A cis-acting diversification activator both necessary and sufficient for AID-mediated hypermutation

Abstract

Hypermutation of the immunoglobulin (Ig) genes requires Activation Induced cytidine Deaminase (AID) and transcription, but it remains unclear why other transcribed genes of B cells do not mutate. We describe a reporter transgene crippled by hypermutation when inserted into or near the Ig light chain (IgL) locus of the DT40 B cell line yet stably expressed when inserted into other chromosomal positions. Step-wise deletions of the IgL locus revealed that a sequence extending for 9.8 kilobases downstream of the IgL transcription start site confers the hypermutation activity. This sequence, named DIVAC for diversification activator, efficiently activates hypermutation when inserted at non-Ig loci. The results significantly extend previously reported findings on AID-mediated gene diversification. They show by both deletion and insertion analyses that cis-acting sequences predispose neighboring transcription units to hypermutation.

Figure 1. Hypermutation of the GFP2 reporter at various distances from the rearranged IgL locus.

(A) A physical map of the rearranged IgL locus, a targeting construct including the GFP2 reporter and the IgL locus after targeted insertion of the GFP2 reporter. (B) Locations of GFP2 insertion relative to the IgL locus on chromosome 15. The reference point is the insertion site of GFP2 in the IgL^GFP2 transfectant. (C) FACS analysis of primary transfectants having integrated the transfected construct targeted. (D) Fluctuation analysis of subclones. Each dot represents the analysis of a single subclone and the median of all subclones from the same primary transfectant is indicated by a bar.

Figure 2. GFP2 hypermutation after deletion and reconstitution of the rearranged IgL locus.

(A) Design of deletions and insertions in the rearranged IgL locus using GFP2 targeting constructs. Upper part: Physical maps of the rearranged IgL locus after the deletion of the ψV locus, a targeting construct including the GFP2 reporter and the locus after targeted integration. Middle part: Physical maps of the deleted rearranged IgL locus, a targeting construct including the GFP2 reporter and the GFP2 insertion site after targeted integration. Lower part: Physical maps of the deleted rearranged IgL locus, a targeting construct including the GFP2 reporter together with the ‘W’ fragment and the locus after targeted integration. (B) FACS analysis of primary transfectants. (C) Sorting of GFP-high and GFP-low cells from a ψV⁻IgL^GFP2 primary transfectant, and semi-quantitative RT-PCR of GFP and EF1α messages from sorted and non-sorted cells. (D) Fluctuation analysis of subclones. (E) The frequencies of particular nucleotide substitutions within the GFP open reading frame of GFP2.

Figure 3. Analysis of the ‘W’ fragment deletion series.

(A) A physical map of the deleted IgL locus and the aligned targeting constructs leading to the insertion of the GFP2 reporter together with parts of the ‘W’ fragment. (B) FACS analysis of representative primary transfectants. (C) Fluctuation analysis of subclones. Only the letter of the reconstituted fragment and not the full name of the transfectants is indicated for clarity.

Figure 4. Insertions of the GFP2 reporter into non-Ig loci.

(A) Chromosomal locations of the GFP2 insertions. (B) FACS analysis of primary transfectants. (C) Fluctuation analysis of subclones.

Figure 5. Hypermutation of the GFP2 reporter at non-Ig loci in the presence of the ‘W’ fragment.

(A) Physical maps of the non-Ig loci, the targeting constructs including the GFP2 reporter together with the ‘W’ fragment and the loci after targeted insertion of the GFP2 reporter. (B) FACS analysis of primary AID positive and AID negative transfectants. (C) Fluctuation analysis of subclones.