Figure 8.
AID cooperates with TET2 for efficient Irf4 enhancer/promoter demethylation in GC B cells. (A) Percentage of IRF4hiPAX5lo cells in GC at day 7 and upon stimulation for 2 h. Left: Representative FACS plots (pregated on GC B cells). Right: Quantification, WT, anti-BCR versus unstimulated, P < 0.05 (t test), nonsignificant for other stimulations; AIDR112H, anti-BCR versus unstimulated, P < 0.01 (t test), nonsignificant for other stimulations. Two-way ANOVA between genotypes (n = 4 per condition, two experiments). (B)Bcl6 and Irf4 mRNA level in purified GC B cells. Upper panels: GC purity by flow cytometry. Lower panels: Quantification of Bcl6 and Irf4 mRNA level by RT-PCR; each dot represents an average value from an individual animal (technical triplicates for each animal), Bcl6 mRNA, WT versus AIDR112H, nonsignificant; Irf4 mRNA, WT versus AIDR112H, P < 0.05, ratio-paired t test (n = 3–4, two experiments). Value and error bar: mean ± SD. (C) CpG DNA methylation status at the Irf4 locus in iGB cells derived from WT and AIDR112H cells on day 7. Top: Schematics for Irf4 gene locus and the region examined for DNA methylation using bisulfite sequencing. DNA methylation level at CpG sites; number above the graph shows the CpG position relative to the first exon. For each CpG site, 15–30 clones were analyzed, WT versus AIDR112H, P < 0.0001, paired t test, data from two independent experiments. (D) IRF4hiPAX5lo cell differentiation in TET2 KO iGB cells. Representative FACS for day 0, day 3, and day 6. Quantification, day 6, WT versus TET2 KO, P < 0.01, two-way ANOVA (n = 3, one experiment). (E) Co-immunoprecipitation of endogenous AID and TET2 protein from iGB cells. Anti-TET2 pulldown and immunoblot to detect TET2 (upper panel) and AID (lower panel, uncropped blot image in Fig. S4 B in the same order). One representative blot from four independent experiments. (F) CUT&RUN sequencing to detect global chromatin association of AID, TET2, and H3K4me3 in iGB cells on day 3. Upper: Average enrichment profiles near TSS (−1.5 to +1.5 kb). (G) CUT&RUN and qPCR to detect association of AID, TET2, and H3K4me3 with the Irf4 locus in iGB cells on day 3. CUT&RUN detection region Irf4-3, Irf4-4, and Irf4-5 as indicated in the bottom of C. Black column: WT; red column: AIDR112H; y axis: % relative to WT, *P < 0.05, **P < 0.01, anti-AID (Active Motif and eBioscience), anti-TET2, anti-DNMT1 and anti-H3K4me3, two-way ANOVA (n = 4, four experiments). (H) CUT&RUN and qPCR to detect association of AID with the Switch mu region and the Irf4 enhancer/promoter in TET2 KO iGB cells on day 3. Black column: WT; blue column: TET2 KO; y axis: % relative to WT, ***P < 0.001, ****P < 0.0001, two-way ANOVA (n = 3, three experiments). Source data are available for this figure: SourceData F8. Refer to the image caption for details. Panel A: Flow cytometry plots show the percentage of IRF4hiPAX5lo cells in GC B cells at day 7 and upon stimulation for 2 hours. The left side displays representative flow cytometry plots pre-gated on GC B cells, while the right side quantifies the data. Wild-type (WT) cells up-regulate IRF4hiPAX5lo cells upon BCR activation, whereas AIDR112H cells do not. Panel B: Flow cytometry plots and bar graphs show the mRNA levels of Bcl6 and Irf4 in purified GC B cells. The upper panels display GC purity by flow cytometry, and the lower panels quantify mRNA levels by RT-PCR. Bcl6 mRNA levels are not significantly different between WT and AIDR112H, but Irf4 mRNA levels are significantly lower in AIDR112H cells. Panel C: The top schematic shows the Irf4 gene locus and the region examined. DNA methylation levels at CpG sites are significantly higher in AIDR112H cells and a scatter plot shows the CpG DNA methylation status at the Irf4 locus in iGB cells. Panel D: Flow cytometry plots and a bar graph show the differentiation of IRF4hiPAX5lo cells in TET2 KO iGB cells. Representative FACS plots for day 0, day 3, and day 6 are shown, with quantification indicating a significant reduction in IRF4hiPAX5lo cells in TET2 KO cells on day 6. Panel E: A co-immunoprecipitation (Co-IP) experiment shows the interaction between endogenous AID and TET2 proteins in iGB cells. Immunoblots detect TET2 and AID. Panel F: Line graphs show the global chromatin association of AID, TET2, and H3K4me3 in iGB cells on day 3, with average enrichment profiles near the transcription start site (TSS). Panel G: Bar graphs show the association of AID, TET2, and H3K4me3 with the Irf4 locus in iGB cells on day 3, detected by Cut and Run and q-PCR. Significant differences are observed between WT and AIDR112H cells. Panel H: Bar graphs show the association of AID with the Switch mu region and the Irf4 enhancer/promoter in TET2 KO iGB cells on day 3, detected by Cut and Run and q-PCR. Significant differences are observed between WT and TET2 KO cells.

AID cooperates with TET2 for efficient Irf4 enhancer/promoter demethylation in GC B cells. (A) Percentage of IRF4hiPAX5lo cells in GC at day 7 and upon stimulation for 2 h. Left: Representative FACS plots (pregated on GC B cells). Right: Quantification, WT, anti-BCR versus unstimulated, P < 0.05 (t test), nonsignificant for other stimulations; AIDR112H, anti-BCR versus unstimulated, P < 0.01 (t test), nonsignificant for other stimulations. Two-way ANOVA between genotypes (n = 4 per condition, two experiments). (B)Bcl6 and Irf4 mRNA level in purified GC B cells. Upper panels: GC purity by flow cytometry. Lower panels: Quantification of Bcl6 and Irf4 mRNA level by RT-PCR; each dot represents an average value from an individual animal (technical triplicates for each animal), Bcl6 mRNA, WT versus AIDR112H, nonsignificant; Irf4 mRNA, WT versus AIDR112H, P < 0.05, ratio-paired t test (n = 3–4, two experiments). Value and error bar: mean ± SD. (C) CpG DNA methylation status at the Irf4 locus in iGB cells derived from WT and AIDR112H cells on day 7. Top: Schematics for Irf4 gene locus and the region examined for DNA methylation using bisulfite sequencing. DNA methylation level at CpG sites; number above the graph shows the CpG position relative to the first exon. For each CpG site, 15–30 clones were analyzed, WT versus AIDR112H, P < 0.0001, paired t test, data from two independent experiments. (D) IRF4hiPAX5lo cell differentiation in TET2 KO iGB cells. Representative FACS for day 0, day 3, and day 6. Quantification, day 6, WT versus TET2 KO, P < 0.01, two-way ANOVA (n = 3, one experiment). (E) Co-immunoprecipitation of endogenous AID and TET2 protein from iGB cells. Anti-TET2 pulldown and immunoblot to detect TET2 (upper panel) and AID (lower panel, uncropped blot image in Fig. S4 B in the same order). One representative blot from four independent experiments. (F) CUT&RUN sequencing to detect global chromatin association of AID, TET2, and H3K4me3 in iGB cells on day 3. Upper: Average enrichment profiles near TSS (−1.5 to +1.5 kb). (G) CUT&RUN and qPCR to detect association of AID, TET2, and H3K4me3 with the Irf4 locus in iGB cells on day 3. CUT&RUN detection region Irf4-3, Irf4-4, and Irf4-5 as indicated in the bottom of C. Black column: WT; red column: AIDR112H; y axis: % relative to WT, *P < 0.05, **P < 0.01, anti-AID (Active Motif and eBioscience), anti-TET2, anti-DNMT1 and anti-H3K4me3, two-way ANOVA (n = 4, four experiments). (H) CUT&RUN and qPCR to detect association of AID with the Switch mu region and the Irf4 enhancer/promoter in TET2 KO iGB cells on day 3. Black column: WT; blue column: TET2 KO; y axis: % relative to WT, ***P < 0.001, ****P < 0.0001, two-way ANOVA (n = 3, three experiments). Source data are available for this figure: SourceData F8.

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