Mobilization of RAG-generated signal ends by transposition and insertion in vivo

Abstract

In addition to their essential roles in V(D)J recombination, the RAG proteins have been found to catalyze transposition in vitro, but it has been difficult to demonstrate transposition by the RAG proteins in vivo in vertebrate cells. As genomic instability and chromosomal translocations are common outcomes of transposition in other species, it is critical to understand if the RAG proteins behave as a transposase in vertebrate cells. To facilitate this, we have developed an episome-based assay to detect products of RAG-mediated transposition in the human embryonic kidney cell line 293T. Transposition events into the target episome, accompanied by characteristic target site duplications, were detected at a low frequency using RAG1 and either truncated "core" RAG2 or full-length RAG2. More frequently, insertion of the RAG-generated signal end fragment into the target was accompanied by deletions or more complex rearrangements, and our data indicate that these events occur by a mechanism that is distinct from transposition. An assay to detect transposition from an episome into the human genome failed to detect bona fide transposition events but instead yielded chromosome deletion and translocation events involving the signal end fragment mobilized by the RAG proteins. These assays provide a means of assessing RAG-mediated transposition in vivo, and our findings provide insight into the potential for the products of RAG-mediated DNA cleavage to cause genome instability.

FIG. 1.

(A) Episomal assay for RAG-mediated transposition. 293T cells were transfected with four plasmids: RAG1 and RAG2 expression vectors, a donor plasmid harboring two RSSs flanking a tetracycline resistance gene (pTetRSS*), and a target plasmid (pECFP-1*). RSSs are indicated as triangles and genes as rectangles. DNA isolated from transfected cells was digested with BglII (which cuts only in the donor backbone), transformed into bacteria, and grown on agar plates containing KTS. Under these conditions, only bacteria harboring a target plasmid into which the Tet^r gene of the donor was inserted should survive. Bacteria harboring both the donor and target plasmids should be eliminated by streptomycin by virtue of the rpsL gene of the donor. Plasmid DNA extracted from KTS^r bacterial colonies was analyzed by restriction digestion and sequenced to determine the nature and site of signal end integration into the target plasmid. (B) Schematic representation of the transposition product.

FIG. 2.

Map of sites of transposition in the episomal assay. Transposition sites are marked with black squares. ECFP, promoterless enhanced cyan fluorescent protein gene; SV40 ori, SV40 origin of replication; f1 ori, f1 single-stranded DNA origin; P KanR, bacterial promoter for expression of the Kan^r gene; KanR, kanamycin resistance gene; pUC ori, pUC plasmid replication origin. The nucleotides of the target plasmid are numbered as indicated outside the circle. The basis of kanamycin resistance for the transposition event that occurred within KanR is unclear.

FIG. 3.

Schematic representation of the mixing experiment. Three different types of mixing experiments were performed and labeled R1R2D/T, R1R2DT, and R1R2/DT. Each oval depicts a plate of 293T cells transfected with the indicated plasmids. Twenty-four hours after transfection, cells from the two plates were harvested, mixed, and replated and after another 24 h, harvested and processed as indicated in Fig. 1. See text, especially Materials and Methods, for details. R1, RAG1; R2, RAG2; D, donor; T, target; pBSK, pBluescript SK(+).

FIG. 4.

Assay for RAG-mediated transposition into the genome. (A) The donor plasmid, pBSK-12puro23-2DT, contained a puromycin resistance gene (Puro^r) flanked by two RSSs (triangles) and two DT expression cassettes. 293T cells were transfected with pBSK-12puro23-2DT and RAG expression vectors and subsequently grown in puromycin to select for acquisition of the puro^r gene. Random integration of the donor plasmid was selected against by the DT cassettes. In puro^r colonies, PCR with primers E3 and C2 was used to confirm the presence of puro^r while PCR with T7-2 and IE or with T3-2 and IC was used to detect the presence of the vector flanks (which should be absent in cells that have taken up the signal end fragment due to transposition or to insertion). For DC-PCR, restriction enzyme-digested genomic DNA was recircularized under conditions favoring intramolecular ligation and PCR amplified as described in Materials and Methods. (B and C) Schematic representations of the signal end fragment insertion events in clones 105 and 74/77.