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Research Projects
The APOBEC-1 homolog AID is required for the mutation and recombination of immunoglobulin genes

Recent studies have linked class switch recombination (CSR) and somatic hypermutation (SHM) through the expression of Activation Induced Deaminase (AID). AID induced CSR and SHM in transgenic AID-/- mouse splenic B cells, suggesting that AID has a determinant and perhaps common role in these pathways. AID also induced CSR on reporter constructs in fibroblasts, thereby demonstrating the ubiquitous expression of factors that are targets for AID regulation and downstream events. Based on AID's homology to APOBEC-1 (the catalytic subunit involved in apoB mRNA editing) and AID's cytidine deaminase activity, it has been predicted that AID deaminates (edits) cytidine to form uridine on a yet-to-be identified mRNA. However, evidence to date favors AID as a deoxycytidine deaminase (DNA editing enzyme). AID modifies dC to dU of single stranded DNA within transcription ‘bubbles’ in experimental systems. It is hypothesized that AID activity recruits proteins involved in DNA repair and DNA recombination to specific sites within the Ig gene and thereby promotes SHM and CSR.

A role for AID in SHM and CSR as an RNA editing enzyme has not been ruled out. The strongest support for this hypothesis is that SHM and CSR require de novo protein synthesis is following AID activity. Editing could either enabling the expression of a non-genomically encoded protein variant activator or impair the expression of a suppressor protein by editing the sequence of an mRNA. Editing could therefore thereby induce critical events in the generation of CSR and SHM.

Research in the Smith lab is evaluating the DNA and RNA editing hypotheses and with a specific focus on factors that regulate targeting of AID activity to select sequences and its trafficking within the cell. The significance of this research is that it addresses critical mechanisms of human B cell CSR and SHM, which are essential processes in human antibody responses. In addition, the results have significance for understanding human immunodeficiency in which CSR and/or SHM is affected and the development of human B-cell malignancies that arise from a contribution from these processes.

 

      Immunoglobulin VDJ recombination occurs in B lymphocytes in the absence of AID expression. The expression of AID controls antibody diversity through multiple gene rearrangements involving mutation of DNA sequence and recombination. The initial expression of antibodies requires immunoglobulin (Ig) gene rearrangement that is AID-independent (1). This occurs in immature B lymphocytes developing in fetal liver or adult bone marrow and requires DNA double strand breaks at the Ig heavy chain locus whose ends are rejoined by non-homologous end joining. The rearranged immunoglobulin V (variable), D (diversity) and J (joining) gene segments encode a variable region that is expressed initially with the mu (µ) constant region (Cµ) to form a primary antibody repertoire composed of IgM antibodies. In humans and many mammals, AID-dependent gene alterations occur in B lymphocytes that are growing in germinal centers of secondary lymphoid organs following antigen activation. This involves multiple mutations of the variable region through Somatic Hypermutation (SHM) as well as removing the Cµ and replacing it with one of several other constant regions (Ca, Cd, Ce or Cg) through a recombination process known as Class Switch Recombination, CSR. In sheep, rabbits and chickens, pre-immune Ig gene diversification is mediated by an AID-dependent process known as gene conversion (GC) in which stretches of nucleotide sequences from one of several pseudogene V elements are recombined into the VDJ exon to generate diversity (for diagrams and further review see references 2 and 3). The mechanism of action of AID and its targets remain controversial. Although evidence is compelling that AID mutates DNA during SHM and CSR a role for AID in mRNA has not been ruled out (4,5).

  • (1) Muramatsu, M., Kinoshita, K., Fagarasan, S., Yamada, S., Shinkai, Y. and Honjo, T. (2000). Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102:553-563.
  • (2) Fugmann, S.D. and States, D.G. (2002). Immunology. One AID to unite them all. Science 295:1244-1245.
  • (3) Honjo, T., Kinoshita, K. and Muramatsu, M. (2002). Molecular Mechanism of Class Switch Recombination: Linkage with Somatic Hypermutation. Ann. Rev. Immunol. 20:165-196.
  • (4) Galloway, C.A., Sowden, M.P. & Smith, H.C. Increasing the Yield of Soluble Recombinant Protein Expressed in E. coli by Induction During Late Log Phase. BioTechniques 34: 524-530 (2003).
  • (5) Smith, H.C., Bottaro, A, Sowden, M.P. & Wedekind, J.E. Activation induced deaminase: the importance of being specific. Trends in Genetics 20:224-227 (2004).