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 3 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.Affinity maturation of the humoral response proceeds by diversification of Ig genes (1, 2). Diversification requires AID 3 -initiated somatic hypermutation (SHM) within IgV regions and concomitant class switch recombination in Ig switch regions (S regions) (3). AID initiates SHM and class switch recombination by deaminating deoxycytidine residues during transcription of the Ig locus (4, 5). AID deaminates dC 3 dU on single-stranded DNA (ssDNA) and during the transcription of double-stranded DNA (6 -8), presumably on the non-transcribed strand. AID favors deamination in WRC (where W represents A/T, and R is A/G) hot spot motifs (7). The observation of strongly enhanced C 3 T mutations in variable regions and S regions, which conform to the mutational signature of AID (7), offers convincing evidence that AID is acting directly on the transcribed DNA. AID is located predominantly in the cytoplasm of activated B-cells (9) and has a nuclear localization signal motif in its N-terminal region and a nuclear export signal at its C terminus (10 -12). Deletion of the nuclear export signal results in nuclear accumulation (11, 12) but does not result in an increase in mutations in S regions (11). There must be a means to regulate the import of AID into the nucleus and then to direct its access to transcribed Ig loci. Presumably, AID is recruited to specific Ig regions by a variety of targeting mechanisms that could include cis-acting transcription factors, proteins that associate with AID, and regions of ssDNA formed by transcription bubbles (4, 5). There are coimmunoprecipitation (co-IP) data showing interactions between AID and potential recruiter molecules, including RNA polymerase II (13), a spliceosome-associated factor CTNNBL-1 (14), ssDNA-binding protein (RPA) (8, 15), and protein kinase A (16). However, it remains unclear how AID is targeted preferentially to actively transcribed Ig genes; no...

Lyn Signaling To Upregulate GANP Is Critical for the Survival of High-Affinity B Cells in Germinal Centers of Lymphoid Organs

“…GANP interacts with AID in the cytoplasm and is involved in recruiting AID to the nucleus and to the IgV H -region DNA in B cells (29). GANP is involved in mRNA export in mammalian cells (30) and particularly in the selective export of Shugoshin-1 mRNA required for cell cycle progression through the M phase (20).…”

Activation-induced cytidine deaminase (AID)-dependent somatic hypermutation requires a splice isoform of the serine/arginine-rich (SR) protein SRSF1

“…S6). With respect to AID recruitment to the IgV gene, in vivo evidence has been limited except for experiments using Ramos human lymphoma cells (44). By careful optimization of ChIP conditions, a weak AID signal was detected on the IgV gene, and this was not further enhanced by SRSF1-3 expression (Fig.…”

GANP Regulates the Choice of DNA Repair Pathway by DNA-PKcs Interaction in AID-Dependent IgV Region Diversification

“…For GFP or GANP overexpression (GANP O/E ) transfectants, a GFP or GFP-GANP gene was introduced into pCXN2 (chicken b-actin promoter) vector containing a neomycin-resistant cassette (33). The AID expression vector (pFB-AID-IRES-GFP) was a gift from Dr. Tasuku Honjo (Kyoto University, Kyoto, Japan).…”

“…Recently, we demonstrated that GANP interacts with cytoplasmic AID and assists its nuclear localization to augment its transport to the transcribed IgV region segment in B cells (33). In this process, the histone acetyltransferase region of GANP is involved in the selective recruitment of GANP toward the terminal region of the rearranged IgV region exons and the site-selective acetylation of the linker histone H1 (34).…”