Loss of Pax5 binding at the TSS correlates with loss of gene expression in Pax5 ^R31Q/− pro-B cells. (A and B) Scatter plot of gene expression differences between ex vivo–sorted Pax5^+/− and Pax5^+/+ pro-B cells (A) as well as between ex vivo–sorted Pax5^−/− (Vav-Cre Pax5^fl/fl) and Pax5^+/+ pro-B cells (B). The expression data of individual genes (dots) are plotted as mean normalized regularized logarithm (rlog) values, based on two RNA-seq experiments per genotype. Genes with an expression difference of >3-fold, an adjusted P value of <0.05, and a TPM value of >5 (in at least one cell type) are colored in blue or red, corresponding to Pax5-activated or Pax5-repressed genes, respectively (Tables S4 and S5). (C) Binding density at Pax5 peaks identified by ChIP-seq in Pax5^+/+ (blue) and Pax5^R31Q/− (red) pro-B cells and displayed from −1.5 to +1.5 kb relative to the summit of the Pax5 peaks that were common to both pro-B cell types or unique to Pax5^+/+ pro-B cells (Fig. 3, B and D). (D) Crystal structure of the Pax5 paired domain bound to DNA (Garvie et al., 2001). The β-sheets (arrows) and α-helices are indicated together with Arg31 (R31), which interacts with the phosphate backbone in the minor groove of the DNA. The 5′-to-3′ orientation of the Pax5-binding sequence (Fig. 3 E) is indicated. For clarity, the ETS domain structure of Ets1, which is also part of the published x-ray structure, is not shown. (E) Correlation of loss of Pax5 binding at the TSS region and loss of mRNA expression of the indicated genes in Pax5^R31Q/− pro-B cells compared with Pax5^+/+ pro-B cells. Horizontal bars indicate MACS-called Pax5 peaks. RPM, reads per million. (F) Correlation of gene activation with differential Pax5 binding at the TSS. The binding difference at Pax5 peaks in 232 TSS regions of Pax5-activated genes (>2-fold) was calculated as a log₂-fold ratio of the ChIP-seq normalized read CPM determined in Pax5^R31Q/− over Pax5^+/+ pro-B cells (see Materials and methods). The cumulative log₂-fold ratios were plotted on the y axis for the 232 TSS regions of activated genes (black dots), which were ranked from high to low expression differences on the x axis (upper part). The fold activation of the ranked genes is shown below. The ranking of the activated genes was 100 times randomly shuffled to generate the randomized dataset of binding differences (gray). (G) Functional classification of the proteins encoded by the activated and repressed genes identified in Pax5^+/+ versus Pax5^R31Q/− pro-B cells (Fig. 3 A and Table S3). Bar size indicates the percentage of activated or repressed genes in each functional class relative to the total activated or repressed genes, respectively. The gene number in each class is shown within the bar.