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. 2013 Apr 16;8(4):e61433.
doi: 10.1371/journal.pone.0061433. Print 2013.

In vivo analysis of Aicda gene regulation: a critical balance between upstream enhancers and intronic silencers governs appropriate expression

Le Thi Huong  1 , Maki Kobayashi, Mikiyo Nakata, Go Shioi, Hitoshi Miyachi, Tasuku Honjo, Hitoshi Nagaoka
Affiliations

In vivo analysis of Aicda gene regulation: a critical balance between upstream enhancers and intronic silencers governs appropriate expression

Le Thi Huong et al. PLoS One. .

Abstract

The Aicda gene encodes activation-induced cytidine deaminase (AID). Aicda 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 in vivo functions of Aicda 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 Aicda-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 Aicda promoter was active, indicating that E2F and c-Myb act as silencers in vivo. Interestingly, the silencer deletion did not cause ectopic activation of the Aicda promoter, indicating that Aicda activation requires enhancer-specific stimulation. In summary, precise regulation of the Aicda promoter appears to depend on a coordinated balance of activities between enhancer and silencer elements.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Generation of BAC transgenic mice.
(A) Aicda on the BAC clone (RP24-68I7) was modified as described in Methods. Open boxes with numbers denote the first to third exons. An expression cassette for cre and human CD2 was inserted into the second exon as indicated. In the enlarged views of regulatory elements in regions 2 or 4 that were deleted or replaced by Frt sites (closed triangle), the number indicates the relative position (base pair) from the transcription initiation site. Binding elements are marked by a symbol showing the name of the factor. A thick line indicates the probe used for Southern blotting. The HindIII restriction sites adjacent to the probe are marked by an H. pA, poly A signal. (B) Characteristic cellular phenotype of Aid-cre-cd2 TG mice crossed with rosa-tdRFP mice. When stimulation induces the Aicda promoter (pAicda), the BAC transgene expresses cre recombinase and the hCD2 marker. Cre recombines loxP and loxP2227 sites in the indicator gene, rosa-tdRFP, and thereby irreversibly expresses RFP. RFP expression is maintained after pAicda activity declines. (C) Southern blot analysis with the probe defined in A: Genomic DNA from wild-type (lane wt), Aid-cre-cd2 (lane 1), dR2-ac-cre (lane 2), dME-ac-cre (lane 3), dR4-ac-cre (lane 4), dCEBP-ac-cre (lane 5), and Aicda-cre (lane 6) mice was digested by HindIII; 3.2- and 3.5-kb bands correspond to endogenous and transgenic loci, respectively.
Figure 2
Figure 2. Dual-reporter hCD2 and RFP expressed in in vivo-activated B cells of each strain of rosa-tdRFP-crossed BAC transgenic mice.
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.
Figure 3
Figure 3. Positive correlation between hCD2 mRNA and the number of RFP+ cells among germinal-center B cells in dME-ac-cre mice.
(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 Gapdh 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, p = 0.042; T1, p = 0.02; T2, p = 0.014; GC, p = 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.
Figure 4
Figure 4. FACS analysis of hCD2 and RFP expression in Aid-cre-cd2 and dME-ac-cre B cell-lineage lymphocytes.
(A) Spleen (SPL) and (B) bone marrow (BM) B cells of 8-week-old Aid-cre-cd2 line 1 and dME-ac-cre line 1 mice were stained for the indicated markers for FACS analysis. Numbers shown in each gate indicate the percentage of cells. FACS results are representative of three independent experiments with three mice each.
Figure 5
Figure 5. FACS analysis of hCD2 and RFP expression in immunized mice.
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.
Figure 6
Figure 6. Lack of hCD2 and RFP expression in the peripheral T cells of dME-ac-cre mice.
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.
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Supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan, KAKENHI Grant-in-AID for Special Promoted Research 17002015 (TH), Grant-in-AID for Sicentific Research 11016207 (HN), and Takeda Science Foundation (HN) (http://www.takeda-sci.or.jp). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.