Supplementary Figure 2: STAT5, PAX5, EBF, PU.1, IRF4, and IKAROS bind to many common target genes in progenitor B cell leukemia

(a) STAT5 (left panel), IKAROS (middle panel) and RELA (right panel) ChIP-qPCR at Myc super-enhancer in Stat5b-CA x Blnk^+/- leukemias stimulated with (+) or without (-) IL7 at 37°C for 30 minutes. (b) Activating/repressive function prediction of STAT5 by ChIP-BETA. The red and the purple lines represent the upregulated and downregulated genes, respectively. The dashed line indicates the non-differentially expressed genes as background. Genes are cumulated by the rank on the basis of the regulatory potential score from high to low. (c) Occupancy of STAT5, PAX5, EBF, PU.1, IRF4 and IKAROS by ChIP-Seq at Bcl2l1, Igll1, Vpreb1, Pim1, Ccnd3, and Bcl6 loci in Stat5b-CA x Blnk^+/- leukemia (STAT5), Rag^-/- pro-B cells (PAX5, EBF, PU.1, IRF4) or WT pre-B cells (IKAROS). Progenitor B cell super-enhancers are annotated as “SE”. (d) (Top panel) Occupancy of STAT5 and IKAROS by ChIP-Seq at Socs2 locus in Stat5b-CA x Blnk^+/- leukemia and WT pre-B cells, respectively. (Middle panel) Diagram of the Socs2 luciferase constructs. STAT5 binding sites are underlined in red while IKAROS binding sites are underlined in blue. Sites of mutation are indicated with asterisks. Base pair (bp) positions indicate distances relative to the Socs2 transcriptional start site. (Bottom panel) Luciferase assay of WT or mutant Socs2 promoter in Ba/F3 progenitor B cells transfected with Empty, Stat5b-CA, or Stat5b-CA and Ikzf1 retroviruses. (e) STAT5 (left panel) and p300 (right panel) ChIP-qPCR at Pim1 super-enhancer in Stat5b-CA x Blnk^+/- leukemias stimulated with (+) or without (-) IL7 at 37°C for 30 minutes. (f) H3K27Ac ChIP-qPCR at Bcl2l1 super-enhancer in Ba/F3 cells transduced with empty (-) or Stat5b-CA (+) retroviruses. (g) STAT5, PAX5, EBF, and IKAROS ChIP-qPCR at the Myc, Bcl2l1, and Igll1 super-enhancers or a negative control locus located downstream of Igll1 in Stat5b-CA x Blnk^+/- leukemias. (a,d,e,f,g) Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 n.s. = not significant (one-way ANOVA with Bonferroni’s Multiple Comparison post-test, a,d,e,f). P = 6.3x10^-26 (upregulated), P = 5.1x10^-18 downregulated (Kolmogorov-Smirnov test, b). *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA with Dunnett’s Multiple Comparison post-test, g) Data are representative of three (a,d,e,f), one (b, c (STAT5, PAX5, EBF, PU.1, IRF4), two (c, IKAROS), or five (g) independent experiments. Data for PEPII ChIP-Seq experiments in d,c came from^30,36-38. Defined super-enhancers in c came from³⁹.