Portal de Periódicos da CAPES

The Origin of B Cell Recurrent Chromosomal Translocations: Proximity versus DNA Damage

2013; Elsevier BV; Volume: 51; Issue: 3 Linguagem: Inglês

10.1016/j.molcel.2013.07.020

1097-4164

Rafael Casellas, Wolfgang Resch, Ofir Hakim, M C Nussenzweig,

Genomic variations and chromosomal abnormalities

B cell lymphomas are characterized by recurrent chromosomal translocations. Why these events are so prevalent is an area of active investigation. Several ideas have been put forward to try to explain this phenomenon, including nuclear proximity between translocating genes, repeated DNA damage by enzymes that mediate Ig gene recombination (AID and RAGs), and selection for deregulated oncogenes. To test the contribution of these parameters, the Alt and Nussenzweig laboratories developed genome-wide techniques to map chromosomal rearrangements (Chiarle et al., 2011Chiarle R. Zhang Y. Frock R.L. Lewis S.M. Molinie B. Ho Y.J. Myers D.R. Choi V.W. Compagno M. Malkin D.J. et al.Cell. 2011; 147: 107-119Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, Klein et al., 2011Klein I.A. Resch W. Jankovic M. Oliveira T. Yamane A. Nakahashi H. Di Virgilio M. Bothmer A. Nussenzweig A. Robbiani D.F. et al.Cell. 2011; 147: 95-106Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), and the Casellas laboratory developed a method to measure AID-mediated damage (Hakim et al., 2012Hakim O. Resch W. Yamane A. Klein I. Kieffer-Kwon K.-R. Jankovic M. Oliveira T. Bothmer A. Voss T.C. Ansarah-Sobrinho C. et al.Nature. 2012; 484: 69-74PubMed Google Scholar, Yamane et al., 2013Yamane A. Robbiani D.F. Resch W. Bothmer A. Nakahashi H. Oliveira T. Rommel P.C. Brown E.J. Nussenzweig A. Nussenzweig M.C. Casellas R. Cell Rep. 2013; 3: 138-147Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Genomic interactions were computed either by 4C or Hi-C. These studies uncovered two kinds of translocations: AID- or RAG-dependent and independent. Translocations that occur in the absence of recurrent DNA damage (e.g., AID−/−) are widespread and join interacting loci that are epigenetically accessible. The location and frequency of these events correlate with nuclear interactions (Hakim et al., 2012Hakim O. Resch W. Yamane A. Klein I. Kieffer-Kwon K.-R. Jankovic M. Oliveira T. Bothmer A. Voss T.C. Ansarah-Sobrinho C. et al.Nature. 2012; 484: 69-74PubMed Google Scholar). Furthermore, because these events are randomly and broadly distributed across the genome (Figure S1A), they cannot be subtracted from sample to sample. In the presence of AID, ∼90% of translocations in trans recapitulate those obtained in AID−/−, in both their distribution and frequency. In contrast, the remaining 10% are AID dependent, i.e., they are recurrent and can be subtracted between samples because they reproducibly accumulate near transcription start sites (TSSs) of Ig and a subset of non-Ig genes (Figure S1A and Hakim et al., 2012Hakim O. Resch W. Yamane A. Klein I. Kieffer-Kwon K.-R. Jankovic M. Oliveira T. Bothmer A. Voss T.C. Ansarah-Sobrinho C. et al.Nature. 2012; 484: 69-74PubMed Google Scholar). Unlike AID-independent events, the frequency of translocations at hotspots does not correlate with target loci proximity but with the amount of damage inflicted by AID and measured by RPA or Rad51 accumulation during DNA end resection by homologous recombination (Figure 5A in Hakim et al., 2012Hakim O. Resch W. Yamane A. Klein I. Kieffer-Kwon K.-R. Jankovic M. Oliveira T. Bothmer A. Voss T.C. Ansarah-Sobrinho C. et al.Nature. 2012; 484: 69-74PubMed Google Scholar and Figure 3C in Yamane et al., 2013Yamane A. Robbiani D.F. Resch W. Bothmer A. Nakahashi H. Oliveira T. Rommel P.C. Brown E.J. Nussenzweig A. Nussenzweig M.C. Casellas R. Cell Rep. 2013; 3: 138-147Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, Spearman’s ρ > 0.6). These ideas were independently confirmed in germinal center cells by 3D FISH (Gramlich et al., 2012Gramlich H.S. Reisbig T. Schatz D.G. PLoS ONE. 2012; 7: e39601Crossref PubMed Scopus (5) Google Scholar). In contrast to these studies, Rocha et al. reanalyzed our published TC-Seq only from AID+/+ samples and concluded that the frequency of translocations correlates with nuclear proximity (Rocha et al., 2012Rocha P.P. Micsinai M. Kim J.R. Hewitt S.L. Souza P.P. Trimarchi T. Strino F. Parisi F. Kluger Y. Skok J.A. Mol. Cell. 2012; 47: 873-885Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The discrepancy is not explained by the application of different statistical analyses, as they claim, but by the authors’ failure to include essential controls and selective data analysis. Rocha et al. failed to analyze AID−/− translocation profiles; consequently, they could not distinguish AID-dependent from AID-independent rearrangements. This is a serious error because, as stated above, only a small fraction (∼10%) of translocations outside Igh are recurrent and overlap with hypermutation hotspots and can thus be credited to AID (Klein et al., 2011Klein I.A. Resch W. Jankovic M. Oliveira T. Yamane A. Nakahashi H. Di Virgilio M. Bothmer A. Nussenzweig A. Robbiani D.F. et al.Cell. 2011; 147: 95-106Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Consequently, Rocha et al.’s “genome-wide correlation” between Igh interactions and translocations reflects the overwhelming profiles of AID-independent translocations and says nothing about AID-dependent events (Chiarle et al., 2011Chiarle R. Zhang Y. Frock R.L. Lewis S.M. Molinie B. Ho Y.J. Myers D.R. Choi V.W. Compagno M. Malkin D.J. et al.Cell. 2011; 147: 107-119Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, Hakim et al., 2012Hakim O. Resch W. Yamane A. Klein I. Kieffer-Kwon K.-R. Jankovic M. Oliveira T. Bothmer A. Voss T.C. Ansarah-Sobrinho C. et al.Nature. 2012; 484: 69-74PubMed Google Scholar, Klein et al., 2011Klein I.A. Resch W. Jankovic M. Oliveira T. Yamane A. Nakahashi H. Di Virgilio M. Bothmer A. Nussenzweig A. Robbiani D.F. et al.Cell. 2011; 147: 95-106Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Figure S1B illustrates this point by comparing Myc rearrangements in cis in the presence and absence of AID. In both cases AID-independent translocation density decreases with increasing distance from the engineered I-SceI site, consistent with the folding preferences of mammalian chromosomes (Lieberman-Aiden et al., 2009Lieberman-Aiden E. van Berkum N.L. Williams L. Imakaev M. Ragoczy T. Telling A. Amit I. Lajoie B.R. Sabo P.J. Dorschner M.O. et al.Science. 2009; 326: 289-293Crossref PubMed Scopus (4865) Google Scholar). Conversely, the frequency and location of AID-dependent translocations in the same chromosome cannot be predicted based on distance from the breakpoint (Figure S1B). Ascribing translocations of nontranscribed DNA (including intergenic domains, e.g., Figure S1A) to AID activity, as Rocha et al. imply, goes against the well-established mechanism of AID-mediated DNA deamination. Rocha et al. claim that their interpretation differs from previous studies because they calculated nuclear interactions using 4C genomic windows centered on TSSs, while we used fixed nonoverlapping windows. This is a factual inaccuracy. Our analysis of translocation frequency of AID-mediated translocations versus 4C (Figure 5 in Hakim et al., 2012Hakim O. Resch W. Yamane A. Klein I. Kieffer-Kwon K.-R. Jankovic M. Oliveira T. Bothmer A. Voss T.C. Ansarah-Sobrinho C. et al.Nature. 2012; 484: 69-74PubMed Google Scholar) makes use of 200 kb windows centered on RPA islands, which are in turn centered on TSSs of AID targets. Regardless, their argument is invalid because a direct comparison between fixed and TSS-centered windows shows highly correlated results (Figure S1C). This is so because long-range interchromosomal interactions occur between large genomic domains rather than individual genes, and the resolution of 4C experiments is no better than 200 kb (Simonis et al., 2007Simonis M. Kooren J. de Laat W. Nat. Methods. 2007; 4: 895-901Crossref PubMed Scopus (242) Google Scholar). In this regard, Rocha et al. selectively use a 20 kb window to analyze one AID target (IL4rα) in an attempt to improve the correlation between proximity and translocations. However, we found no such improvement when all AID targets are included (Figure S1D). Instead, a large fraction of 20 kb windows lack 4C-Seq reads altogether due to their small size (Figure S1D), a confirmation that this approach is incompatible with the resolution of 4C. Furthermore, because translocations are relatively infrequent, 200 kb genomic windows in some instances lack translocations. The Rocha et al. study is further confounded by their separate treatment of events that occur in cis and in trans. For example, in their analysis of chr.12, they plot 4C values using a linear scale and set an arbitrary cutoff of 60 Mb from Igh. They indicate that within this window, distance to Igh determines the frequency of AID-mediated translocations, and that this feature explains the absence of translocation hotspots in cis beyond 60 Mb. This claim, however, contradicts their main conclusion, because when the same criterion is applied in trans, essentially all recurrent translocation hotspots are excluded (Figure S1E). Furthermore, a side-by-side comparison between cis and trans targets dismisses the alleged correlation between interactions and translocation frequency at hotpots. For instance, AID targets Gpr132, Klhdc2, and Satb1 are located at 0.4 Mb, 44 Mb, or in trans relative to Igh. Despite the fact that Igh contact frequency with Klhdc2 and Satb1 is 10-fold and 10,000-fold lower than with Gpr132, all three genes translocate to Igh at comparable frequencies (Figure S1E). As discussed in our study (Hakim et al., 2012Hakim O. Resch W. Yamane A. Klein I. Kieffer-Kwon K.-R. Jankovic M. Oliveira T. Bothmer A. Voss T.C. Ansarah-Sobrinho C. et al.Nature. 2012; 484: 69-74PubMed Google Scholar), Rocha et al. notice that AID targets tend to interact more frequently with Igh than would be expected in a random model (Figure S1E). They interpret this observation as direct evidence that close proximity to Igh predisposes genes to AID-mediated damage. This is an over-interpretation, however, because only a minority of genes interacting frequently with Igh are bona fide AID targets (Chiarle et al., 2011Chiarle R. Zhang Y. Frock R.L. Lewis S.M. Molinie B. Ho Y.J. Myers D.R. Choi V.W. Compagno M. Malkin D.J. et al.Cell. 2011; 147: 107-119Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, Hakim et al., 2012Hakim O. Resch W. Yamane A. Klein I. Kieffer-Kwon K.-R. Jankovic M. Oliveira T. Bothmer A. Voss T.C. Ansarah-Sobrinho C. et al.Nature. 2012; 484: 69-74PubMed Google Scholar, Klein et al., 2011Klein I.A. Resch W. Jankovic M. Oliveira T. Yamane A. Nakahashi H. Di Virgilio M. Bothmer A. Nussenzweig A. Robbiani D.F. et al.Cell. 2011; 147: 95-106Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). For instance, while nearly 1,500 genes outrank Myc in Igh interaction frequency, few of these are translocated to Igh in an AID-dependent manner (Figure S1E). Rocha et al. fail to consider that the tendency of translocation hotspots to comingle with Igh results from the fact that most AID targets are highly transcribed (Figure S1F), and that Igh, as other constitutively active genes, preferentially interacts with euchromatin (Figure S1G). In summary, our studies do not contend that AID-mediated translocations occur independently of interactions, since genes must be in contact to rearrange. Rather, the data clearly demonstrate that the translocation frequency of AID targets (including Myc) is neither determined nor predicted by their proximity to Igh but by the amount of AID-mediated damage. This phenomenon is likely explained by the fact that DNA damage limits the incidence of translocations (Robbiani et al., 2009Robbiani D.F. Bunting S. Feldhahn N. Bothmer A. Camps J. Deroubaix S. McBride K.M. Klein I.A. Stone G. Eisenreich T.R. et al.Mol. Cell. 2009; 36: 631-641Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Download .pdf (4.57 MB) Help with pdf files Document S1. Figure S1 Response to Casellas et al.Rocha et al.Molecular CellAugust 08, 2013In BriefCasellas and colleagues challenge the conclusions from our recent paper and claim that AID-dependent translocations occur independent of Igh proximity. In contrast to our study (Rocha et al., 2012), they focus solely on hotspots, which they claim represent the only “true” AID-mediated translocations (Hakim et al., 2012). However, their contention that other sites with translocation capture (TC) reads reflect AID-independent rearrangements is not supported by any genome-wide subtraction analysis examining signal enrichment in the IghI-SceI AID-sufficient versus deficient sample. Full-Text PDF Open Archive