain reaction (left panel) and immunoblot analysis (right panel) of Tfap4 mRNA and TFAP4 protein in BM B220+ IgM− cells of Eμ-Myc−Tfap4+/+ (non-Tg) and Eμ-Myc+ Tfap4+/+ and Tfap4+/− mice. Tfap4 mRNA expression was normalized to spike-in control RNA, ERCC-00108. Histone H3 serves as a loading control in immunoblot. (F) Frequency of coding TFAP4 mutations registered across BL subtypes.26 (G) Mapping of recurrent (≥4 independent) somatic missense mutations of TFAP4 identified in primary human tumors from TCGA, PeCan, and COSMIC databases. Each mutation is shown by an asterisk and is mapped to its position in the TFAP4 protein; PeCan lymphoid tumors are shown in blue, and Duke BL cases are shown in red. The basic region (b) functioning as a DNA binding domain and the helix-loop-helix (HLH) domain are shown. A caret (^) indicates that the R129W mutation is found outside of this region. (H) List of recurrent somatic mutations in the DNA binding region and their cancer types in panel G with hematopoietic malignancies shown in bold. (I) Assay to determine the function of somatic TFAP4 variants in upregulation of CD25, which is a direct TFAP4 target (supplemental Figure 1C-E), in Tfap4−/− CD8 T cells (left panel). Expression of CD25 in Tfap4−/− CD8 T cells retrovirally expressing each TFAP4 somatic variant (right panel). Data are representative of 3 independent experiments . *P < .05 by 1-way ANOVA. Ctrl, control; MFI, median fluorescence intensity; rIL-2, recombinant interleukin-2; RV; retrovirus; α, anti-.

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