Eukaryotes have evolved complex mechanisms to repair DNA double-strand breaks (DSBs) through coordinated actions of protein sensors, transducers, and effectors. Here we show that ∼21-nucleotide small RNAs are produced from the sequences in the vicinity of DSB sites in Arabidopsis and in human cells. We refer to these as diRNAs for DSB-induced small RNAs. In Arabidopsis, the biogenesis of diRNAs requires the PI3 kinase ATR, RNA polymerase IV (Pol IV), and Dicer-like proteins. Mutations in these proteins as well as in Pol V cause significant reduction in DSB repair efficiency. In Arabidopsis, diRNAs are recruited by Argonaute 2 (AGO2) to mediate DSB repair. Knock down of Dicer or Ago2 in human cells reduces DSB repair. Our findings reveal a conserved function for small RNAs in the DSB repair pathway. We propose that diRNAs may function as guide molecules directing chromatin modifications or the recruitment of protein complexes to DSB sites to facilitate repair.

RAG endonuclease initiates antibody heavy chain variable region exon assembly from V, D, and J segments within a chromosomal V(D)J recombination center (RC) by cleaving between paired gene segments and flanking recombination signal sequences (RSSs). The IGCR1 control region promotes DJ intermediate formation by isolating Ds, Js, and RCs from upstream Vs in a chromatin loop anchored by CTCF-binding elements (CBEs). How Vs access the DJRC for V to DJ rearrangement was unknown. We report that CBEs immediately downstream of frequently rearranged V-RSSs increase recombination potential of their associated V far beyond that provided by RSSs alone. This CBE activity becomes particularly striking upon IGCR1 inactivation, which allows RAG, likely via loop extrusion, to linearly scan chromatin far upstream. V-associated CBEs stabilize interactions of D-proximal Vs first encountered by the DJRC during linear RAG scanning and thereby promote dominant rearrangement of these Vs by an unanticipated chromatin accessibility-enhancing CBE function.

The authors declare no competing financial interests. Data availability HTGTS V(D)J-seq, Hi-C, 3C-HTGTS, GRO-Seq and ChIP-Seq sequencing data reported in this study has been deposited in the GEO database under the accession number GSE130224. Specifically, HTGTS V(D)J-seq data is deposited in the GEO database under the accession number GSE130216 and is related to Fig. 1e-h; 2b, c; 3a-c, e; 4c; Extended Data Fig. 2c-e, g, h; 3e, f; 4a, c, d; 5a-c; 6c; 7d, e; 9b; and Supplementary Information Table 1&2. Hi-C data is deposited in the GEO database under accession number GSE134543 and is related to Extended Data Fig. 8a. 3C-HTGTS data is deposited in the GEO database under the accession number GSE130214 and is related to Fig. 3f; 4d; and Extended Data Fig. 8a; 9c; 10q, r. GRO-Seq data is deposited in the GEO database under the accession number GSE130215 and is related to Fig. 3d; 4b; and Extended Data Fig. 4e; 6d; 7f; 9b. ChIP-Seq data is deposited in the GEO database under the accession number GSE130213 and is related to Extended Data Fig. 8c, d; 9d. Code availability. HTGTS V(D)J-seq and 3C-HTGTS data was processed through published pipeline available at (http://robinmeyers.github.io/ transloc_pipeline/). Code for Hi-C data process is available at (github.com/aidenlab). GRO-Seq and ChIP-Seq were aligned to mm9 genome with bowtie2 v2.2.8 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml), processed by samtools v1.8 (https:// sourceforge.net/projects/samtools/files/samtools/1.8/) and generated graph files via RSeQC tool v2.6 (http://rseqc.sourceforge.net/ #bam2wig-py).

DNA double-strand breaks (DSBs) are highly cytotoxic lesions and pose a major threat to genome stability if not properly repaired. We and others have previously shown that a class of DSB-induced small RNAs (diRNAs) is produced from sequences around DSB sites. DiRNAs are associated with Argonaute (Ago) proteins and play an important role in DSB repair, though the mechanism through which they act remains unclear. Here, we report that the role of diRNAs in DSB repair is restricted to repair by homologous recombination (HR) and that it specifically relies on the effector protein Ago2 in mammalian cells. Interestingly, we show that Ago2 forms a complex with Rad51 and that the interaction is enhanced in cells treated with ionizing radiation. We demonstrate that Rad51 accumulation at DSB sites and HR repair depend on catalytic activity and small RNA-binding capability of Ago2. In contrast, DSB resection as well as RPA and Mre11 loading is unaffected by Ago2 or Dicer depletion, suggesting that Ago2 very likely functions directly in mediating Rad51 accumulation at DSBs. Taken together, our findings suggest that guided by diRNAs, Ago2 can promote Rad51 recruitment and/or retention at DSBs to facilitate repair by HR.

Antibody class switch recombination (CSR) in B lymphocytes replaces immunoglobulin heavy chain locus (Igh) Cμconstant region exons (C H s) with one of 6 C H s lying 100-200kb downstream 1 . Each C H is flanked upstream by an I-promoter and long repetitive switch (S) region 1 . Cytokines/ activators induce Activation-Induced Cytidine Deaminase (AID) 2 and I-promoter transcription, with 3'IgH regulatory region (3'IgHRR) enhancers controlling the latter via I-promoter competition for long-range 3'IgHRR interactions [3][4][5][6][7][8] . Transcription through donor Sμ and an activated downstream acceptor S region targets AID-generated deamination lesions at, potentially, any of 100s of individual S region deamination motifs [9][10][11] . General DNA repair pathways convert these lesions to DSBs and join an Sμ upstream DSB-end to an acceptor S region downstream DSB-end for deletional CSR 12 . AID-initiated DSBs at targets spread across activated S regions routinely participate in such deletional CSR joining 11 . Here, we report that chromatin loop Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

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RAG endonuclease initiates IgH V(D)J recombination in pro-B cells by binding a J H -recombination signal sequence (RSS) within a recombination center (RC) and then linearly scanning upstream chromatin, presented by cohesin-mediated loop extrusion, for convergent D-RSSs 1 , 2 . Utilization of convergently-oriented RSSs and cryptic RSSs is intrinsic to long-range RAG scanning 3 . RAG scanning from the DJ H -RC-RSS to upstream convergent V H -RSSs is impeded by D-proximal CTCF-binding elements (CBEs) 2 – 5 . Primary pro-B cells undergo a mechanistically-undefined V H locus contraction proposed to provide distal V H s access to the DJ H -RC 6 – 9 . Here, we report that a 2.4 mega-base V H locus inversion in primary pro-B cells abrogates rearrangement of both V H -RSSs and normally convergent cryptic RSSs, even though locus contraction still occurs. In addition, this inversion activated both utilization of cryptic V H -locus RSSs normally in opposite orientation and RAG scanning beyond the V H locus through multiple convergent-CBE domains to the telomere. Together, these findings imply that broad deregulation of CBE impediments in primary pro-B cells promotes loop extrusion-mediated RAG V H locus-scanning. We further found that expression of Wapl 10 , a cohesin-unloading factor, is low in primary pro-B cells versus v-Abl -transformed pro-B lines that lack contraction and RAG-scanning of the V H locus. Correspondingly, Wapl depletion in v-Abl -tranformed lines activated both processes, further implicating loop extrusion in the locus contraction mechanism.

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...