Understanding the Immunoglobulin Locus Specificity of Hypermutation

Abstract

The immunoglobulin (Ig) genes of B cells are diversified at high rate by point mutations whereas the non-Ig genes of B cells accumulate no or significantly fewer mutations. Ig hypermutations are critical for the affinity maturation of antibodies for most of jawed vertebrates and also contribute to the primary Ig diversity repertoire formation in some species. How the hypermutation activity is specifically targeted to the Ig loci is a long-standing debate. Here we describe a new experimental approach to investigate the locus specificity of Ig hypermutation using the chicken B-cell line DT40. One feature is the use of a green fluorescent protein (GFP) gene as a mutation reporter. Some nucleotide changes produced by somatic hypermutation can cripple the GFP gene which leads to a decrease or loss of the green fluorescence. Therefore such changes can be easily quantified by fluorescence-activated cell sorting (FACS). Another advantage of this approach is the targeted integration of the mutation reporter into a defined chromosomal position. This system allowed us to identify a 10 kb sequence within the Ig light chain (IgL) locus, which is both necessary and sufficient to activate hypermutation in the neighboring reporter gene. We have called this sequence Diversification Activator (DIVAC) and postulated that similar cis-acting sequences exist in the heavy and light chain Ig loci of all jawed vertebrate species. Our experimental system promises further insight into the molecular mechanism of Ig hypermutation. For example, it may be possible to identify smaller functional motifs within DIVAC and address the role of putative transacting binding factors by gene knock-outs.

Network properties derived from deep sequencing of human B-cell receptor repertoires delineate B-cell populations

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Recombinase-Mediated Cassette Exchange as a Novel Method To Study Somatic Hypermutation in Ramos Cells

“…This is preferably done within the endogenous locus, since all of the cis -acting sequences required for this process have yet to be determined. Antibody-forming B cell lines in tissue culture allow the examination of multiple different engineered versions of parts of the Ig gene, but such experiments require the repeated replacement of the wild-type (WT) Ig gene by the modified H or L chain genes (14). Currently, this is most often done using the DT40 cell line, which has a high frequency of homologous recombination (15, 16).…”

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“…Complementarity-determining region 3 (CDR3) is a part of the rearranged BCR critical for antigen recognition. Antigen-specific BCRs can develop improved affinity through somatic hypermutation and clonal affinity selection resulting in increased diversity, and the sequences of these clones will be highly related (11,12). Analysis of these antibodies could reveal clonal families of B cells that persist over time and show evidence of progressive affinity maturation and class switching.…”