Figure 5.
OBF1 regulates the GC transcriptional program. (A) Differentially expressed genes between anti-CD40/IL4 and LPS-stimulated CD19+ mouse splenic B cells. GC-related genes with significantly higher expression under anti-CD40/IL4 stimulation are indicated (yellow dots). (B) Heatmap showing OBF1 ChIP enrichment at promoter regions (TSS ± 1 kb) of selected GC-related genes under LPS or anti-CD40/IL4 stimulations (red dots, OCT1-binding regions showing significantly higher binding in B cells treated with anti-CD40/IL4; green dots, OCT1-binding regions showing significantly higher binding in B cells treated with LPS). (C) OCT2, OBF1, and OCT1 read densities at 3 individual gene loci, as indicated. (D) Anti-CD40/IL4-induced OBF1 binding to the Bcl6 and Myc gene promoters. (E) Average expression (log2 counts per million, x-axis) and change of expression (log2-fold change, y-axis) for genes in WT and OBF1 knockout CD19+ splenic B cells stimulated with anti-CD40/IL4. Differentially expressed genes are color-coded (DEGs fold change ≥1, FDR <0.01, logCPM >2): red data points represent genes with higher expression level in OBF1 knockout cells, green data points represent genes with higher expression in WT cells. (F) Gene ontology analysis was performed with the genes up- and downregulated in OBF1 knockout vs WT splenic B cells stimulated with anti-CD40/IL4. (G) GSEA of relative gene expression in OBF1 knockout vs WT anti-CD40/IL4 stimulated splenic B cells against the genes downregulated in GC B cells vs plasma cells. NES, normalized enrichment score. (H) Heatmaps showing differentially expressed genes between OBF1 knockout vs WT B cells. Differentially expressed genes involved in GC reaction are selected. (I) Boxplot showing the percentages of germinal center B cells in WT and Pou2af1KO/KO mice by FACS at day 7 post-SRBC challenge. n = 5; mean ± standard deviation; *P < .05; **P < .01; ***P < .001; ****P < .0001 (2-sided Student t test). (J) Top, workflow of germinal center induction by intraperitoneal injection of 10% SRBC solution and the isolation of GC B cells for Bio-ChIP-seq. Bottom, OBF1 and H3K27ac reads densities at six individual gene loci, as indicated, in purified mouse germinal center B cells induced by SRBCs.

OBF1 regulates the GC transcriptional program. (A) Differentially expressed genes between anti-CD40/IL4 and LPS-stimulated CD19+ mouse splenic B cells. GC-related genes with significantly higher expression under anti-CD40/IL4 stimulation are indicated (yellow dots). (B) Heatmap showing OBF1 ChIP enrichment at promoter regions (TSS ± 1 kb) of selected GC-related genes under LPS or anti-CD40/IL4 stimulations (red dots, OCT1-binding regions showing significantly higher binding in B cells treated with anti-CD40/IL4; green dots, OCT1-binding regions showing significantly higher binding in B cells treated with LPS). (C) OCT2, OBF1, and OCT1 read densities at 3 individual gene loci, as indicated. (D) Anti-CD40/IL4-induced OBF1 binding to the Bcl6 and Myc gene promoters. (E) Average expression (log2 counts per million, x-axis) and change of expression (log2-fold change, y-axis) for genes in WT and OBF1 knockout CD19+ splenic B cells stimulated with anti-CD40/IL4. Differentially expressed genes are color-coded (DEGs fold change ≥1, FDR <0.01, logCPM >2): red data points represent genes with higher expression level in OBF1 knockout cells, green data points represent genes with higher expression in WT cells. (F) Gene ontology analysis was performed with the genes up- and downregulated in OBF1 knockout vs WT splenic B cells stimulated with anti-CD40/IL4. (G) GSEA of relative gene expression in OBF1 knockout vs WT anti-CD40/IL4 stimulated splenic B cells against the genes downregulated in GC B cells vs plasma cells. NES, normalized enrichment score. (H) Heatmaps showing differentially expressed genes between OBF1 knockout vs WT B cells. Differentially expressed genes involved in GC reaction are selected. (I) Boxplot showing the percentages of germinal center B cells in WT and Pou2af1KO/KO mice by FACS at day 7 post-SRBC challenge. n = 5; mean ± standard deviation; *P < .05; **P < .01; ***P < .001; ****P < .0001 (2-sided Student t test). (J) Top, workflow of germinal center induction by intraperitoneal injection of 10% SRBC solution and the isolation of GC B cells for Bio-ChIP-seq. Bottom, OBF1 and H3K27ac reads densities at six individual gene loci, as indicated, in purified mouse germinal center B cells induced by SRBCs.

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