Activation-Induced Cytidine Deaminase Expression in CD4+ T Cells is Associated with a Unique IL-10-Producing Subset that Increases with Age

Activation-induced cytidine deaminase (AID), produced by the Aicda gene, is essential for the immunoglobulin gene (Ig) alterations that form immune memory. Using a Cre-mediated genetic system, we unexpectedly found CD4+ T cells that had expressed Aicda (exAID cells) as well as B cells. ExAID cells increased with age, reaching up to 25% of the CD4+ and B220+ cell populations. ExAID B cells remained IgM+, suggesting that class-switched memory B cells do not accumulate in the spleen. In T cells, AID was expressed in a subset that produced IFN-γ and IL-10 but little IL-4 or IL-17, and showed no evidence of genetic mutation. Interestingly, the endogenous Aicda expression in T cells was enhanced in the absence of B cells, indicating that the process is independent from the germinal center reaction. These results suggest that in addition to its roles in B cells, AID may have previously unappreciated roles in T-cell function or tumorigenesis

Activation-induced cytidine deaminase shuttles between nucleus and cytoplasm like apolipoprotein B mRNA editing catalytic polypeptide 1

Activation-induced cytidine deaminase (AID) is a molecule central to initiating class switch recombination, somatic hypermutation, and gene conversion of Ig genes. However, its mechanism to initiate these genetic alterations is still unclear. AID can convert cytosine to uracil on either mRNA or DNA and is involved in DNA cleavage. Although these events are expected to take place in the nucleus, overexpressed AID was found predominantly in the cytoplasm. Here, we demonstrated that AID is a nucleocytoplasmic shuttling protein with a bipartite nuclear localization signal and a nuclear export signal in its N and C termini, respectively. In addition to previously identified genetic, structural, and biochemical similarities of AID with apolipoprotein B mRNA editing catalytic polypeptide 1, an RNA editing enzyme of ApoB100 mRNA, the present finding provides another aspect to their resemblance, suggesting that both may have homologous reaction mechanisms

HNRNPU facilitates antibody class-switch recombination through C-NHEJ promotion and R-loop suppression

Summary: B cells generate functionally different classes of antibodies through class-switch recombination (CSR), which requires classical non-homologous end joining (C-NHEJ) to join the DNA breaks at the donor and acceptor switch (S) regions. We show that the RNA-binding protein HNRNPU promotes C-NHEJ-mediated S-S joining through the 53BP1-shieldin DNA-repair complex. Notably, HNRNPU binds to the S region RNA/DNA G-quadruplexes, contributing to regulating R-loop and single-stranded DNA (ssDNA) accumulation. HNRNPU is an intrinsically disordered protein that interacts with both C-NHEJ and R-loop complexes in an RNA-dependent manner. Strikingly, recruitment of HNRNPU and the C-NHEJ factors is highly sensitive to liquid-liquid phase separation inhibitors, suggestive of DNA-repair condensate formation. We propose that HNRNPU facilitates CSR by forming and stabilizing the C-NHEJ ribonucleoprotein complex and preventing excessive R-loop accumulation, which otherwise would cause persistent DNA breaks and aberrant DNA repair, leading to genomic instability

HNRNPU facilitates antibody class-switch recombination through C-NHEJ promotion and R-loop suppression

B cells generate functionally different classes of antibodies through class-switch recombination (CSR), which requires classical non-homologous end joining (C-NHEJ) to join the DNA breaks at the donor and acceptor switch (S) regions. We show that the RNA-binding protein HNRNPU promotes C-NHEJ-mediated S-S joining through the 53BP1-shieldin DNA-repair complex. Notably, HNRNPU binds to the S region RNA/DNA G-quadruplexes, contributing to regulating R-loop and single-stranded DNA (ssDNA) accumulation. HNRNPU is an intrinsically disordered protein that interacts with both C-NHEJ and R-loop complexes in an RNA-dependent manner. Strikingly, recruitment of HNRNPU and the C-NHEJ factors is highly sensitive to liquid-liquid phase separation inhibitors, suggestive of DNA-repair condensate formation. We propose that HNRNPU facilitates CSR by forming and stabilizing the C-NHEJ ribonucleoprotein complex and preventing excessive R-loop accumulation, which otherwise would cause persistent DNA breaks and aberrant DNA repair, leading to genomic instability

<i>In Vivo</i> Analysis of <i>Aicda</i> Gene Regulation: A Critical Balance between Upstream Enhancers and Intronic Silencers Governs Appropriate Expression

<div><p>The <i>Aicda</i> gene encodes activation-induced cytidine deaminase (AID). <i>Aicda</i> is strongly transcribed in activated B cells to diversify immunoglobulin genes, but expressed at low levels in various other cells in response to physiological or pathological stimuli. AID’s mutagenic nature has been shown to be involved in tumor development. Here, we used a transgenic strategy with bacterial artificial chromosomes (BACs) to examine the <i>in vivo</i> functions of <i>Aicda</i> regulatory elements, which cluster in two regions: in the first intron (region 2), and approximately 8-kb upstream of the transcription start site (region 4). Deleting either of these regions completely abolished the expression of <i>Aicda</i>-BAC reporters, demonstrating these elements’ critical roles. Furthermore, we found that selectively deleting two C/EBP-binding sites in region 4 inactivated the enhancer activity of the region despite the presence of intact NF-κB-, STAT6- and Smad-binding sites. On the other hand, selectively deleting E2F- and c-Myb-binding sites in region 2 increased the frequency of germinal-center B cells in which the <i>Aicda</i> promoter was active, indicating that E2F and c-Myb act as silencers <i>in vivo</i>. Interestingly, the silencer deletion did not cause ectopic activation of the <i>Aicda</i> promoter, indicating that <i>Aicda</i> activation requires enhancer-specific stimulation. In summary, precise regulation of the <i>Aicda</i> promoter appears to depend on a coordinated balance of activities between enhancer and silencer elements.</p></div

Dual-reporter hCD2 and RFP expressed in <i>in vivo</i>-activated B cells of each strain of rosa-tdRFP-crossed BAC transgenic mice.

<p>RFP and hCD2 in B cells (B220+) or germinal-center (GC) B cells (B220+CD38–) in Peyer’s Patches (mice at 8 weeks of age) from Aid-cre-cd2 line 1; dME-ac-cre line 1; dR2-ac-cre line 1; dR4-ac-cre line 1; or dCEBP-ac-cre line 1 mice. All mice were crossed with the rosa-tdRFP reporter strain. The mean fluorescent intensity (MFI) of hCD2 staining of the gated population and the percentage within the gate are indicated. FACS results are representative of three independent experiments with three mice each.</p

FACS analysis of hCD2 and RFP expression in immunized mice.

<p>Aid-cre-cd2 line 1 and dME-ac-cre line 1 mice (12 weeks of age) were immunized with SRBC and sacrificed on day 7 after immunization. The percentage of hCD2+ and RFP+ cells among B cells or germinal-center (GC) B cells is indicated. SPL, spleen. FACS results are representative of two independent experiments.</p

Percentage of RFP+ Lymphocytes (mean ± SEM).

<p>All animals were crossed with rosa-tdRFP.</p><p>Three mice from each line were analyzed.</p><p>Percentage of RFP+ B cells in mice carrying the dME-ac-cre transgene was significantly higher than in mice carrying the Aid-cre-cd2 transgene. P = 0.0067 (Spleen), P = 0.0034 (mLN), P = 0.0021 (Peyer’s patch) by two-tailed unpaired Student’s t-test. mLN, mesenteric lymph node.</p

Positive correlation between hCD2 mRNA and the number of RFP+ cells among germinal-center B cells in dME-ac-cre mice.

<p>(A) Representative sorting and gating strategies for samples from the germinal centers of Peyeŕs patches (PP), bone marrow (BM), and spleen (SPL) of 8-week-old Aid-cre-RFP mice. Cells were stained by the indicated markers for FACS analysis. Sorting gates are indicated. (B) RT-qPCR analysis of hCD2 in sorted cells. Aid-cre-cd2 line 1 and dME-ac-cre line 1 mice (8 weeks old) were used; values were normalized to the <i>Gapdh</i> expression. Data represent means ± s.d. (bars) of three independent experiments. Asterisk indicates a mild statistical difference between Aid-cre-cd2 and dME-ac-cre mice, assessed by a two-tailed Student’s t-test (preB, <i>p</i> = 0.042; T1, <i>p = </i>0.02; T2, <i>p = </i>0.014; GC, <i>p = </i>0.024). T1, transitional B cell; T2, transitional B cell 2; MZ, marginal zone B cell; FO, follicular B cell; GC, germinal-center B cell; n.d., not detectable.</p

Lack of hCD2 and RFP expression in the peripheral T cells of dME-ac-cre mice.

<p>FACS analysis of hCD2 and RFP expression in naïve CD3+ or CD4+CD3+ T cells isolated from (A) spleen (SPL), (B) mesenteric lymph nodes (mLN), (C) Peyer’s patches (PP), and CD4+CD8+ T cells isolated from the thymus (D). Data are representative of three independent experiments. Data shown are from a 24-week-old mouse. Numbers in panels show the percentage of gated cells of each gate.</p