Somatic Rearrangement in B Cells: It’s (Mostly) Nuclear Physics
2015; Cell Press; Volume: 162; Issue: 4 Linguagem: Inglês
10.1016/j.cell.2015.07.034
ISSN1097-4172
Autores Tópico(s)Chromosomal and Genetic Variations
ResumoWe discuss how principles of nuclear architecture drive typical gene rearrangements in B lymphocytes, whereas translocation hot spots and recurrent lesions reflect the extent of AID-mediated DNA damage and selection. We discuss how principles of nuclear architecture drive typical gene rearrangements in B lymphocytes, whereas translocation hot spots and recurrent lesions reflect the extent of AID-mediated DNA damage and selection. Chromosome rearrangements are essential for human development. At immunoglobulin (Ig) gene loci in bone marrow B cells, the recombination-activating gene (RAG) proteins mediate joining of V, D, and J segments to create a diverse array of antibodies. In the periphery, the activation-induced cytidine deaminase AID initiates class switch recombination (CSR) of Igh constant domains, leading to different antibody isotypes. These tightly regulated processes lie at the heart of the adaptive immune response. Non-targeted rearrangements are important for cellular homeostasis and genomic integrity. Spontaneous DNA double-strand breaks (DSBs) are usually repaired in cis by non-homologous end joining (NHEJ) or in trans by homologous recombination (HR). Although largely beneficial, these processes can generate cancer-causing translocations that juxtapose oncogenes (e.g., myc) to potent Ig enhancers. We review three principles of nuclear architecture that influence patterns of recombination in B cells: polymer folding, looping between convergent CTCF motifs, and A-B compartmentalization. We discuss that while Ig gene recombination evolved to exploit nuclear architecture, DNA damage and selection determine the location and frequency of recurrent cancer-causing rearrangements. A simple estimate for the rearrangement frequency between two loci, A and B, is the probability that they are in close spatial proximity within the cell nucleus. In the absence of local folding features (such as loops), this probability ought to decline monotonically as A and B are positioned further apart in the genome. This decline is evident using Hi-C experiments (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 (4893) Google Scholar). The decline passes through multiple scaling regimes. For inter-locus distances between 500 kb and 7 Mb, Hi-C experiments showed that the frequency of contact is related to the distance by a power law with an exponent of approximately −1.0. Polymer theory provides a rationale for these observations. In such studies, theoretical and physical simulations of condensed chromatin are used to deduce the relationship between 1D proximity-in-sequence and 3D proximity-in-space. Presently, the most commonly employed model is the fractal globule, which predicts the frequency of contact (or recombination) between two loci scales roughly as the reciprocal of the distance between them (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 (4893) Google Scholar). The resulting predictions closely match the empirical values obtained by Hi-C. Many studies have confirmed polymer model predictions. The simplest, that most DSBs ought to be resolved in cis, was confirmed by studies in which breaks were induced by RAGs or by ionizing radiation. More quantitative predictions have been made by using next-generation sequencing of chromosomal rearrangements. These rely on the insertion of restriction sites for I-SceI, a yeast endonuclease, at loci of interest such as Igh and myc. By transducing activated B cells with I-SceI, DSBs form at the targeted loci, and the resulting rearrangements can be profiled in a high-throughput fashion (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 (359) 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 (292) Google Scholar). Monotonic declines in I-SceI recombination frequency were characterized by power law scalings (−1.3) resembling those estimated from Hi-C and polymer modeling. Thus, mammalian genomes rearrange in cis with a profile that mimics the polymer behavior of chromatin. CSR benefits from this propensity. During CSR, activated B cells replace the IgM constant domain (Cμ) with that of a downstream isotype (Cγ, Cα, or Cε). In the mouse, CHs are confined to a relatively small region (160 Kb) within the vast Igh locus (2.8 Mb, Figure 1A). Recombination is facilitated by transcription of switch (S) regions upstream of each CH domain, which imparts accessibility to AID and leads to DSBs. In experiments in which switch regions were replaced by I-SceI sites (Gostissa et al., 2014Gostissa M. Schwer B. Chang A. Dong J. Meyers R.M. Marecki G.T. Choi V.W. Chiarle R. Zarrin A.A. Alt F.W. Proc. Natl. Acad. Sci. USA. 2014; 111: 2644-2649Crossref PubMed Scopus (28) Google Scholar, Zarrin et al., 2007Zarrin A.A. Del Vecchio C. Tseng E. Gleason M. Zarin P. Tian M. Alt F.W. Science. 2007; 315: 377-381Crossref PubMed Scopus (86) Google Scholar), the induction of I-SceI breaks promoted CSR. Proximal I-SceI breaks recombined at higher frequencies than distant ones, consistent with the idea that, at least in part, the monotonic decline of Igh interactions influences these rearrangements. Thus, CSR appears to exploit the polymer behavior of chromosomes, bringing about recombination events between switch regions through the repair of DSBs that come into spatial proximity. V(D)J recombination occurs over a broader range of distances than CSR. The first recombination event joins D and JH segments separated in the mouse genome by a maximum distance of 100 Kb (Figure 1A). In contrast, VH-DJH recombination deletes at least 45–150 Kb for the most proximal V segment (IghV5-1) and up to ∼2.6 Mb for the most distal one (IghV1-86, Figure 1A). Thus, if VH-DJH recombination relied exclusively on the monotonic behavior of chromatin polymers, proximal VH segments would dominate the mature Ig repertoire, drastically curtailing antibody diversity. However, microscopy studies have shown that the Igh locus undergoes conformational changes during V(D)J recombination (Jhunjhunwala et al., 2008Jhunjhunwala S. van Zelm M.C. Peak M.M. Cutchin S. Riblet R. van Dongen J.J. Grosveld F.G. Knoch T.A. Murre C. Cell. 2008; 133: 265-279Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). The entire VH domain associates with D-JH segments in cells undergoing VH-DJH recombination (Subrahmanyam and Sen, 2012Subrahmanyam R. Sen R. Curr. Top. Microbiol. Immunol. 2012; 356: 39-63Crossref PubMed Scopus (12) Google Scholar). Clearly, mechanisms beyond local polymer folding are at work. A recent Hi-C map of human B lymphoblastoid cells shed light on these mechanisms revealing ∼10,000 loops between pairs of CTCF sites whose motifs are convergent (i.e., facing one another [Rao et al., 2014Rao S.S. Huntley M.H. Durand N.C. Stamenova E.K. Bochkov I.D. Robinson J.T. Sanborn A.L. Machol I. Omer A.D. Lander E.S. Aiden E.L. Cell. 2014; 159: 1665-1680Abstract Full Text Full Text PDF PubMed Scopus (3627) Google Scholar]). The Igh locus illustrates this. Its VH region contains >100 CTCF sites, all pointing downstream. They face a single motif, pointing upstream, that is situated at the 5′ end of the D region (Figure 1A). In this configuration CTCF looping likely facilitates recombination between distant VHs and rearranged DJHs. In support of this, deletion of CTCF-binding sites at the D region decreased recombination with distal VHs (Guo et al., 2011Guo C. Yoon H.S. Franklin A. Jain S. Ebert A. Cheng H.L. Hansen E. Despo O. Bossen C. Vettermann C. et al.Nature. 2011; 477: 424-430Crossref PubMed Scopus (202) Google Scholar, Lin et al., 2015Lin S.G. Guo C. Su A. Zhang Y. Alt F.W. Proc. Natl. Acad. Sci. USA. 2015; 112: 1815-1820Crossref PubMed Scopus (44) Google Scholar). Thus, V(D)J recombination appears to rely on CTCF-mediated looping to bring about long-range rearrangements. Besides CTCF, other factors have also been implicated in Igh locus contraction, including E2A, Pax5, cohesin, Brg1, and YY-1 (reviewed by Alt et al., 2013Alt F.W. Zhang Y. Meng F.L. Guo C. Schwer B. Cell. 2013; 152: 417-429Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). How these factors complement CTCF-mediated looping remains unclear. The DSBs that form during Ig gene recombination are not always properly repaired in cis and can instead lead to incidental rearrangements, both in cis and especially in trans. In B cell tumors chromosomal translocations frequently join oncogenes to potent Ig enhancers that deregulate their expression. The origin of these events has been debated for several decades; proximity, patterns of DSB formation, and selection have all been proposed as the driving mechanism. At first, the relationship between nuclear architecture and translocations was explored using microscopy. These studies tested the hypothesis that oncogenes and Ig loci translocate frequently because they preferentially associate in B cell nuclei. Fluorescence in situ hybridization (FISH) showed that Igh and myc are within 1 μm of each other in a fraction of activated B cells, but the overlap frequency varied greatly across studies. When B cells were analyzed shortly after activation ( 20% of lymphocytes (Osborne et al., 2007Osborne C.S. Chakalova L. Mitchell J.A. Horton A. Wood A.L. Bolland D.J. Corcoran A.E. Fraser P. PLoS Biol. 2007; 5: e192Crossref PubMed Scopus (311) Google Scholar), while at later stages of activation (72 hr) or in germinal center B cells, values were in the 3%–5% range (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, Gramlich et al., 2012Gramlich H.S. Reisbig T. Schatz D.G. PLoS ONE. 2012; 7: e39601Crossref PubMed Scopus (5) Google Scholar, Wang et al., 2009Wang J.H. Gostissa M. Yan C.T. Goff P. Hickernell T. Hansen E. Difilippantonio S. Wesemann D.R. Zarrin A.A. Rajewsky K. et al.Nature. 2009; 460: 231-236Crossref PubMed Scopus (106) Google Scholar). These experiments were interpreted to suggest that, at least under some conditions, Igh and myc preferentially associate in B cells. However, the studies did not consider the frequency at which gene loci overlap at random. For 3D-FISH in a diploid nucleus using a 1 μm overlap threshold, the random overlap frequency between two genes is given by the volume of two spheres of radius 1 μm (∼8.4 μm3) divided by the total volume of the nucleus. For B cells at rest or shortly after activation (15 min), a volume of 55 μm3 is typical (Figure 1B), implying that two genes overlap at random in 15% of the cells. In contrast, the average volume of activated B cells >24 hr post-activation is ∼250 μm3 (Figure 1B), resulting in a random overlap frequency of roughly 3.5%. (For 2D FISH, the probabilities are 14% before activation and 5% afterward.) These considerations suggest that the reported differences in Igh-myc overlap may be explained by differences in nuclear volume rather than by preferential association. Recent contact mapping experiments have provided more definitive results. In particular, kilobase resolution Hi-C mapping did not reveal peaks of contact frequency between pairs of loci lying in trans (Rao et al., 2014Rao S.S. Huntley M.H. Durand N.C. Stamenova E.K. Bochkov I.D. Robinson J.T. Sanborn A.L. Machol I. Omer A.D. Lander E.S. Aiden E.L. Cell. 2014; 159: 1665-1680Abstract Full Text Full Text PDF PubMed Scopus (3627) Google Scholar). Furthermore, 4C studies found that Igh and myc do not form preferential locus-specific associations beyond those between any pair of transcriptionally active genes (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). Crucially, the observation that there are no biases in trans between individual gene loci does not imply that there are no biases in trans whatsoever. The existence of chromosome territories and their preferential associations is well known, as is the fact that A-B compartmentalization leads to spatial segregation of open and closed chromatin (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 (4893) Google Scholar, de Laat and Grosveld, 2007de Laat W. Grosveld F. Curr. Opin. Genet. Dev. 2007; 17: 456-464Crossref PubMed Scopus (50) Google Scholar). Consequently, Igh and myc, on chromosomes 12 and 15 respectively, colocalize with transcriptionally active loci genome wide at similar frequencies (Figure 1C; 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). These broad patterns of spatial proximity influence rearrangement patterns. In AID−/− B cells or in irradiated pro-B cells—where the distribution of DNA breaks is relatively uniform across the genome—translocation patterns reflect both chromosome territories and A-B compartmentalization (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). Thus, in the absence of recurrent DNA damage, incidental rearrangements—those that do not arise due to targeted mechanisms such as VDJ recombination—mostly follow the broad contours of nuclear compartmentalization. The findings discussed so far highlight the significant influence of nuclear architecture on patterns of rearrangement. Yet, another key feature that impacts recombination is the frequency of DNA DSBs. In the presence of DNA-damaging enzymes such as RAGs and AID, the rate of formation of such lesions is not constant across the genome but is instead targeted to specific sites. In cancer, yet another factor must be considered: tumorigenesis may select for or against particular rearrangements. A primary role of AID-mediated deamination is to promote the formation of DSBs during CSR. However, AID is also a significant driver of rearrangement hot spots. This tendency has been clearly observed in activated and germinal center B cells. In addition to the rearrangements seen in the AID−/− background, activated B cells with intact AID exhibit translocation hot spots at myc and other oncogenes implicated in B cell transformation. By monitoring the accumulation of the repair factors RPA and Rad51 at resected DNA breaks, these hot spots were confirmed to be sites of recurrent AID-induced DNA damage. Indeed, there is a direct proportionality between the extent of AID-mediated DSB formation at hot spots and the absolute number of Igh translocations. These translocation frequencies dramatically exceed predictions based on nuclear architecture alone. For instance, Igh is 20-fold more likely to spatially co-locate with Gpr132, which lies 400 kb away on chromosome 12, than with Il4ra, which lies, in trans, on chromosome 7 (Figure 1D). But Igh is 20-fold more likely to rearrange with Il4ra than with Gpr132. Similarly, myc is not particularly likely to spatially co-locate with Igh: >2,000 genes are more likely to do so. Yet myc is one of a relatively small fraction of genes that recurrently rearrange to Igh (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). Thus, patterns of AID-mediated DSBs are a principal factor in determining sites of recurrent translocation in B cells, irrespective of topology. This also applies to translocations induced by other forms of recurrent DNA damage, such as Rag1/2 activity or the CRISPR-Cas9 system (Frock et al., 2015Frock R.L. Hu J. Meyers R.M. Ho Y.J. Kii E. Alt F.W. Nat. Biotechnol. 2015; 33: 179-186Crossref PubMed Scopus (468) Google Scholar). These arguments highlight the need to understand the factors that make loci vulnerable to each damage modality. In the case of AID, several proposals have been made, including the presence of super-enhancers, convergent gene transcription, and high interconnectivity between regulatory elements (Alinikula and Schatz, 2014Alinikula J. Schatz D.G. Cell. 2014; 159: 1490-1492Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). Different factors are likely to be relevant for each damage modality. For instance, the mere presence of H3K4me3 appears to be sufficient for RAG recruitment (Teng et al., 2015Teng G. Maman K. Resch W. Kim M. Yamane A. Qian J. Kieffer-Kwon K.-R. Mandal M. Meffre E. Clark M. et al.Cell. 2015; 162 (this issue): 751-765Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). In tumors, the rate at which a particular rearrangement is found does not reflect its rate of formation in the tumor's cell of origin. Instead, there is strong positive selection for incidental translocations that deregulate oncogene expression. For instance, Il4ra translocations form 10-fold more frequently than myc translocations in activated B cells (Figure 1D), but—unlike myc—Il4ra translocations are yet to be reported in B cell tumors. In Ig translocations, oncogene deregulation tends to be the result of the potent Igκ, Igλ, or Igh enhancers. Because these enhancers can work at a distance, they are believed to rely on spatial proximity to drive their biological function. Yet this same architecture imposes constraints on their activity. For instance, when mouse B cells lacking AID-mediated damage were cultured under non-selective conditions, incidental translocations placed the Igh 3′ enhancer, known as 3′RR, at a wide range of distances from myc (Kovalchuk et al., 2012Kovalchuk A.L. Ansarah-Sobrinho C. Hakim O. Resch W. Tolarová H. Dubois W. Yamane A. Takizawa M. Klein I. Hager G.L. et al.Proc. Natl. Acad. Sci. USA. 2012; 109: 10972-10977Crossref PubMed Scopus (21) Google Scholar). Following selection (during mouse plasmacytomagenesis), the same element was found no more than 500 kb from myc. Beyond this point, no local changes were observed in the level of Pol II recruitment, gene expression, or epigenetic modifications. Given the above-noted findings about CTCF orientation, it is likely that the transformational potency of the 3′RR enhancer is related to the fact that it lies adjacent to what we dub a CTCF "superanchor": a series of ten CTCF sites, all of which point in the same direction. We predict that the selection of an Igh translocation depends on the capacity of this superanchor to form long-range functional loops with translocating CTCF motifs. At least in the setting of myc translocations, the upper limit of these loops appears to be half a megabase. Thus, nuclear architecture influences the location and frequency of rearrangements as well as which of these events are selectively favored during transformation. This observation may have implications for tumors that are not derived from B and T cells and that typically lack targeted, recurrent DNA damage. In the past 5 years, technological advances have helped to unravel many longstanding issues relating to both physiological and pathological rearrangement in B lymphocytes. As the field of 3D genomics advances, we anticipate that it will continue to illuminate these mechanisms and their implications for tumorigenesis, both within the immune system and beyond. We thank Wouter de Laat, Neva Durand, Suhas Rao, and Adrian Sanborn for helpful discussions and Wolfgang Resch, Marei Dose, Suhas Rao, and Sigrid Knemeyer for figure assistance.
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