Figure 6.
P2/CD23–cells are pre-PBs rather than totally committed PBs. (A) Distribution on the RNA-seq PCA graph of genes upregulated in P1 vs P2/CD23– (green dots) or upregulated in P2/CD23– (purple dots). Note that although expression levels are lower in P2/CD23– than in less differentiated cell subsets, the genes upregulated in this population (relative to P1) are mainly B-cell identity genes. The position of the CD38-encoding gene is indicated. The green dot on the lower left side is FCER2 (encoding CD23), which is weakly expressed by P1 cells. (B) A volcano plot of ATAC-seq reads comparing P2/CD23– and P1 cells for all regions (gray dots). The fold changes and P values for DO regions associated with genes upregulated in P1 vs P2/CD23– are indicated by green dots (including a region close to CD38, red arrow). Regions associated with genes upregulated in P2/CD23– vs P1 are indicated by violet diamonds. (C) Boxplots of read densities for regions associated with genes upregulated in P2/CD23– cells (left side, 288 regions) or P1 cells (right side, 59 regions). The statistical significance of the difference between P2/CD23– and P1 populations was assessed in a 2-tailed Wilcoxon rank sum test with continuity correction. Wilcoxon test: 48 933; 95% confidence interval [CI], 1.8e−4 to 5.4e−4; location shift estimate, 3.7e−4; P = 1.3e−4 for regions associated with genes upregulated in P2/CD23– cells. W: 719; 95% CI, −1.97e−3 to −9.4e−4; location shift estimate, −1.41e−3; P = 3.9e−8 for regions associated with genes upregulated in P1 cells. (D) Chromatin accessibility at IGH locus during differentiation. The boxplot shows DO region cluster C6 read densities (n = 1074) in NBCs and in the 4 cell populations of interest. Dots indicate the read densities of the IGHM locus (red dots), IGHA1 locus (red triangles), and the enhancer hs3 in the 3′ RR2 regulatory region (red diamonds). IGHM is the locus that varied most and was fully open in P1 cells only. (E) An IGV browser window showing RNA-seq and ATAC-seq signals in the different cell populations for the regions depicted in D close to the IGH locus. The left panel shows the hs3 enhancer of the 3′ RR2 regulatory region58; the middle panel shows the first coding exons of IGHA1 and an upstream potential regulatory ATAC-seq region that opens in PBs only (Region_48456); the right panel shows the first coding exons of IGHM and an upstream potential regulatory ATAC-seq region that opens in PBs only (Region_48462).

P2/CD23–cells are pre-PBs rather than totally committed PBs. (A) Distribution on the RNA-seq PCA graph of genes upregulated in P1 vs P2/CD23– (green dots) or upregulated in P2/CD23– (purple dots). Note that although expression levels are lower in P2/CD23– than in less differentiated cell subsets, the genes upregulated in this population (relative to P1) are mainly B-cell identity genes. The position of the CD38-encoding gene is indicated. The green dot on the lower left side is FCER2 (encoding CD23), which is weakly expressed by P1 cells. (B) A volcano plot of ATAC-seq reads comparing P2/CD23– and P1 cells for all regions (gray dots). The fold changes and P values for DO regions associated with genes upregulated in P1 vs P2/CD23– are indicated by green dots (including a region close to CD38, red arrow). Regions associated with genes upregulated in P2/CD23– vs P1 are indicated by violet diamonds. (C) Boxplots of read densities for regions associated with genes upregulated in P2/CD23– cells (left side, 288 regions) or P1 cells (right side, 59 regions). The statistical significance of the difference between P2/CD23– and P1 populations was assessed in a 2-tailed Wilcoxon rank sum test with continuity correction. Wilcoxon test: 48 933; 95% confidence interval [CI], 1.8e−4 to 5.4e−4; location shift estimate, 3.7e−4; P = 1.3e−4 for regions associated with genes upregulated in P2/CD23– cells. W: 719; 95% CI, −1.97e−3 to −9.4e−4; location shift estimate, −1.41e−3; P = 3.9e−8 for regions associated with genes upregulated in P1 cells. (D) Chromatin accessibility at IGH locus during differentiation. The boxplot shows DO region cluster C6 read densities (n = 1074) in NBCs and in the 4 cell populations of interest. Dots indicate the read densities of the IGHM locus (red dots), IGHA1 locus (red triangles), and the enhancer hs3 in the 3′ RR2 regulatory region (red diamonds). IGHM is the locus that varied most and was fully open in P1 cells only. (E) An IGV browser window showing RNA-seq and ATAC-seq signals in the different cell populations for the regions depicted in D close to the IGH locus. The left panel shows the hs3 enhancer of the 3′ RR2 regulatory region58 ; the middle panel shows the first coding exons of IGHA1 and an upstream potential regulatory ATAC-seq region that opens in PBs only (Region_48456); the right panel shows the first coding exons of IGHM and an upstream potential regulatory ATAC-seq region that opens in PBs only (Region_48462).

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