Core enhancers of the 3'RR optimize IgH nuclear position and loop conformation for successful oriented class switch recombination.

In B lymphocytes, class switch recombination (CSR) is an essential process that adapts immunoglobulin (Ig) subtypes to antigen response. Taking place within the Ig heavy chain (IgH) locus, CSR needs controlled transcription of targeted regions governed by the IgH 3' regulatory region (3'RR). This super-enhancer is composed of four core enhancers surrounded by inverted repeated sequences, forming a quasi-palindrome.

Molecular basis for differential Igk versus Igh V(D)J joining mechanisms.

In developing B cells, V(D)J recombination assembles exons encoding IgH and Igκ variable regions from hundreds of gene segments clustered across Igh and Igk loci. V, D and J gene segments are flanked by conserved recombination signal sequences (RSSs) that target RAG endonuclease. RAG orchestrates Igh V(D)J recombination upon capturing a J-RSS within the J-RSS-based recombination centre (RC).

Contribution of the IGCR1 regulatory element and the 3' CTCF-binding elements to regulation of V(D)J recombination.

Immunoglobulin heavy chain variable region exons are assembled in progenitor-B cells, from V, D, and J gene segments located in separate clusters across the locus. RAG endonuclease initiates V(D)J recombination from a J-based recombination center (RC). Cohesin-mediated extrusion of upstream chromatin past RC-bound RAG presents Ds for joining to Js to form a DJ-RC.

Contribution of the IGCR1 regulatory element and the 3 CBEs to Regulation of V(D)J Recombination.

Unlabelled: Immunoglobulin heavy chain variable region exons are assembled in progenitor-B cells, from V , D, and J gene segments located in separate clusters across the locus. RAG endonuclease initiates V(D)J recombination from a J -based recombination center (RC). Cohesin-mediated extrusion of upstream chromatin past RC-bound RAG presents Ds for joining to J s to form a DJ -RC.

Loop extrusion mediates physiological Igh locus contraction for RAG scanning.

RAG endonuclease initiates Igh V(D)J recombination in progenitor B cells by binding a J-recombination signal sequence (RSS) within a recombination centre (RC) and then linearly scanning upstream chromatin, presented by loop extrusion mediated by cohesin, for convergent D-RSSs. The utilization of convergently oriented RSSs and cryptic RSSs is intrinsic to long-range RAG scanning. Scanning of RAG from the DJ-RC-RSS to upstream convergent V-RSSs is impeded by D-proximal CTCF-binding elements (CBEs).

CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning.

The RAG endonuclease initiates Igh locus V(D)J recombination in progenitor (pro)-B cells. Upon binding a recombination centre-based J, RAG scans upstream chromatin via loop extrusion, potentially mediated by cohesin, to locate Ds and assemble a DJ-based recombination centre. CTCF looping factor-bound elements (CBEs) within IGCR1 upstream of Ds impede RAG scanning; however, their inactivation allows scanning to proximal Vs, where additional CBEs activate rearrangement and impede scanning any further upstream.

BCR selection and affinity maturation in Peyer's patch germinal centres.

The antigen-binding variable regions of the B cell receptor (BCR) and of antibodies are encoded by exons that are assembled in developing B cells by V(D)J recombination. The BCR repertoires of primary B cells are vast owing to mechanisms that create diversity at the junctions of V(D)J gene segments that contribute to complementarity-determining region 3 (CDR3), the region that binds antigen. Primary B cells undergo antigen-driven BCR affinity maturation through somatic hypermutation and cellular selection in germinal centres (GCs).

Fundamental roles of chromatin loop extrusion in antibody class switching.

Antibody class switch recombination (CSR) in B lymphocytes replaces immunoglobulin heavy chain locus (Igh) Cμ constant region exons (Cs) with one of six Cs lying 100-200 kb downstream. Each C is flanked upstream by an I promoter and long repetitive switch (S) region. Cytokines and activators induce activation-induced cytidine deaminase (AID) 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.

The fundamental role of chromatin loop extrusion in physiological V(D)J recombination.

The RAG endonuclease initiates Igh V(D)J assembly in B cell progenitors by joining D segments to J segments, before joining upstream V segments to DJ intermediates. In mouse progenitor B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain at the 3' end of Igh contains an internal subdomain that spans the 5' CBE anchor (IGCR1), the D segments, and a RAG-bound recombination centre (RC). The RC comprises the J-proximal D segment (DQ52), four J segments, and the intronic enhancer (iEμ).

RAG Chromatin Scanning During V(D)J Recombination and Chromatin Loop Extrusion are Related Processes.

An effective adaptive immune system depends on the ability of developing B and T cells to generate diverse immunoglobulin (Ig) and T cell receptor repertoires, respectively. Such diversity is achieved through a programmed somatic recombination process whereby germline V, D, and J segments of antigen receptor loci are assembled to form the variable region V(D)J exons of Ig and TCRs. Studies of this process, termed V(D)J recombination, have provided key insights into our understanding of a variety of general gene regulatory and DNA repair processes over the last several decades.

CTCF-Binding Elements Mediate Accessibility of RAG Substrates During Chromatin Scanning.

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.

IDN2 Interacts with RPA and Facilitates DNA Double-Strand Break Repair by Homologous Recombination in Arabidopsis.

Repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genome integrity. We previously showed that DSB-induced small RNAs (diRNAs) facilitate homologous recombination-mediated DSB repair in Here, we show that INVOLVED IN DE NOVO2 (IDN2), a double-stranded RNA binding protein involved in small RNA-directed DNA methylation, is required for DSB repair in Arabidopsis. We find that IDN2 interacts with the heterotrimeric replication protein A (RPA) complex.

Highly sensitive and unbiased approach for elucidating antibody repertoires.

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.

Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination.

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.

Small RNAs: emerging key players in DNA double-strand break repair.

DNA double-strand break (DSB) is the most deleterious form of DNA damage and poses great threat to genome stability. Eukaryotes have evolved complex mechanisms to repair DSBs through coordinated actions of protein sensors, transducers, and effectors. DSB-induced small RNAs (diRNAs) or Dicer/Drosha-dependent RNAs (DDRNAs) have been recently discovered in plants and vertebrates, adding an unsuspected RNA component into the DSB repair pathway.

A role for small RNAs in DNA double-strand break repair.

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.