Switch Tandem Repeats Influence the Choice of the Alternative End-Joining Pathway in Immunoglobulin Class Switch Recombination.

Immunoglobulin class switch recombination (CSR) plays an important role in humoral imm\une responses by changing the effector functions of antibodies. CSR occurs between highly repetitive switch (S) sequences located upstream of immunoglobulin constant gene exons. Switch sequences differ in size, the nature of their repeats, and the density of the motifs targeted by the activation-induced cytidine deaminase (AID), the enzyme that initiates CSR. CSR involves double-strand breaks (DSBs) at the universal Sµ donor region and one of the acceptor S regions. The DSBs ends are fused by the classical non-homologous end-joining (C-NHEJ) and the alternative-NHEJ (A-NHEJ) pathways. Of the two pathways, the A-NHEJ displays a bias towards longer junctional micro-homologies (MHs). The Sµ region displays features that distinguish it from other S regions, but the molecular basis of Sµ specificity is ill-understood. We used a mouse line in which the downstream Sγ3 region was put under the control of the Eµ enhancer, which regulates Sµ, and analyzed its recombination activity by CSR-HTGTS. Here, we show that provision of Eµ enhancer to Sγ3 is sufficient to confer the recombinational features of Sµ to Sγ3, including efficient AID recruitment, enhanced internal deletions and robust donor function in CSR. Moreover, junctions involving Sγ3 display a bias for longer MH irrespective of sequence homology with switch acceptor sites. The data suggest that the propensity for increased MH usage is an intrinsic property of Sγ3 sequence, and that the tandem repeats of the donor site influence the choice of the A-NHEJ.

Tissue and Temperature-Specific RNA-Seq Analysis Reveals Genomic Versatility and Adaptive Potential in Wild Sea Turtle Hatchlings (Caretta caretta).

Background: Digital transcriptomics is rapidly emerging as a powerful new technology for modelling the environmental dynamics of the adaptive landscape in diverse lineages. This is particularly valuable in taxa such as turtles and tortoises (order Testudines) which contain a large fraction of endangered species at risk due to anthropogenic impacts on the environment, including pollution, overharvest, habitat degradation, and climate change. Sea turtles (family Cheloniidae) in particular invite a genomics-enabled approach to investigating their remarkable portfolio of adaptive evolution. The sex of the endangered loggerhead sea turtle (Caretta caretta) is subject to temperature-dependent sex determination (TSD), a mechanism by which exposure to temperatures during embryonic development irreversibly determines sex. Higher temperatures produce mainly female turtles and lower temperatures produce mainly male turtles. Incubation temperature can have long term effects on the immunity, migratory ability, and ultimately longevity of hatchlings. We perform RNA-seq differential expression analysis to investigate tissue- and temperature-specific gene expression within brain (n = 7) and gonadal (n = 4) tissue of male and female loggerhead hatchlings. Results: We assemble tissue- and temperature-specific transcriptomes and identify differentially expressed genes relevant to sexual development and life history traits of broad adaptive interest to turtles and other amniotic species. We summarize interactions among differentially expressed genes by producing network visualizations, and highlight shared biological pathways related to migration, immunity, and longevity reported in the avian and reptile literature. Conclusions: The measurement of tissue- and temperature-specific global gene expression of an endangered, flagship species such as the loggerhead sea turtle (Caretta caretta) reveals the genomic basis for potential resiliency and is crucial to future management and conservation strategies with attention to changing climates. Brain and gonadal tissue collected from experimentally reared loggerhead male and female hatchlings comprise an exceedingly rare dataset that permits the identification of genes enriched in functions related to sexual development, immunity, longevity, and migratory behavior and will serve as a large, new genomic resource for the investigation of genotype-phenotype relationships in amniotes.

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 JH-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-RSSs1,2. The utilization of convergently oriented RSSs and cryptic RSSs is intrinsic to long-range RAG scanning3. Scanning of RAG from the DJH-RC-RSS to upstream convergent VH-RSSs is impeded by D-proximal CTCF-binding elements (CBEs)2-5. Primary progenitor B cells undergo a mechanistically undefined contraction of the VH locus that is proposed to provide distal VHs access to the DJH-RC6-9. Here we report that an inversion of the entire 2.4-Mb VH locus in mouse primary progenitor B cells abrogates rearrangement of both VH-RSSs and normally convergent cryptic RSSs, even though locus contraction still occurs. In addition, this inversion activated both the utilization of cryptic VH-RSSs that are normally in opposite orientation and RAG scanning beyond the VH locus through several convergent CBE domains to the telomere. Together, these findings imply that broad deregulation of CBE impediments in primary progenitor B cells promotes RAG scanning of the VH locus mediated by loop extrusion. We further found that the expression of wings apart-like protein homologue (WAPL)10, a cohesin-unloading factor, was low in primary progenitor B cells compared with v-Abl-transformed progenitor B cell lines that lacked contraction and RAG scanning of the VH locus. Correspondingly, depletion of WAPL in v-Abl-transformed lines activated both processes, further implicating loop extrusion in the locus contraction mechanism.

Physiological role of the 3'IgH CBEs super-anchor in antibody class switching.

IgH class switch recombination (CSR) replaces Cµ constant region (CH) exons with one of six downstream CHs by joining transcription-targeted double-strand breaks (DSBs) in the Cµ switch (S) region to DSBs in a downstream S region. Chromatin loop extrusion underlies fundamental CSR mechanisms including 3'IgH regulatory region (3'IgHRR)-mediated S region transcription, CSR center formation, and deletional CSR joining. There are 10 consecutive CTCF-binding elements (CBEs) downstream of the 3'IgHRR, termed the "3'IgH CBEs." Prior studies showed that deletion of eight 3'IgH CBEs did not detectably affect CSR. Here, we report that deletion of all 3'IgH CBEs impacts, to varying degrees, germline transcription and CSR of upstream S regions, except that of Sγ1. Moreover, deletion of all 3'IgH CBEs rendered the 6-kb region just downstream highly transcribed and caused sequences within to be aligned with Sµ, broken, and joined to form aberrant CSR rearrangements. These findings implicate the 3'IgH CBEs as critical insulators for focusing loop extrusion-mediated 3'IgHRR transcriptional and CSR activities on upstream CH locus targets.

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 cells1. Upon binding a recombination centre-based JH, RAG scans upstream chromatin via loop extrusion, potentially mediated by cohesin, to locate Ds and assemble a DJH-based recombination centre2. CTCF looping factor-bound elements (CBEs) within IGCR1 upstream of Ds impede RAG scanning3-5; however, their inactivation allows scanning to proximal VHs, where additional CBEs activate rearrangement and impede scanning any further upstream5. Distal VH utilization is thought to involve diffusional access to the recombination centre following large-scale Igh locus contraction6-8. Here we test the potential of linear RAG scanning to mediate distal VH usage in G1-arrested v-Abl pro-B cell lines9, which undergo robust D-to-JH but little VH-to-DJH rearrangements, presumably owing to lack of locus contraction2,5. Through an auxin-inducible approach10, we degraded the cohesin component RAD2110-12 or CTCF12,13 in these G1-arrested lines. Degradation of RAD21 eliminated all V(D)J recombination and interactions associated with RAG scanning, except for reecombination centre-located DQ52-to-JH joining, in which synapsis occurs by diffusion2. Remarkably, while degradation of CTCF suppressed most CBE-based chromatin interactions, it promoted robust recombination centre interactions with, and robust VH-to-DJH joining of, distal VHs, with patterns similar to those of 'locus-contracted' primary pro-B cells. Thus, downmodulation of CTCF-bound scanning-impediment activity promotes cohesin-driven RAG scanning across the 2.7-Mb Igh locus.

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 recombination1. 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 antigen1. Primary B cells undergo antigen-driven BCR affinity maturation through somatic hypermutation and cellular selection in germinal centres (GCs)2,3. Although most GCs are transient3, those in intestinal Peyer's patches (PPs)-which depend on the gut microbiota-are chronic4, and little is known about their BCR repertoires or patterns of somatic hypermutation. Here, using a high-throughput assay that analyses both V(D)J segment usage and somatic hypermutation profiles, we elucidate physiological BCR repertoires in mouse PP GCs. PP GCs from different mice expand public BCR clonotypes (clonotypes that are shared between many mice) that often have canonical CDR3s in the immunoglobulin heavy chain that, owing to junctional biases during V(D)J recombination, appear much more frequently than predicted in naive B cell repertoires. Some public clonotypes are dependent on the gut microbiota and encode antibodies that are reactive to bacterial glycans, whereas others are independent of gut bacteria. Transfer of faeces from specific-pathogen-free mice to germ-free mice restored germ-dependent clonotypes, directly implicating BCR selection. We identified somatic hypermutations that were recurrently selected in such public clonotypes, indicating that affinity maturation occurs in mouse PP GCs under homeostatic conditions. Thus, persistent gut antigens select recurrent BCR clonotypes to seed chronic PP GC responses.

Induction of recurrent break cluster genes in neural progenitor cells differentiated from embryonic stem cells in culture.

Mild replication stress enhances appearance of dozens of robust recurrent genomic break clusters, termed RDCs, in cultured primary mouse neural stem and progenitor cells (NSPCs). Robust RDCs occur within genes ("RDC-genes") that are long and have roles in neural cell communications and/or have been implicated in neuropsychiatric diseases or cancer. We sought to develop an in vitro approach to determine whether specific RDC formation is associated with neural development. For this purpose, we adapted a system to induce neural progenitor cell (NPC) development from mouse embryonic stem cell (ESC) lines deficient for XRCC4 plus p53, a genotype that enhances DNA double-strand break (DSB) persistence to enhance detection. We tested for RDCs by our genome-wide DSB identification approach that captures DSBs via their ability to join to specific genomic Cas9/single-guide RNA-generated bait DSBs. In XRCC4/p53-deficient ESCs, we detected seven RDCs, all of which were in genes and two of which were robust. In contrast, in NPCs derived from these ESC lines we detected 29 RDCs, a large fraction of which were robust and associated with long, transcribed neural genes that were also robust RDC-genes in primary NSPCs. These studies suggest that many RDCs present in NSPCs are developmentally influenced to occur in this cell type and indicate that induced development of NPCs from ESCs provides an approach to rapidly elucidate mechanistic aspects of NPC RDC formation.

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 (CHs) with one of six CHs lying 100-200 kb downstream1. Each CH is flanked upstream by an I promoter and long repetitive switch (S) region1. Cytokines and 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 interactions3-8. Transcription through donor Sµ and an activated downstream acceptor S-region targets AID-generated deamination lesions at, potentially, any of hundreds of individual S-region deamination motifs9-11. General DNA repair pathways convert these lesions to double-stranded breaks (DSBs) and join an Sµ-upstream DSB-end to an acceptor S-region-downstream DSB-end for deletional CSR12. AID-initiated DSBs at targets spread across activated S regions routinely participate in such deletional CSR joining11. Here we report that chromatin loop extrusion underlies the mechanism11 by which IgH organization in cis promotes deletional CSR. In naive B cells, loop extrusion dynamically juxtaposes 3' IgHRR enhancers with the 200-kb upstream Sµ to generate a CSR centre (CSRC). In CSR-activated primary B cells, I-promoter transcription activates cohesin loading, leading to generation of dynamic subdomains that directionally align a downstream S region with Sµ for deletional CSR. During constitutive Sα CSR in CH12F3 B lymphoma cells, inversional CSR can be activated by insertion of a CTCF-binding element (CBE)-based impediment in the extrusion path. CBE insertion also inactivates upstream S-region CSR and converts adjacent downstream sequences into an ectopic S region by inhibiting and promoting their dynamic alignment with Sµ in the CSRC, respectively. Our findings suggest that, in a CSRC, dynamically impeded cohesin-mediated loop extrusion juxtaposes proper ends of AID-initiated donor and acceptor S-region DSBs for deletional CSR. Such a mechanism might also contribute to pathogenic DSB joining genome-wide.

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 JH segments, before joining upstream VH segments to DJH intermediates1. In mouse progenitor B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain2 at the 3' end of Igh contains an internal subdomain that spans the 5' CBE anchor (IGCR1)3, the DH segments, and a RAG-bound recombination centre (RC)4. The RC comprises the JH-proximal D segment (DQ52), four JH segments, and the intronic enhancer (iEµ)5. Robust RAG-mediated cleavage is restricted to paired V(D)J segments flanked by complementary recombination signal sequences (12RSS and 23RSS)6. D segments are flanked downstream and upstream by 12RSSs that mediate deletional joining with convergently oriented JH-23RSSs and VH-23RSSs, respectively6. Despite 12/23 compatibility, inversional D-to-JH joining via upstream D-12RSSs is rare7,8. Plasmid-based assays have attributed the lack of inversional D-to-JH joining to sequence-based preference for downstream D-12RSSs9, as opposed to putative linear scanning mechanisms10,11. As RAG linearly scans convergent CBE-anchored chromatin loops4,12-14, potentially formed by cohesin-mediated loop extrusion15-18, we revisited its scanning role. Here we show that the chromosomal orientation of JH-23RSS programs RC-bound RAG to linearly scan upstream chromatin in the 3' Igh subdomain for convergently oriented D-12RSSs and, thereby, to mediate deletional joining of all D segments except RC-based DQ52, which joins by a diffusion-related mechanism. In a DQ52-based RC, formed in the absence of JH segments, RAG bound by the downstream DQ52-RSS scans the downstream constant region exon-containing 3' Igh subdomain, in which scanning can be impeded by targeted binding of nuclease-dead Cas9, by transcription through repetitive Igh switch sequences, and by the 3' Igh CBE-based loop anchor. Each scanning impediment focally increases RAG activity on potential substrate sequences within the impeded region. High-resolution mapping of chromatin interactions in the RC reveals that such focal RAG targeting is associated with corresponding impediments to the loop extrusion process that drives chromatin past RC-bound RAG.