Chromosomal Loop Domains Direct the Recombination of Antigen Receptor Genes

Hu, Jiazhi; Zhang, Yu; Zhao, Lijuan; Frock, Richard L.; Du, Zhou; Meyers, Robin M.; Meng, Fei-Long; Schatz, David G.; Alt, Frederick W.

doi:10.1016/j.cell.2015.10.016

Cited by 141 publications

(288 citation statements)

References 47 publications

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“…Because V(D)J recombination generates rearrangements with junctions at borders of V, D, and J segments, we can use primers for any of these gene segments as LAM-HTGTS bait to identify sites of RAG-generated DSBs, both in progenitor or precursor lymphocytes undergoing V(D)J recombination, as well as in mature lymphocytes to retrospectively identify V(D)J recombination events that occurred earlier in development. Notably, LAM-HTGTS using endogenous RAG-generated DSBs identified RAG-generated DJ H joins, RSS joins in excision circles, and off-target junctions in developing B-lineage cells that were not detected by prior assays (22), illustrating the high sensitivity of the assay. Based on these earlier studies, we now describe an adaptation of LAM-HTGTS as a robust repertoire-sequencing assay that we term "HTGTS-adapted repertoire sequencing" (HTGTS-Rep-seq).…”

Section: Significancementioning

confidence: 99%

“…LAM-HTGTS, like its predecessor HTGTS (17), employs a single primer for a DSB-associated bait sequence to perform linear amplification across bait-prey junctions to identify all prey sequences joined to the bait DSBs in an unbiased manner (16,18). We have used various types of DSBs as bait for LAM-HTGTS, including those generated by engineered nucleases and endogenous DSBs (17)(18)(19)(20)(21)(22). Because V(D)J recombination generates rearrangements with junctions at borders of V, D, and J segments, we can use primers for any of these gene segments as LAM-HTGTS bait to identify sites of RAG-generated DSBs, both in progenitor or precursor lymphocytes undergoing V(D)J recombination, as well as in mature lymphocytes to retrospectively identify V(D)J recombination events that occurred earlier in development.…”

Section: Significancementioning

confidence: 99%

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Highly sensitive and unbiased approach for elucidating antibody repertoires

Lin

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Developing B lymphocytes undergo V(D)J recombination to assemble germ-line V, D, and J gene segments into exons that encode the antigen-binding variable region of Ig heavy (H) and light (L) chains. IgH and IgL chains associate to form the B-cell receptor (BCR), which, upon antigen binding, activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. The ability of V(D)J recombination to generate vast primary B-cell repertoires results from a combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B-cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (HTGTS-Repseq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJ H intermediates and V(D)J exons in either productive or nonproductive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions, and also for following antibody affinity maturation processes. 1). In this process, the V, D, and J coding ends are generated as covalent hairpins that must be opened and that are often further processed, before being joined by classical nonhomologous end joining (2). Processing of V, D, J coding ends can involve generation of deletions or insertions of nucleotides at the junction regions (2), including the frequent de novo addition of nucleotides by the terminal deoxynucleotidyl transferase component of the V(D)J recombination process (3). Notably the V(D)J junctional region encodes a major antigen contact region of the antibody variable region, known as complementarity determining region 3 (CDR3), and thus these junctional diversification processes make a huge contribution to antibody diversity.The mouse IgH locus spans 2.7 megabases (Mb). There are 100s of V H s in the several megabase distal portion of the IgH, with the number varying substantially in certain mouse strains (4). The V H s lie ∼100 kb upstream from a 50-kb region containing 13 D H s, which is followed several kilobases downstream by a 2-kb region containing four J H s. The IgH constant region (C H ) exons lie downstream of the J H s. After assembly of a V H DJ H exon, transcription initiates upstream of the V H and terminates downstream of the C H exons, with V(D)J and C H portions being fused...

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Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Highly sensitive and unbiased approach for elucidating antibody repertoires

Lin

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…9 and this study). Because of increased contact frequency, translocation frequency is increased further between sequences on the same cis chromosome (3,28,29). HTGTS detects only those DSBs that translocate.…”

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confidence: 99%

“…Because of cellular heterogeneity in 3D genome organization (3,27,29), HTGTS detects, with great sensitivity, recurrent DSBs in any given genomic location (4,9,(26)(27)(28)(29) or recurrent classes of DSBs, such as DSBs at TSSs, that may occur at much lower frequency in any given location (ref. 9 and this study).…”

mentioning

confidence: 99%

“…We previously developed an unbiased, high-throughput, genomewide, translocation sequencing (HTGTS) approach to identify DSBs via their translocation to bait DSBs at defined genomic sites in primary progenitor and activated mature B cells, NSPCs, and human cell lines (4,9,(26)(27)(28). Because of cellular heterogeneity in 3D genome organization (3,27,29), HTGTS detects, with great sensitivity, recurrent DSBs in any given genomic location (4,9,(26)(27)(28)(29) or recurrent classes of DSBs, such as DSBs at TSSs, that may occur at much lower frequency in any given location (ref.…”

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confidence: 99%

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Transcription-associated processes cause DNA double-strand breaks and translocations in neural stem/progenitor cells

Schwer

Wei

Chang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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High-throughput, genome-wide translocation sequencing (HTGTS) studies of activated B cells have revealed that DNA double-strand breaks (DSBs) capable of translocating to defined bait DSBs are enriched around the transcription start sites (TSSs) of active genes. We used the HTGTS approach to investigate whether a similar phenomenon occurs in primary neural stem/progenitor cells (NSPCs). We report that breakpoint junctions indeed are enriched around TSSs that were determined to be active by global run-on sequencing analyses of NSPCs. Comparative analyses of transcription profiles in NSPCs and B cells revealed that the great majority of TSS-proximal junctions occurred in genes commonly expressed in both cell types, possibly because this common set has higher transcription levels on average than genes transcribed in only one or the other cell type. In the latter context, among all actively transcribed genes containing translocation junctions in NSPCs, those with junctions located within 2 kb of the TSS show a significantly higher transcription rate on average than genes with junctions in the gene body located at distances greater than 2 kb from the TSS. Finally, analysis of repair junction signatures of TSS-associated translocations in wild-type versus classical nonhomologous end-joining (C-NHEJ)–deficient NSPCs reveals that both C-NHEJ and alternative end-joining pathways can generate translocations by joining TSS-proximal DSBs to DSBs on other chromosomes. Our studies show that the generation of transcription-associated DSBs is conserved across divergent cell types.

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FGFR3 preferentially colocalizes with IGH in the interphase nucleus of multiple myeloma patient B‐cells when FGFR3 is located outside of CT4

Martin

Harizanova

Mai

et al. 2016

Genes Chromosomes & Cancer

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Many B-cell malignancies are characterized by chromosomal translocations involving IGH and a proto-oncogene. For translocations to occur, spatial proximity of translocation-prone genes is necessary. Currently, it is not known how such genes are brought into proximity with one another. Although decondensed chromosomes occupy definitive, non-random spaces in the interphase nucleus known as chromosome territories (CTs), chromatin at the edges of CTs can intermingle, and specific genomic regions from some chromosomes have been shown to "loop out" of their respective CTs. This extra-territorial positioning of specific genomic regions may provide a mechanism whereby translocation-prone genes are brought together in the interphase nucleus. FGFR3 and MAF recurrently participate in translocations with IGH at different frequencies. Using 3D, 4-color FISH, and 3D analysis software, we show frequent extra-territorial positioning of FGFR3 and significantly less frequent extra-territorial positioning of MAF. Frequent extra-territorial positioning may be characteristic of FGFR3 in B-cells from healthy adult donors and non-malignant B-cells from patients, but not in hematopoietic stem cells from patients with translocations. The frequency of extra-territorial positioning of FGFR3 and MAF in B-cells correlates with the frequency of translocations in the patient population. Most importantly, in patient B-cells, we demonstrate a significant proportion of extra-territorial FGFR3 participating in close loci pairs and/or colocalizing with IGH. This preliminary work suggests that in patient B-cells, extra-territorial positioning of FGFR3 may provide a mechanism for forming close loci pairs and/or colocalization with IGH; indirectly facilitating translocation events involving these two genes. © 2016 Wiley Periodicals, Inc.

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Chromosomal Loop Domains Direct the Recombination of Antigen Receptor Genes

Cited by 141 publications

References 47 publications

Highly sensitive and unbiased approach for elucidating antibody repertoires

Highly sensitive and unbiased approach for elucidating antibody repertoires

Transcription-associated processes cause DNA double-strand breaks and translocations in neural stem/progenitor cells

FGFR3 preferentially colocalizes with IGH in the interphase nucleus of multiple myeloma patient B‐cells when FGFR3 is located outside of CT4

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