In vivo transposition mediated by V(D)J recombinase in human T lymphocytes

Abstract

The rearrangement of immunoglobulin (Ig) and T-cell receptor (TCR) genes in lymphocytes by V(D)J recombinase is essential for immunological diversity in humans. These DNA rearrangements involve cleavage by the RAG1 and RAG2 (RAG1/2) recombinase enzymes at recombination signal sequences (RSS). This reaction generates two products, cleaved signal ends and coding ends. Coding ends are ligated by non-homologous end-joining proteins to form a functional Ig or TCR gene product, while the signal ends form a signal joint. In vitro studies have demonstrated that RAG1/2 are capable of mediating the transposition of cleaved signal ends into non-specific sites of a target DNA molecule. However, to date, in vivo transposition of signal ends has not been demonstrated. We present evidence of in vivo inter-chromosomal transposition in humans mediated by V(D)J recombinase. T-cell isolates were shown to contain TCRalpha signal ends from chromosome 14 inserted into the X-linked hypo xanthine-guanine phosphoribosyl transferase locus, resulting in gene inactivation. These findings implicate V(D)J recombinase-mediated transposition as a mutagenic mechanism capable of deleterious genetic rearrangements in humans.

V(D)J recombinase mediated inter-chromosomalHPRT alterations at cryptic recombination signal sequences in peripheral human T cells

“…The 9p21 deletions found in both T and B cell ALL also occur by a similar mechanism (Kitagawa, et al, 2002). In addition, we have reported inter-chromosomal in vivo RAG1/2 mediated transposition of TCRα signal ends in intron 1 of HPRT (Messier, et al, 2003) providing evidence that RAG1/2 are able to transpose signal ends in vivo. This observation supports the model that has been proposed for chromosomal translocation as a consequence of RAG1/2 processing of transposition intermediates (Gellert, et al, 1999;Hoim, et al, 1998;Lee, et al, 2002).…”

“…1). We have also observed V(D)J mediated transposition at the HPRT locus providing evidence that signal ended fragments, once thought to be non-reactive DNA lost during cell division, are reactive and can be used by the V(D)J recombinase system as substrates not only in vitro (Agrawal, et al, 1998;Hoim, et al, 1998;Lee, et al, 2002) but also in vivo (Messier, et al, 2003).…”

“…Cell pellets were used to generate cDNA by reverse transcription using a RNA PCR core kit (Applied Biosystems, Foster City CA USA; www.appliedbiosystems.com) utilizing MuLV reverse transcriptase with oligo d(T) 16 primers with subsequent PCR amplification as previously described (Messier, et al, 2003).…”

“…For example, in cases of follicular lymphoma, a non-B DNA structure within the BCL2 breakpoint cluster region can be cleaved by RAG1/2 even though the region has no observable RSS (Raghavan, et al, 2004). Lastly, RAG1/2 cleaved signal ends can integrate into other DNA regions by transposition adding another layer of complexity in categorizing these V(D)J mediated rearrangements (Agrawal, et al, 1998;Hoim, et al, 1998;Messier, et al, 2003). The V(D)J mediated rearrangements described can result in changes in the expression of existing regulatory genes and/or the creation of new fusion proteins that lead to the dysregulation of pathways involved with cell cycle control, apoptosis or the maintenance of genomic integrity, and thereby contribute to the development of lymphoid malignancies (Rabbitts, 1994).…”

DNA structures at chromosomal translocation sites

“…(42,62) and, upon HPRT selection of millions of T lymphocytes, transposition has only been found at the hprt locus in two cases thus far. (68) Hence, it appears to be a very rare occurrence.…”