Figure 5.
Differential Pax5 binding and gene regulation in Pax5R31Q/−pro-B cells. (A) Scatter plot of gene expression differences between ex vivo–sorted Pax5+/+ and Pax5R31Q/− pro-B cells. The expression data of individual genes (dots) are plotted as mean normalized regularized logarithm (rlog) values and are based on two RNA-seq experiments per genotype (Table S3). Genes with an expression difference of >3-fold, an adjusted P value of <0.05, and TPM value of >5 (in at least one cell type) are colored in blue or red. (B) Overlap of Pax5 peaks between Pax5+/+ and Pax5R31Q/− pro-B cells, as determined by ChIP-seq analysis of Pax5+/+ and Pax5R31Q/− pro-B cells. Numbers refer to common (black) and unique (white) Pax5 peaks identified by MACS peak calling (P value <10−10). (C) Common (green) and unique (red) Pax5 peaks at Nedd9 and Siglecg, visualized as reads per million (RPM). Horizontal bars indicate MACS-called peaks. (D) Density heatmaps of common and unique Pax5 peaks. (E) Pax5-binding motifs identified by de novo motif discovery analysis in common and unique peaks with E values of 8.1 × 10−17 and 2.4 × 10−27, respectively. Arrows point to nucleotide positions in the Pax5 motif of unique peaks that deviate from that of common peaks. (F) Correlation of gene activation with differential Pax5 binding at TSSs. The Pax5-binding difference at Pax5 peaks in 232 TSS regions of Pax5-activated (>2-fold) genes was calculated as a log2-fold ratio of the ChIP-seq normalized read CPM determined in Pax5R31Q/− over Pax5+/+ pro-B cells (see Materials and methods). The distribution of the Pax5-binding difference at the TSS regions is shown for activated genes (>2-fold; blue) and nonregulated genes (expression >5 TPM, gray). The median (black line) and middle 50% (boxes) of the data and values within the 1.5 × interquartile range (whiskers) are shown; P value determined by two-tailed Student’s t test. (G) Expression of selected Pax5 target genes in Pax5+/+ (blue), Pax5R31Q/− (red), and Pax5−/− (Vav-Cre Pax5fl/fl; gray) pro-B cells, shown as mean TPM values of two RNA-seq experiments per genotype.

Differential Pax5 binding and gene regulation in Pax5 R31Q/− pro-B cells. (A) Scatter plot of gene expression differences between ex vivo–sorted Pax5+/+ and Pax5R31Q/− pro-B cells. The expression data of individual genes (dots) are plotted as mean normalized regularized logarithm (rlog) values and are based on two RNA-seq experiments per genotype (Table S3). Genes with an expression difference of >3-fold, an adjusted P value of <0.05, and TPM value of >5 (in at least one cell type) are colored in blue or red. (B) Overlap of Pax5 peaks between Pax5+/+ and Pax5R31Q/− pro-B cells, as determined by ChIP-seq analysis of Pax5+/+ and Pax5R31Q/− pro-B cells. Numbers refer to common (black) and unique (white) Pax5 peaks identified by MACS peak calling (P value <10−10). (C) Common (green) and unique (red) Pax5 peaks at Nedd9 and Siglecg, visualized as reads per million (RPM). Horizontal bars indicate MACS-called peaks. (D) Density heatmaps of common and unique Pax5 peaks. (E) Pax5-binding motifs identified by de novo motif discovery analysis in common and unique peaks with E values of 8.1 × 10−17 and 2.4 × 10−27, respectively. Arrows point to nucleotide positions in the Pax5 motif of unique peaks that deviate from that of common peaks. (F) Correlation of gene activation with differential Pax5 binding at TSSs. The Pax5-binding difference at Pax5 peaks in 232 TSS regions of Pax5-activated (>2-fold) genes was calculated as a log2-fold ratio of the ChIP-seq normalized read CPM determined in Pax5R31Q/− over Pax5+/+ pro-B cells (see Materials and methods). The distribution of the Pax5-binding difference at the TSS regions is shown for activated genes (>2-fold; blue) and nonregulated genes (expression >5 TPM, gray). The median (black line) and middle 50% (boxes) of the data and values within the 1.5 × interquartile range (whiskers) are shown; P value determined by two-tailed Student’s t test. (G) Expression of selected Pax5 target genes in Pax5+/+ (blue), Pax5R31Q/− (red), and Pax5−/− (Vav-Cre Pax5fl/fl; gray) pro-B cells, shown as mean TPM values of two RNA-seq experiments per genotype.

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