Regulation of aicda expression and AID activity: relevance to somatic hypermutation and class switch DNA recombination

Abstract

Expression and activity of activation-induced cytidine deaminase (AID) encoded by the aicda gene are essential for immunoglobulin (Ig) gene somatic hypermutation (SHM) and class switch DNA recombination (CSR). SHM and CSR unfold, in general, in germinal centers and/are central to the maturation of effective antibody responses. AID expression is induced by activated B-cell CD40 signaling, which is critical for the germinal center reaction, and is further enhanced by other stimuli, including interleukin-4 (IL-4) secreted from CD4+ T cells or Toll-like receptor (TLR)-activating bacterial and/or viral molecules. Integration of different intracellular signal transduction pathways, as activated by these stimuli, leads to a dynamic aicda-regulating program, which involves both positively acting trans-factors, such as Pax5, HoxC4, E47, and Irf8, and negative modulators, such as Blimp1 and Id2, to restrict aicda expression primarily to germinal center B cells. The phosphatidylinositol 3-kinase (PI 3-K), which functions downstream of activated B-cell receptor (BCR) signaling, likely plays an important role in triggering the downregulation of aicda expression in postgerminal center B cells and throughout plasmacytoid differentiation. In B cells undergoing SHM and CSR, AID activity, and, possibly, AID targeting to the Ig locus are regulated at a posttranslational level, including AID dimerization/oligomerization, nuclear/cytoplasmic AID translocation, and phosphorylation of the AID Ser38 residue by protein kinase A (PKA). Here, we discuss the role of B-cell activation signals, transcription regulation programs, and posttranslational modifications in controlling aicda expression and AID activity, thereby delineating an integrated model of modulation of SHM and CSR in the germinal center reaction.

FIGURE 1

Induction of aicda expression by activated B cell surface molecules. From left to right: (i) the TLR4-MD2-CD14 complex is the receptor for LPS or other bacterial glycoproteins; TLR9 is located in endosomes and recognizes CpG DNA from microbial pathogens. TLR4 and TLR9 associate with adaptor protein MyD88 or Trif through the Tir domain, depicted in yellow; (ii) the BCR complex containing BCR, Igα and Igβ is crosslinked by physically-linked antigens. The CD19/CD21/CD81 co-receptor is activated by the C3d(g) complement cleavage product and modulates the BCR signaling threshold. CD81 is a co-receptor for HCV E2 envelope protein; (iii) cytokine receptors IL-4R and TGF-βR mediate aicda induction and CSR. IL-4R-triggered signaling can be inhibited by activated CD45; and (iv) among the TNF receptor family of B cell surface molecules, CD40 is engaged by CD154 expressed on CD4⁺ T cell surface and exists as a trimer at B cell surface. CD30, when engaged by CD153 expressed CD40⁺ T_H cell surface, negatively regulates CD40 signaling. TACI, BAFF-R and BCMA are receptors for BAFF and/or APRIL secreted by dentritic cells and can mediate CSR in B cells of mucosal areas. Activated intracellular signal transduction pathways likely converge to assemble an enhanceosome, which would include NF-κB, Stat6, Pax5, E47, the Irf8/SpiB complex and the HoxC4/Oct1/2/OcaB complex, at the aicda locus to induce efficient aicda expression. The arrows depict the transcription initiation of aicda or other germinal center B cell-specific genes.

FIGURE 2

Germinal center B cell differentiation, including SHM and CSR, stage-specific expression of AID, and positive regulators (depicted in blue) and negative modulators (depicted in gray) of aicda gene transcription. AID expression initiates in early centroblasts, is maximal in full-blown centroblasts, significantly decreases in centrocytes and extinguishes in plasma cells. The expression profile of AID and transcription factors in memory B cells is not shown here. The dual role of OcaB and Irf4 is dependent on different germinal center reaction stages. Most data are derived from studies in human tonsil or mouse spleen B cells and plasma cells. The expression profile of Id2 was not available and is deduced from those of Irf4 and Bcl6, two putative id2 transcription regulators.

FIGURE 3

Downregulation of aicda expression by Blimp1 and B cell plasmacytoid differentiation. (a) Expression profiles of genes that regulate aicda expression in germinal center B cells (left panel) and plasmacytes (right panel). Positive regulators and negative regulators of aicda transcription at each stage of the germinal center reaction are depicted in solid green and red circles, respectively. Broken circles depict genes with a low steady-state expression level. Solid and broken arrows/lines indicate direct and indirect regulation of gene expression, respectively. In germinal center B cells, multiple transcription factors synergize to induce aicda expression. In plasmacytes, the transcription suppressor Blimp1 downregulates expression of both aicda and the positive regulators of aicda expression, such as pax5, e2a and irf8 and upregulates negative regulators of aicda expression, such as id2. Irf4 switches from a positive to a negative regulator of aicda expression during plasmacytoid differentiation and its gradient-like expression pattern in light zone B cells is depicted as a light green circle. c-myc is one of the germinal center B cells-specific genes and cd138 (syndecan1); other genes involved in protein folding and secretion are highly expressed in plasmacytes. (b) Blimp1-binding site in the promoter of its direct target genes: “*” denotes predicted Blimp1-binding sites and “**” denotes the consensus Blimp1-binding motif. Underlined nucleotides are present in all Blimp1-binding sites and likely contribute to Blimp1-DNA interaction that is essential for the transcription regulation activity of Blimp1.

FIGURE 4

Predicted three-dimensional structure of the human AID dimer (Egest, Xu and Casali, unpublished data). The structure of amino acid residues 11 to 180 of AID is modeled, as based on the solved crystal structure of the human Apobec2 (PDB No: 2NYT) (33.3% identity and 44% homology). The structure of the carboxyl-terminal NES (amino acid residues 184–198) is modeled, as based on the solved crystal structure of the human p53 tetramerization domain (PDB No: 1C26) (33% homology), which contains a helical NES. The Pro 183 of AID likely hampers the formation of a long helix spanning carboxyl-terminal amino acid residues 168–198. Shown are the side chains of His56, Glu58, Cys87 and Cys90, which forms the active site in the AID cytidine deamination domain. His56, Cys87 and Cys90 coordinate with a Zn²⁺ ion, which is essential for the AID activity. The conformational change of the β1′-turn from a loop to a hairpin, as shown in purple, likely controls the access of the AID active site to DNA/RNA substrates. The β2 strand (amino acid residues Ser 41 to Asn 51) of each AID monomer mediates the dimerization. Shown is the side chain of Ser38, the PKA phosphorylation site.

FIGURE 5

An integrated model of AID post-translational modifications. AID forms dimers/oligomers spontaneously. Most AID molecules localize in the cytoplasm, likely through active nuclear export, as effected by CRM1. Phosphorylation of cytoplasmic AID dimers by PKA would result in efficient nuclear import. After localizing to the nucleus, phosphorylated AID dimers binds to RPA and a putative AID cofactor, depicted as a dimer, to effect its DNA deaminase functions. Alternatively, PKA-mediated phosphorylation of cytoplasmic AID dimers increases AID binding to the putative cofactor before translocation into the nucleus, where the AID-cofactor complex binds to RPA and then deaminates dC in DNA. After deamination, AID is released, possibly dephosphorylated by a phosphatase and then exported out of the nucleus to replenish the cytoplasmic AID reservoir.