AID-mediated diversification within the IgL locus of chicken DT40 cells is restricted to the transcribed IgL gene

Abstract

Somatic hypermutation (SHM) and gene conversion (GCV) are closely related processes that increase the diversity the primary immunoglobulin repertoire. In both processes the activationinduced cytidine deaminase (AID) converts cytosine residues to uracils within the DNA of the immunoglobulin (Ig) genes in a transcription-dependent manner, and subsequent error-prone repair processes lead to changes in the antigen recognition site of the encoded receptors. This activity is specifically recruited to the Ig loci by unknown mechanisms. Our analyses of the chicken B-cell line DT40, and derivatives thereof, now revealed that even the closest neighbors of the Ig light chain (IgL) gene are protected from AID activity, albeit being transcribed and thus acting as potential targets of AID. Our findings are in support of a model in which cis-acting DNA boundary elements restrict AID activity to the IgL locus and guard the genome in the vicinity of the IgL gene from deleterious mutations.

Classical Mus musculus Igκ Enhancers Support Transcription but not High Level Somatic Hypermutation from a V-Lambda Promoter in Chicken DT40 Cells

“…As some of the GCV and non-templated SHM events do not result in a reversion of the stop codon, we also determined the frequency of mutation events by sequencing the VJ exon of the IGL locus at the end of the four week culture. The mutation frequency of 0.363×10 −4 events/bp is dramatically lower than that observed in the wild-type cells (4.07×10 −4 events/bp, [13] ), and is even below the background level of our system (0.5×10 −4 events/bp, observed in AID-deficient DT40 cells, [23] ) ( Fig. 2E ).…”

“…E) Mutation event frequencies in the IGL (filled bars) and IGH (open bars) loci were determined by sequencing the VJ or VDJ exon from two independent subclones after four weeks of continuous culture. The background level of 0.5×10–5 events/bp for this system was determined in AID-deficient DT40 cells [23] , and is shown as a dotted line. The data for wild type (WT) and ΔME6K were obtained from [13] .…”

Separation of Mutational and Transcriptional Enhancers in Ig Genes

“…DNA sequencing confirmed this absence of SHM and GCV (Fig. 2C), with mutation frequencies being measured below the background of 5×10 −5 events/bp observed in AID −/− DT40 cells (23). The IGH locus mutation frequency of 3.47×10 −4 events/bp (Fig.…”

“…The compiled frequencies are obtained from the analyses of two independent clones of each genotype. The background levels of 5×10 −5 events/bp is shown as a dotted line and was obtained by sequencing the IGL locus of AID −/− DT40 cells after four weeks of culture (23). (D) Surface IGM reversion analysis was done by flow cytometry using PE-labeled anti chicken IGM antibodies.…”

A cis-Acting Diversification Activator Both Necessary and Sufficient for AID-Mediated Hypermutation

“…The hypermutation activity of DT40 appeared however to be limited to the IgL locus, because no mutations were found in the highly transcribed Elongation Factor 1 alpha gene (NP_989488) [19]. This was confirmed by a recent study showing that neither the VpreB3 (NC_006102) nor the Carbonic Anhydrase (XP_415218) gene, immediate upstream and downstream neighbors of the IgL locus respectively, showed sequence heterogeneity in DT40 [21].…”