Figure 5(A) A schematic diagram of the Pax5-Jak2 protein containing a C-terminal biotin acceptor sequence that can be biotinylated in vivo by the E. coli biotin ligase BirA upon expression from the Rosa26BirA allele (B) Genome-wide binding of Pax5-Jak2 in in vitro cultured pro-B cells from Pax5Jak2/+ Rosa26BirA/+ mice at the age of 3 weeks (expressing Pax5) and ex vivo Pax5Jak2/+ Rosa26BirA/+ B-ALL tumors (lacking Pax5), as determined by Bio-ChIP-seq analysis The DNA-binding pattern of full-length Pax5 was determined by Bio-ChIP-seq analysis of ex vivo sorted Pax5Bio/Bio pro-B cells, which carried a C‐terminal biotin acceptor sequence together with an IRES‐BirA gene insertion in the 3' untranslated region of Pax5 Two independent Bio-ChIP-seq experiments were performed for each cell type. Representative binding patterns of Pax5 and Pax5-Jak2 in the three B cell types are shown for a selected genomic region, with horizontal bars indicating Pax5 or Pax5-Jak2 peaks that were identified by MACS peak calling (left). The number of Pax5 (white) and Pax5-Jak2 (grey or black) peaks, which were defined by stringent MACS peak calling with a P value of < 10-10 in the three B cell types, are shown to the right. (C) Pax5 recognition motifs identified in the three different B cell types by de novo motif discovery analysis of the top 300 peaks (D) Bio-ChIP-qPCR analysis of Pax5 binding at four sites, which were selected for their differential Pax5 binding in the three cell types, as determined by Bio-ChIP-seq analysis The amount of precipitated DNA was determined as percentage relative to input and is shown as mean value with SEM based on 3-4 independent biological experiments. A gene-less region on Chr.1 was used as a negative control. (E) Schematic diagram of the mutant Pax5-Jak2 protein carrying the indicated amino acid changes that were introduced in the N-terminal part of the paired domain by CRISPR/Cas9-mediated mutagenesis in the Pax5Prd*-Jak2 allele (F, G) Flow-cytometric analysis of the bone marrow and spleen from Pax5Prd*-Jak2 /+ (black; n = 6) and control Pax5+/+ (grey; n = 5) mice at the age of 6-7 weeks. The frequency of the indicated B cell types in each organ is indicated in (F), and the flow-cytometric analysis of CD19 and B220 expression on bone marrow B cells is shown in (G). Statistical data are shown as mean percentages with SEM and were analyzed by multiple t-tests (unpaired and two-tailed with Holm-Šídák's correction): ns > 0.05; *P < 0.05; **P < 0.01. Each dot corresponds to one mouse. (H) Kaplan-Meier survival analysis of Pax5Prd*-Jak2 /+ (black) and Pax5+/+ (grey) mice. A P value of < 0.0001 was determined for the survival curves by statistical analysis with the log-rank (Mantel-Cox) test. n, number of mice analyzed. (I) Flow-cytometric analysis and intracellular Pax5 staining of lymph node tumor cells from two moribund Pax5Prd*-Jak2 /+ mice (black line), which differed by the absence (mouse #1) or presence (mouse #2) of Pax5 expression in the tumor cells. Pax5+/+ (WT) B cells (filled grey) were used as controls. The apparently higher Pax5 expression level in the blasting tumor cells of mouse # 2 compared with that of quiescent Pax5+/+ B cells may reflect a difference in cell size rather than in Pax5 expression. The percentage and number of Pax5+ and Pax5- Pax5Prd*-Jak2 /+ tumors analyzed is shown to the right.