Research ArticleIMMUNOGENOMICS

TET enzymes augment activation-induced deaminase (AID) expression via 5-hydroxymethylcytosine modifications at the Aicda superenhancer

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Science Immunology  26 Apr 2019:
Vol. 4, Issue 34, eaau7523
DOI: 10.1126/sciimmunol.aau7523
  • Fig. 1 Dynamic changes in 5hmC during B cell activation.

    (A) Flowchart of experiments. (B) Comparison between 5hmC-enriched regions in unstimulated and activated B cells (72 hours). One hundred ninety-three regions represented only in naïve B cells were not shown. (C) Number of DhmRs. (D) Kinetics of 5hmC modification in the DhmR72h-up (left). There is no increase in the same number of randomly chosen common 5hmC-marked regions (middle) and random regions (right). 5hmC enrichment is shown as normalized reads per 100-bp bin. (E) The 85 and 1953 regions with increased 5hmC in 24-hour- and 48-hour-activated B cells relative to naïve B cells show decreased “methylation” (bisulfite-resistant cytosine, 5mC + 5hmC) at their centers 48 hours after activation. Average methylation was calculated for each 200-bp bin across 6 kb. (F) Motif enrichment analysis of DhmR72h-up. Common 5hmC-enriched regions were used as background for analysis. The y axis indicates the fold enrichment versus background, circle size indicates the percentage of regions containing the respective motif, and the color indicates the significance (log10 P value). (G) 5hmC is enriched at active (H3K4me1+ H3K27Achi) relative to poised (H3K4me1+ H3K27Aclo) enhancers in both activated and naïve B cells. The y and x axes indicate the levels (log2) of H3K4me1 and H3K27Ac relative to input, respectively. (H) A substantial fraction of superenhancers (352 of 459, 76.7%) identified by high H3K27Ac enrichment overlap with DhmR72h-up. Fisher’s exact test was used to analyze the significance. ***P < 0.01 (P = 8.9 × 10−266). n.s., not significant. (I) Ccr4 locus (mm10; chr9:114,484,000 to 114,501,000) as an example of a region with increased 5hmC, increased H3K27Ac, and decreased CpG methylation after activation. The red track indicates CpGs that were included for analysis based on coverage. (J) Kinetics Ccr4 mRNA expression (by RNA-seq) in activated B cells. See also fig. S1.

  • Fig. 2 Comparison of 5hmC modification in WT and Tet2/3 DKO B cells.

    (A) Mean mRNA expression for TET family members (from RNA-seq). TPM, transcripts per million. (B) Description of mice and flowchart of experiment. (C) Tet2 and Tet3 are efficiently deleted. Tet2 and Tet3 expression in B cells from tamoxifen-treated WT control and Tet2/3 DKO mice [as described in (B)] were analyzed by qRT-PCR. Data were normalized to Gapdh within sample and subsequently to the value from WT. A representative experiment is shown (two independent experiments with three technical replicates). ***P < 0.01. (D) Number of differential 5hmC-enriched regions (DhmR) between WT and Tet2/3 DKO B cells. (E) Kinetics of 5hmC enrichment from WT (left) and Tet2/3 DKO (right) at DhmRs72h between WT and DKO [72 hours, (D)]. Regions with decreased 5hmC in DKO are shown above (WT > DKO) and those with increased 5hmC are shown below (DKO > WT). 5hmC enrichment is shown in normalized reads per 100-bp bin. (F) Motif enrichment analysis of DhmR72h-WT > DKO (analyzed as in Fig. 1F). See also fig. S2.

  • Fig. 3 TET proteins facilitate CSR.

    (A) Flowchart of in vivo experiment. f.p., footpad. (B) Top: Flow cytometric analysis of CD19+GL7+Fas+ GC B cells at the draining popliteal lymph nodes from WT and Tet2/3 DKO mice after NP-OVA immunization as in (A). Bottom: Decreased IgG1-switched cells among GC B cells in Tet2/3 DKO (YFP+ GC B–gated) compared with WT mice (GC B–gated). (C to F) Quantification of experiments shown in (B). Data shown are aggregated results from two independent experiments. Means and SEs are shown. WT, n = 11; DKO, n = 12. (G) Flowchart of in vitro IgG1 switching. Cells were labeled with Cell trace violet and activated for 4 days with LPS and IL-4. (H and I) Flow cytometry plots (H) and quantification (I) of IgG1-switched B cells in Tet2/3 DKO (n = 4) and WT (n = 4) mice. Representative of at least three independent experiments. (J) Circular γ1 transcript was quantified by qRT-PCR and normalized to Gapdh and then to the level of WT. Representative experiment of two independent experiments with three technical replicates. (K) Flowchart of in vitro IgA switching. Cells were activated for 5 days with anti-CD40, rmIL-4, rmIL-5, and rhTGFβ. (L to N) Flow cytometry plots (L) and quantification (M and N) of IgG1-switched (M) and IgA-switched cells (N). Representative of three independent experiments with three technical replicates. Statistical significance was calculated using unpaired two-tailed t test. *P < 0.05, ***P < 0.01. See also fig. S3.

  • Fig. 4 TET2/3 facilitate CSR by regulating expression of the cytidine deaminase AID.

    (A) qRT-PCR analysis of Aicda mRNA expression in WT and Tet2/3 DKO B cells activated 4 days with LPS and IL-4. Aicda expression was normalized to Gapdh and then to the level in WT. Data shown are representative of two independent experiments with three technical replicates. *P < 0.05. (B) Immunoblotting of whole-cell lysates from day 4 activated WT and Tet2/3 DKO with indicated antibodies. Left lane contains lysate from the AID-KO CH12 B cells as a control for the specificity of anti-AID antibody. β-Actin was used as loading control. Data shown are representative of two independent experiments. See also fig. S4D. (C and D) WT and Tet2/3 DKO B cells were transduced with empty vector (Thy1.1, left), WT AID (AIDWT, middle), or catalytically inactive AID (AIDH56R/E58Q, right). Gated on live-transduced B cells (CD19+ Thy1.1+). Representative flow cytometry plots (C) and quantification (D) are shown. Data shown are representative of three independent experiments. ***P < 0.01. See also fig. S4.

  • Fig. 5 TET2 and TET3 control Aicda expression via TET-responsive elements TetE1 and TetE2.

    Diagram shows two conserved TET-responsive elements TetE1 and TetE2 located 5′ of the Aicda gene (labeled with green rectangles and gray shades). (A) Top two tracks: ChIP-seq analysis showed that TET2 (blue track) specifically bound to multiple elements in the Aicda locus (mm10; chr6:122,523,500 to 122,576,500) after activation when compared with Ig control (gray track). Middle track (green): Increased H3K27Ac after activation. Bottom track: Activation induced DNA demethylation at TetE1 and TetE2. WGBS showing DNA methylation (5mC + 5hmC) in naïve and 48-hour-activated B cells (mCG; black track). CpGs included in the analysis are indicated by red lines (red track). Bottom track indicates the conserved DNA elements among placental animals (“Conserve”). A previously identified superenhancer is indicated. For TET2 and Ig, scales indicate per 10 million reads; H3K27Ac, quantile-normalized reads; BS, percentage of bisulfite-resistant cytosine. (B) Activation induced TET2/3-dependent 5hmC deposition at Aicda distal elements. 5hmC modification was analyzed in WT and Tet2/3 DKO B cells activated as in Fig. 3G. Differentially 5hmC-enriched regions between WT and DKO after 72 hours activation are shown (WT > DKO DhmR72h). Scales indicate quantile-normalized reads. (C) TET2/3 deposit 5hmC and demethylate TetE1. Top: CpG modifications (5hmC, 5mC, and C) at TetE1 were analyzed by oxBS-seq using DNA isolated from WT and Tet2/3 DKO B cells before (0 hours) and after (72 hours) activation. Bottom: Quantification of the probability of 5mC modification on all CpGs in TetE1. The difference between samples was compared by using the Wilcoxon rank-sum test. *P < 0.05, ***P < 0.01. See also fig. S5H.

  • Fig. 6 BATF facilitates TET protein–mediated hydroxymethylation at TetE1.

    (A) Mean mRNA expression (RNA-seq) of Batf family (Batf1 to Batf3). (B and C) BATF binding correlates with 5hmC enrichment. WT BATF peaks were divided into two clusters on the basis of the pattern of 5hmC distribution. (B) Cluster 1 showed a broad 5hmC distribution, with the 5hmC level remaining unchanged after activation and in the absence of TET2/3 (top, compare “5hmC from WT” with “5hmC from DKO”). In contrast, a substantial proportion of regions in cluster 2 showed progressive TET-dependent 5hmC modification after activation (bottom), as further illustrated in (C) as line plots. Data shown are mean enrichment per 100-bp bin. (D) Recruitment of BATF and other transcription factors to Aicda enhancers. Top three tracks: Genome browser view of BATF-binding in unstimulated and 72-hour-activated WT (blue) and Tet2/3 DKO B cells (red) at the Aicda locus. Note that loss of Tet2/3 has no significant effect on BATF recruitment (compare WT and DKO, tracks 2 and 3; see also fig. S8G). Activation also induced E2A and PU.1 binding to Aicda enhancers (orange and purple tracks). Green tracks indicate H3K27Ac. Coordinates are chr6:122,523,500 to 122,576,500 (mm10). See also fig. S8. (E) BATF is required for 5hmC modification at TetE1. Batf-WT and Batf-KO B cells were activated with LPS and IL-4 for 4 days. 5hmC modification at TetE1 was quantified using AbaSI-qPCR.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/34/eaau7523/DC1

    Materials and Methods

    Fig. S1. TET-mediated DNA hydroxymethylation correlates with demethylation and enhancer activity.

    Fig. S2. Phenotypic features of WT and Tet2/3 DKO B cells.

    Fig. S3. TET family proteins are important for B cell–intrinsic CSR.

    Fig. S4. Decreased Aicda expression in Tet2/3 DKO B cells.

    Fig. S5. TET-responsive element TetE1 regulates CSR and Aicda mRNA expression in the CH12 B cell line.

    Fig. S6. TET proteins sustain enhancer accessibility.

    Fig. S7. Analysis of TET-dependent accessible regions.

    Fig. S8. AP-1 proteins in activated B cells.

    Table S1. Differentially expressed genes between WT and Tet2/3 DKO B cells.

    Table S2. Time-series analysis of RNA expression in WT B cells.

    Table S3. Primer sequences.

    Table S4. Reagents and resources.

    Table S5. Raw data.

    References (5973)

  • Supplementary Materials

    The PDF file includes:

    • Materials and Methods
    • Fig. S1. TET-mediated DNA hydroxymethylation correlates with demethylation and enhancer activity.
    • Fig. S2. Phenotypic features of WT and Tet2/3 DKO B cells.
    • Fig. S3. TET family proteins are important for B cell–intrinsic CSR.
    • Fig. S4. Decreased Aicda expression in Tet2/3 DKO B cells.
    • Fig. S5. TET-responsive element TetE1 regulates CSR and Aicda mRNA expression in the CH12 B cell line.
    • Fig. S6. TET proteins sustain enhancer accessibility.
    • Fig. S7. Analysis of TET-dependent accessible regions.
    • Fig. S8. AP-1 proteins in activated B cells.
    • References (5973)

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Differentially expressed genes between WT and Tet2/3 DKO B cells.
    • Table S2 (Microsoft Excel format). Time-series analysis of RNA expression in WT B cells.
    • Table S3 (Microsoft Excel format). Primer sequences.
    • Table S4 (Microsoft Excel format). Reagents and resources.
    • Table S5 (Microsoft Excel format). Raw data.

    Files in this Data Supplement:

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