GANP-mediated recruitment of activation-induced cytidine deaminase to cell nuclei and to immunoglobulin variable region DNA

Abstract

AID (activation-induced cytidine deaminase) catalyzes transcription-dependent deamination of C --> U in immunoglobulin variable (IgV) regions to initiate somatic hypermutation (SHM) in germinal center B-cells. SHM is essential in generating high affinity antibodies. Here we show that when coexpressed with GANP (germinal center-associated nuclear protein) in COS-7 cells, AID is transported from the cytoplasm and concentrated in the nucleus. GANP forms a complex with AID in cotransfected cells in vivo and in vitro. We have isolated AID mutants that bind with reduced affinity to GANP compared with wild type AID. One of these mutants, AID (D143A) binds GANP with a 10-fold lower affinity compared with wild type AID yet retains substantial C-deamination activity in vitro. Mutant AID (D143A) remains localized predominantly in the cytoplasm when coexpressed with GANP. Exogenous expression of GANP in Ramos B-cells promotes binding of AID to IgV DNA and mRNA and increases SHM frequency. These data suggest that GANP may serve as an essential link required to transport AID to B-cell nuclei and to target AID to actively transcribed IgV regions.

FIGURE 1.

GANP interacts with AID in vivo and in vitro. A, AID co-IP with GANP. Lysates from COS-7 cells, transfected with GFP-GANP, AID, GFP-GANP + AID, or GFP-alone constructs, were immunoprecipitated using anti-GFP Ab. Immunoblotting (IB) was carried out with anti-GFP Ab, anti-AID Ab, or anti-β-actin Ab by IP-Western blot analysis. Specific association in the IP with the anti-GFP Ab was confirmed by comparing with experiments using the control IgG (bottom panel). Western blots for whole cell lysates (WCL) are shown. B, reverse IP-Western blot analysis was performed by IP with anti-AID Abs followed by immunoblot with anti-GFP Ab or anti-AID Ab. The specific signal representing 240-kDa GFP-GANP was detected in immunoprecipitates using both mouse anti-AID Abs (M mAb) and rabbit anti-AID Abs (R mAb). The anti-AID (M) mAb contains the IgL band (25-kDa) used for IP as indicated with an asterisk. C, the WCL from COS-7 cells transfected with GFP-GANP and AID constructs was incubated for 10 min with either RNase A (5 μg/ml (+R) or 250 μg/ml (++R)) or DNase I (10 units (+D) or 50 units (++D)) at 4 °C or 37 °C and then immunoprecipitated with anti-GFP Ab followed by immunoblot with anti-GFP or anti-AID (M) Ab. D, interaction of endogenous GANP and AID was examined in Ramos B-cells by co-IP. The asterisk shows the IgL band. E and F, FLAG-GANP and AID proteins were synthesized in vitro using a WGE cell-free system, and coimmunoprecipitated using anti-AID (M) Ab (E) or anti-FLAG Ab (F). Controls for specificity of co-IP experiments using mouse (M) IgG are shown in the bottom panels (E and F). G, the interaction between WGE-produced GANP and AID was still preserved after treatment with RNase A or DNase I. The data were determined from three independent experiments.

FIGURE 2.

Mutant AID (D143A) binds GANP with significantly reduced affinity compared with wild type AID while retaining substantial C-deamination activity. A, schematic representation of the AID domain structure. B, the ability of mutant AID (D143A) to interact with FLAG-GANP was compared with the WT AID by co-IP using anti-FLAG Ab. AID (D143A), AID (WT), and FLAG-GANP were produced using a WGE. The presence of AID in the co-IP precipitate was examined by anti-AID (M) mAb. The asterisk shows the IgL band. AID (D143A) migrated slightly faster than WT AID in our gel condition. C, deamination activity of AID (WT) and AID (D143A) protein was measured using a ³²P-labeled 36-nt ssDNA substrate containing a single C target. ssDNA (30 nm) was incubated with 10 ng of purified Sf9-expressed AID (WT or D143A) protein in the presence of RNase A (20 ng) for 5 min at 37 °C. Left graph, the deaminated product (14 nt) was separated from the 36-nt substrate by denaturing PAGE and visualized by phosphorimaging. Right graph, relative specific activity of mutant AID compared with WT AID. The data were from three independent experiments.

FIGURE 3.

Localization of GANP and AID in COS-7 cells. A, the cells were singly transfected or cotransfected with AID (WT)-DsRed (top panels) or AID (D143A)-DsRed (middle panels) in combination with GFP-GANP. Transfectants were observed under microscopy for 40 h. As a control, HSP25-DsRed (bottom panels) remains strictly cytoplasmic in the absence or presence of coexpressed GFP-GANP. The fluorescence signals were merged with DNA staining after adding Hoechst 33342 in the culture. The scale bar represents 20 μm (right bottom; white) as shown in the merged pictures. B, subcellular localization of GANP, AID (WT or D143A), and HSP25 was shown by the percentage score as predominantly cytoplasmic (C; white bars), nuclear (N; black bars), or in both (N+C; gray bars) from the indicated number of examined transfected cells (n) in multiple microscope fields 40 h after transfection. The significant change (p < 0.05) of the nuclear localization of AID (D143A) is marked by an asterisk. C, nuclear localization of AID in GC B-cell from ganp-deficient mice (CD19-Cre/ganp^F/F) and ganp^Tg mice compared with the littermate control (ganp^F/F). Mice (n = 3 for each genotype) were immunized with sheep red blood cells, and the splenic GC B-cells (B220⁺GL7⁺Fas⁺) were isolated by cell sorting. The nuclear (N) and cytoplasmic (C) fractions were prepared and subjected to Western blotting (IB) with anti-AID (R) (rabbit), anti-β-tubulin, and anti-histone H3 Ab (left gels). The relative level of AID localized in the nucleus is expressed as a ratio of AID/histone H3 integrated band intensities (right graph). The error bars (S.E.) and p values were determined from three independent experiments.

FIGURE 4.

Increased GANP expression in Ramos B-cells promotes binding of AID to IgV DNA and mRNA. A, ChIP was used to detect the binding of GANP and AID to IgV_H mRNA. Aliquots of ChIP samples were treated with DNase I for IgV mRNA (RNA-ChIP) and PCR-amplified with specific primers following reverse transcription. B, RNA-ChIP-qPCR analysis of Ramos cell transfectants with the GFP-GANP or the GFP-alone control construct. C, DNA-ChIP was used to detect the binding of GANP and AID to IgV_H region DNA. Aliquots of ChIP samples were treated with RNase A before PCR amplification with IgV-specific primers. D, DNA-ChIP-qPCR analysis for IgV_H region and control cd4 gene of Ramos cell transfectants with the GFP-GANP or the GFP-alone control construct. Error bars, S.E.

FIGURE 5.

Mutation analysis of SHM of IgV_H region gene in Ramos B-cells transfected with gfp alone or gfp-ganp constructs. The patterns of nucleotide substitutions in the genomic IgV_H region of GFP and GFP-GANP transfectants are shown (input). For GFP-GANP transfectants, IgV_H region DNA, associated with GFP-GANP or AID, was isolated from IP precipitates using anti-GFP Ab or anti-AID Ab and sequenced. The number in the center of the pie chart indicates the number of IgV_H region sequences analyzed, and the color of each part of the pie indicates the mutation number of each sequence as 0 (blue), 1 (red), 2 (yellow), and 3 (green). The percentage frequencies are shown. The mutation frequency at WRC hot spots (WRC and GYW) was counted and estimated for the top and bottom strands.

FIGURE 6.

A model of AID targeting by GANP. AID and GANP are induced in GC B-cells. AID is abundant in the cytoplasm and becomes localized at the nucleus in the presence of cofactors, including GANP. The observation that GANP binds to IgV DNA and RNA and to AID and that the binding of AID to IgV DNA is enhanced in the presence of GANP, accompanied by a 6-fold increase in SHM, suggests that GANP plays an important role in targeting AID to actively transcribed IgV DNA.