Figure 3.
ZMIZ1 induces MYB to convergently activate coherent feedforward loops that turn on the expression of ETP-ALL oncogenes. (A) Volcano plot of significance versus log2FC of RNA-seq data between shControl and shZMIZ1-15 transduced LOUCY cells; n = 4/group. (B) qRT-PCR of the ETP-ALL oncogenes highlighted in A in CUTLL3 cells; n = 3/group. (C) qRT-PCR for murine homologs of the ETP-ALL oncogenes highlighted in A of sorted ETP cells from thymuses of Rosa26-CreERT2Zmiz1f/f mice that had been injected 72 h previously with 0.15 mg/g tamoxifen; n = 3/group. (D) qRT-PCR for murine homologs of the ETP-ALL oncogenes highlighted in A of sorted ETP cells after culturing ZMIZ1-overexpressing LMPPs on OP9-DL1 cultures as described in Fig. 2 A; n = 3/group. (E) Venn diagram showing overlaps of MYB, ZMIZ1, and MEF2C ChIP-seq datasets in LOUCY cells. GSE94000; GSE160407. P values are for both Fisher exact test and Chi-square. (F) Metagene plots and heatmaps showing overlaps of MYB, ZMIZ1, and MEF2C ChIP-seq datasets in LOUCY cells. (G and H) Co-IP assay detecting MYB protein upon ZMIZ1 pulldown in LOUCY (G) and CUTLL3 (H) cells. SMARCB1 was used as a positive control. (I and J) Western blot showing effect of ZMIZ1 shRNAs on MYB protein expression in LOUCY cells (I) and CUTLL3 cells (J). (K) Western blot showing the effect of MYB shRNA on MYB protein expression in LOUCY cells. (L) Volcano plot of significance versus log2FC of RNA-seq data between shControl and shMYB (917)-transduced LOUCY cells; n = 4/group. (M) Venn diagram showing overlap of ZMIZ1- and MYB-induced genes. P values are for both Fisher exact test and Chi-square. (N) Pie chart showing high convergence (green) and low antagonism (red) between ZMIZ1 and MYB in co-regulating gene expression. (O) Coherent feedforward circuit diagram in which ZMIZ1 induces MYB to convergently activate ETP-ALL oncogenes. (P) GSEA of ZMIZ1-induced genes showing the plot of the #1 ranked Hallmark MSigDB list (Hallmark_MYC_V1). Unless noted otherwise, P values were based on two-sided t tests for two samples and one-way ANOVA for more than two samples. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData F3. Refer to the image caption for details.

ZMIZ1 induces MYB to convergently activate coherent feedforward loops that turn on the expression of ETP-ALL oncogenes. (A) Volcano plot of significance versus log2FC of RNA-seq data between shControl and shZMIZ1-15 transduced LOUCY cells; n = 4/group. (B) qRT-PCR of the ETP-ALL oncogenes highlighted in A in CUTLL3 cells; n = 3/group. (C) qRT-PCR for murine homologs of the ETP-ALL oncogenes highlighted in A of sorted ETP cells from thymuses of Rosa26-CreERT2Zmiz1f/f mice that had been injected 72 h previously with 0.15 mg/g tamoxifen; n = 3/group. (D) qRT-PCR for murine homologs of the ETP-ALL oncogenes highlighted in A of sorted ETP cells after culturing ZMIZ1-overexpressing LMPPs on OP9-DL1 cultures as described in Fig. 2 A; n = 3/group. (E) Venn diagram showing overlaps of MYB, ZMIZ1, and MEF2C ChIP-seq datasets in LOUCY cells. GSE94000; GSE160407. P values are for both Fisher exact test and Chi-square. (F) Metagene plots and heatmaps showing overlaps of MYB, ZMIZ1, and MEF2C ChIP-seq datasets in LOUCY cells. (G and H) Co-IP assay detecting MYB protein upon ZMIZ1 pulldown in LOUCY (G) and CUTLL3 (H) cells. SMARCB1 was used as a positive control. (I and J) Western blot showing effect of ZMIZ1 shRNAs on MYB protein expression in LOUCY cells (I) and CUTLL3 cells (J). (K) Western blot showing the effect of MYB shRNA on MYB protein expression in LOUCY cells. (L) Volcano plot of significance versus log2FC of RNA-seq data between shControl and shMYB (917)-transduced LOUCY cells; n = 4/group. (M) Venn diagram showing overlap of ZMIZ1- and MYB-induced genes. P values are for both Fisher exact test and Chi-square. (N) Pie chart showing high convergence (green) and low antagonism (red) between ZMIZ1 and MYB in co-regulating gene expression. (O) Coherent feedforward circuit diagram in which ZMIZ1 induces MYB to convergently activate ETP-ALL oncogenes. (P) GSEA of ZMIZ1-induced genes showing the plot of the #1 ranked Hallmark MSigDB list (Hallmark_MYC_V1). Unless noted otherwise, P values were based on two-sided t tests for two samples and one-way ANOVA for more than two samples. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData F3.

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