Genomic instability due to V(D)J recombination-associated transposition - PubMed (original) (raw)

Genomic instability due to V(D)J recombination-associated transposition

Yeturu V R Reddy et al. Genes Dev. 2006.

Abstract

The first step in assembling immunoglobulin and T-cell receptors by V(D)J recombination has similarities to transposon excision. The excised transposon-like element then integrates into DNA targets at random in vitro, but whether this activity significantly threatens the genomic integrity of its host has been unclear. Here, we recover examples where the putative transposon associated with V(D)J recombination integrated into the genome of a pre-B-cell line. Transposition accounted for a surprisingly high proportion (one-third) of integrations, while most of the remaining events had parallels to other aberrant V(D)J recombination pathways linked to oncogenic translocation. In total, transposition occurred approximately once every 50,000 V(D)J recombinations. Transposition may thus contribute significantly to genomic instability.

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Figures

Figure 1.

Figure 1.

Recovery of integrations of a transposon-like fragment. (A) The assay for recovering potential transposon integrations is described. (Boxes) Coding sequence; (filled triangle) 12-type recombination signal (12-RS); (open triangle) 23-type recombination signal (23-RS); (puror) intact gene for puromycin resistance; (zeorGFP) fusion gene that confers zeocin resistance and green fluorescence. In B and C, DNA from subclones resistant to puromycin and zeocin after induction of V(D)J recombination (numbered as in Table 2) were digested with EcoRI (cuts once 5′ of the puror gene) and PstI (cuts only in flanking chromosomal DNA), Southern blotted, and analyzed with a probe from the puror gene (B) or zeorGFP gene (C). DNA from the parental line (P) and the initial clone with unrearranged substrate (clone A) are included for comparison. The mobility of molecular weight markers (in kilobase pairs) are shown at the right of each panel.

Figure 2.

Figure 2.

Integrations consistent with transpositions. (A) For each of the identified integration events (#, numbered as in Table 2) we report the number of base pairs deleted from 12-RS and 23-RS ends of the zeorGFP fragment (ΔRS 12/23), the chromosome where the fragment integrated (Ch.), a brief description of genomic DNA immediately flanking 12-RS ends and 23-RS ends, and the amount of genomic DNA between integration flanks (Δ genomic DNA). The sequences of flanking target site duplications are in bold. At the bottom is a graphic summarizing the typical integration structure. (*) As described in detail in footnote c of Table 2, the repetitive nature of sequences flanking integration #6 did not allow for unambiguous location of flanks within the region. (B) Pathway for integration by transposition. Ovals represent RAG proteins.

Figure 3.

Figure 3.

Integration into an inverted repeat. (A) The structure of integration #7 is summarized as in Figure 2A. (B) Pathway for integration by transposition into a hairpin-forming sequence. Ovals represent RAG proteins. (C) A 415-bp chromosome 5 fragment that contained the targets for cellular integration #7 is represented as a line. The termini of the cloned fragment are defined by nucleotide numbers that map to the sequence in accession AC115293. The approximate location of (CA)N and (TG)N repeats within this fragment are noted by thick lines (see also footnote d of Table 2). The locations of cellular integration sites are noted above the line, while the locations of in vitro-defined integrations are noted with open triangles below the line. (D) Seven-hundred base pairs of a chromosome 7 fragment (from AC109232) that contained the target for cellular integration #8 are represented as in C. The site of integration of the 12-RS of the zeorGFP fragment is noted above the line. The 23-RS flank could not be located in this region (see footnote e of Table 2).

Figure 4.

Figure 4.

Other nontargeted integrations. The structures of integration #8–11 are summarized as in Figures 2A and 3A. A possible inserted nucleotide (nontemplated) is noted in lowercase. (*) Sequences flanking the 23-RS for #8 and #9 were derived from Moloney Murine leukemia virus genes, and could not be located near the 12-RS flanks. (See also footnote e of Table 2.)

Figure 5.

Figure 5.

Integration by intermolecular V(D)J recombination. (A) The structure of integrations #12–14 are summarized as in Figures 2, 3, 4. Rectangles represent flanking immunoglobulin (Ig) locus coding segments, and triangles represent flanking Ig locus recombination signals. The numbers in parentheses within these symbols refer to the base pairs of the genomic sequence deleted relative to the site of RAG protein cleavage. (*) Integration #14 is the product of two recombinations, as described in Supplementary Figure 2. B and C describe how intermolecular recombination can similarly lead to both integration of the zeorGFP fragment (B) as well as translocations between receptor loci and oncogene (onc) loci (C). Ovals represent RAG proteins, while boxes represent NHEJ proteins.

Figure 6.

Figure 6.

Integration by end donation. (A) The structure of integrations #15–21 are summarized as in Figures 2, 3, 4, 5. Rectangles represent flanking Ig locus coding segments, and triangles represent flanking Ig locus recombination signals. The numbers in parentheses within these symbols refer to the base pairs of the genomic sequence deleted relative to the site of RAG protein cleavage. (*) Sequence flanks a cryptic 23-type signal within the Vκ region: CACtGTG (23 bp) AgAAAAACC (nucleotides that match consensus RS in capital letters). B and C describe how end donation can similarly lead to both integration of the zeorGFP fragment (B) as well as translocations between receptor loci and onc loci (C). Boxes represent NHEJ proteins.

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