Off-Target V(D)J Recombination Drives Lymphomagenesis and Is Escalated by Loss of the Rag2 C Terminus.

Genome-wide analysis of thymic lymphomas from Tp53(-/-) mice with wild-type or C-terminally truncated Rag2 revealed numerous off-target, RAG-mediated DNA rearrangements. A significantly higher fraction of these errors mutated known and suspected oncogenes/tumor suppressor genes than did sporadic rearrangements (p < 0.0001). This tractable mouse model recapitulates recent findings in human pre-B ALL and allows comparison of wild-type and mutant RAG2. Recurrent, RAG-mediated deletions affected Notch1, Pten, Ikzf1, Jak1, Phlda1, Trat1, and Agpat9. Rag2 truncation substantially increased the frequency of off-target V(D)J recombination. The data suggest that interactions between Rag2 and a specific chromatin modification, H3K4me3, support V(D)J recombination fidelity. Oncogenic effects of off-target rearrangements created by this highly regulated recombinase may need to be considered in design of site-specific nucleases engineered for genome modification.

RAG2 mutants alter DSB repair pathway choice in vivo and illuminate the nature of 'alternative NHEJ'.

DNA double-stranded breaks (DSBs) can be repaired by several mechanisms, including classical NHEJ (c-NHEJ) and a poorly defined, error-prone process termed alternative NHEJ (a-NHEJ). How cells choose between these alternatives to join physiologic DSBs remains unknown. Here, we show that deletion of RAG2's C-terminus allows a-NHEJ to repair RAG-mediated DSBs in developing lymphocytes from both c-NHEJ-proficient and c-NHEJ-deficient mice, demonstrating that the V(D)J recombinase influences repair pathway choice in vivo. Analysis of V(D)J junctions revealed that, contrary to expectation, junctional characteristics alone do not reliably distinguish between a-NHEJ and c-NHEJ. These data suggest that a-NHEJ is not necessarily mutagenic, and may be more prevalent than previously appreciated. Whole genome sequencing of a lymphoma arising in a p53(-/-) mouse bearing a C-terminal RAG2 truncation reveals evidence of a-NHEJ and also of aberrant recognition of DNA sequences resembling RAG recognition sites.

Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells.

Whereas chromosomal translocations are common pathogenetic events in cancer, mechanisms that promote them are poorly understood. To elucidate translocation mechanisms in mammalian cells, we developed high-throughput, genome-wide translocation sequencing (HTGTS). We employed HTGTS to identify tens of thousands of independent translocation junctions involving fixed I-SceI meganuclease-generated DNA double-strand breaks (DSBs) within the c-myc oncogene or IgH locus of B lymphocytes induced for activation-induced cytidine deaminase (AID)-dependent IgH class switching. DSBs translocated widely across the genome but were preferentially targeted to transcribed chromosomal regions. Additionally, numerous AID-dependent and AID-independent hot spots were targeted, with the latter comprising mainly cryptic I-SceI targets. Comparison of translocation junctions with genome-wide nuclear run-ons revealed a marked association between transcription start sites and translocation targeting. The majority of translocation junctions were formed via end-joining with short microhomologies. Our findings have implications for diverse fields, including gene therapy and cancer genomics.

Wolf-Hirschhorn syndrome candidate 1 is involved in the cellular response to DNA damage.

Wolf-Hirschhorn syndrome (WHS) is a malformation syndrome associated with growth retardation, mental retardation, and immunodeficiency resulting from a hemizygous deletion of the short arm of chromosome 4, called the WHS critical region (WHSC). The WHSC1 gene is located in this region, and its loss is believed to be responsible for a number of WHS characteristics. We identified WHSC1 in a genetic screen for genes involved in responding to replication stress, linking Wolf-Hirschhorn syndrome to the DNA damage response (DDR). Here, we report that the WHSC1 protein is a member of the DDR pathway. WHSC1 localizes to sites of DNA damage and replication stress and is required for resistance to many DNA-damaging and replication stress-inducing agents. Through its SET domain, WHSC1 regulates the methylation status of the histone H4 K20 residue and is required for the recruitment of 53BP1 to sites of DNA damage. We propose that Wolf-Hirschhorn syndrome results from a defect in the DDR.

The role of mechanistic factors in promoting chromosomal translocations found in lymphoid and other cancers.

Recurrent chromosomal abnormalities, especially chromosomal translocations, are strongly associated with certain subtypes of leukemia, lymphoma and solid tumors. The appearance of particular translocations or associated genomic alterations can be important indicators of disease prognosis, and in some cases, certain translocations may indicate appropriate therapy protocols. To date, most of our knowledge about chromosomal translocations has derived from characterization of the highly selected recurrent translocations found in certain cancers. Until recently, mechanisms that promote or suppress chromosomal translocations, in particular, those responsible for their initiation, have not been addressed. For translocations to occur, two distinct chromosomal loci must be broken, brought together (synapsed) and joined. Here, we discuss recent findings on processes and pathways that influence the initiation of chromosomal translocations, including the generation fo DNA double strand breaks (DSBs) by general factors or in the context of the Lymphocyte-specific V(D)J and IgH class-switch recombination processes. We also discuss the role of spatial proximity of DSBs in the interphase nucleus with respect to how DSBs on different chromosomes are justaposed for joining. In addition, we discuss the DNA DSB response and its role in recognizing and tethering chromosomal DSBs to prevent translocations, as well as potential roles of the classical and alternative DSB end-joining pathways in suppressing or promoting translocations. Finally, we discuss the potential roles of long range regulatory elements, such as the 3'IgH enhancer complex, in promoting the expression of certain translocations that are frequent in lymphomas and, thereby, contributing to their frequent appearance in tumors.

DNA palindromes with a modest arm length of greater, similar 20 base pairs are a significant target for recombinant adeno-associated virus vector integration in the liver, muscles, and heart in mice.

Our previous study has shown that recombinant adeno-associated virus (rAAV) vector integrates preferentially in genes, near transcription start sites and CpG islands in mouse liver (H. Nakai, X. Wu, S. Fuess, T. A. Storm, D. Munroe, E. Montini, S. M. Burgess, M. Grompe, and M. A. Kay, J. Virol. 79:3606-3614, 2005). However, the previous method relied on in vivo selection of rAAV integrants and could be employed for the liver but not for other tissues. Here, we describe a novel method for high-throughput rAAV integration site analysis that does not rely on marker gene expression, selection, or cell division, and therefore it can identify rAAV integration sites in nondividing cells without cell manipulations. Using this new method, we identified and characterized a total of 997 rAAV integration sites in mouse liver, skeletal muscle, and heart, transduced with rAAV2 or rAAV8 vector. The results support our previous observations, but notably they have revealed that DNA palindromes with an arm length of greater, similar 20 bp (total length, greater, similar 40 bp) are a significant target for rAAV integration. Up to approximately 30% of total integration events occurred in the vicinity of DNA palindromes with an arm length of greater, similar 20 bp. Considering that DNA palindromes may constitute fragile genomic sites, our results support the notion that rAAV integrates at chromosomal sites susceptible to breakage or preexisting breakage sites. The use of rAAV to label fragile genomic sites may provide an important new tool for probing the intrinsic source of ongoing genomic instability in various tissues in animals, studying DNA palindrome metabolism in vivo, and understanding their possible contributions to carcinogenesis and aging.

New approaches to the analysis of palindromic sequences from the human genome: evolution and polymorphism of an intronic site at the NF1 locus.

The nature of any long palindrome that might exist in the human genome is obscured by the instability of such sequences once cloned in Escherichia coli. We describe and validate a practical alternative to the analysis of naturally-occurring palindromes based upon cloning and propagation in Saccharomyces cerevisiae. With this approach we have investigated an intronic sequence in the human Neurofibromatosis 1 (NF1) locus that is represented by multiple conflicting versions in GenBank. We find that the site is highly polymorphic, exhibiting different degrees of palindromy in different individuals. A side-by-side comparison of the same plasmids in E.coli versus. S.cerevisiae demonstrated that the more palindromic alleles were inevitably corrupted upon cloning in E.coli, but could be propagated intact in yeast. The high quality sequence obtained from the yeast-based approach provides insight into the various mechanisms that destabilize a palindrome in E.coli, yeast and humans, into the diversification of a highly polymorphic site within the NF1 locus during primate evolution, and into the association between palindromy and chromosomal translocation.