Expression of AID transgene is regulated in activated B cells but not in resting B cells and kidney

Abstract

Activation-induced DNA cytidine deaminase (AID) is required for somatic hypermutation (SHM) and efficient class switch recombination (CSR) of immunoglobulin (Ig) genes. We created AID-transgenic mice that express AID ubiquitously under the control of a beta-actin promoter. When crossed with AID-/- mice, the AID-transgenic,AID-/- mice carried out SHM and CSR, showing that the AID transgenes were functional. However, the frequencies of SHM in V- and switch-regions, and CSR were reduced compared to those in a wild type AID background. Several criteria suggested that the inefficiency of SHM was due to reduced AID activity, rather than lack of recruiting error-prone DNA repair. High levels of AID mRNA were produced in resting B cells and kidney, cells that do not express AID in wild type mice. Compared with these cells, activated B cells expressed about an order of magnitude less AID mRNA suggesting that there may be a post-transcriptional mechanism that regulates AID mRNA levels in professional AID producers but not other cells. The AID protein expressed in resting B cells and kidney was phosphorylated at serine-38. Despite this modification, known to enhance AID activity, resting B cells did not undergo SHM. Apparently, the large amounts of AID in resting B cells are not targeted to Ig genes in vivo, in contrast to findings in vitro.

Fig. 1

AID mRNA levels are higher in PNA-lo B cells and kidney than in PNA-hi B cells of AID-transgenic mice. A, PNA-lo and PNA-hi B cells and kidney from AID-transgenic mice of line #14. B, PNA-lo and PNA-hi B cells from wildtype mice and PNA-lo B cells from AID-transgenic mice of line #14. The total cycles of PCR for β-actin were 29 and for AID were 32. There was no PCR product in the samples without RT (no DNA contamination) (data not shown).

Fig. 2

Phosphorylated serine 38 of AID in AID-transgenic mice. AID was immuno-precipitated from cell lysates with anti-AID antibodies and the immunoprecipitates were revealed in Western blots with either anti-pS38 (top) or anti-AID (bottom). The positions of AID and S38-phosphorylated AID are indicated. The band at the top of the blots probed for pS-38 is nonspecific (it also appears in AID −/− cells). A, rAID, recombinant AID produced in E. coli; the four other lanes contain IPs from 50 million cells: AID−/− and wt, CD43- B cells were stimulated in culture with LPS and IL-4 for 3 days; #18, CD43⁻ or CD43⁺ cells from AID transgenic mice line #18 (the cells were not activated in vitro). B and C, Immuno-precipitation and Western blots as in A. Five wt (.1 to .5) and four different AID transgenic mice each of lines #14 and #18 (.1 to .4) are indicated. The AID−/− and wt.2 CD43- B cells were stimulated for 3 days with LPS and IL4. B, Left six lanes: 40 million sorted, CD43⁻ or CD43⁺ cells/lane. Right six lanes: 60 million cells per lane. C, Left six lanes: 40 million CD43⁻ cells or kidney. Right seven lanes: 20 million cells pre lane.

Fig. 3

Normal mutation pattern in Ig_H gene of AID-transgenic mice. The mutations were collected from PNA-hi B cells of AID-transgenic/AID +/+ (endogenous) (top) and AID-transgenic/AID−/− (bottom) mice (see Table 1 A and B). The adjusted percentage of mutations in A, C, G, and T, respectively was based on the nucleotide composition in the 1.1 kb sequenced DNA fragment. No identical sequences in the same PCR reaction were observed in this study.

Fig. 4

Gradual decline of mutations 3’ of VDJ_H also in AID-transgenic mice. A, Occurrence of cytidine in the 1.1 kb region at the start of the Ig heavy chain JC intron. The bars are Cs on both DNA strands. B, Positions of WRC hotspots in both DNA strands. C, Mutation pattern in the AID-trangenic, AID+/+ mice. D, Mutation pattern in the AID-transgenic, AID−/− mice. E, The Ig heavy chain gene (not to scale). Bent arrow, transcription initiation site; variable region (VDJ), JC intron (the line between VDJ and C), and constant region (C) are indicated. The sequenced region extends from 1 to 1,100 bp of the JC intron.

Fig. 5

PCNA in B cells. Western blot of PNA-lo and PNA-hi B cells from wildtype and AID-transgenic mice. Top, anti-PCNA; bottom, anti-actin. DT40, chicken B cell line as a positive control; in this cell mono-ubiquitination has been shown essential for efficient SHM (Arakawa et al., 2006).

Fig. 6

CSR occurs in AID-transgenic/AID^−/− mice. A, Germline transcripts (GLTs) were analyzed by semi-quantitative RT-PCR using cDNAs derived from splenic B cells from AID-transgenic/AID^−/− and WT mice that were activated with LPS or LPS +IL4 for 48 hours. Gapdh RT-PCR products were harvested after 29 cycles (lanes 1, 4, 7,10), 27 cycles (lanes 2, 5, 8, 11), 25 cycles (lanes 3, 6, 9, 12). GLT and AID RT-PCR products were harvested after 33 cycles (lanes 1, 4, 7, 10), 31 cycles (lanes 2, 5, 8, 11) and 29 cycles (lanes 3, 6, 9, 12). AID originating from the endogenous (e) alone (Muramatsu et al., 2000) or from a combination of endogenous genes and transgenes (e+Tg) (see Materials and Methods) are shown. B, Post-switch transcripts (PSTs) were analyzed by semi-quantitative RT-PCR using cDNAs derived from splenic B cells from AID-transgenic/AID^−/−, AID^+/+ and AID^−/− mice that were activated with LPS or LPS+IL4 for 5 days. Gapdh PCR products were harvested after 29 cycles (lanes 1, 4, 7), 27 cycles (lanes 2, 5, 8) and 25 cycles (lanes 3, 6, 9). PST PCR products were harvested after 33 cycles (lanes 1, 4, 7), 31 cycles (lanes 2, 5, 8), and 29 cycles (lanes 3, 6, 9).

Fig. 7

FACS analysis of μ → γ3 and μ → γ1 CSR in AID^+/+, AID-transgenic/AID^−/−, and AID^−/− mice. Splenic B cells were isolated from age-matched mice and activated with LPS or LPS+IL4 for 5 days then analyzed by FACs for CSR. Results are representative of three independent experiments. Flow cytometric analyses of IgG3- (top) or IgG1- (bottom) expressing B cells and the percentages of positive cells are indicated in each diagram.