Early human IgH gene assembly in Epstein-Barr virus-transformed fetal B cell lines. Preferential utilization of the most JH-proximal D segment (DQ52) and two unusual VH-related rearrangements.
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ABSTRACT:
PROVIDER: S-EPMC2189244 | BioStudies | 1989-01-01T00:00:00Z
REPOSITORIES: biostudies
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Project description: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 (iE?)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.
| S-EPMC6867615 | BioStudies
Project description:RAG endonuclease initiates IgH locus (Igh) V(D)J assembly in progenitor (pro)-B cells by joining Ds to JHs, before joining upstream VHs to DJH intermediates. In mouse pro-B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain at the 3’end of Igh contains an internal sub-domain spanning the 5’CBE anchor (IGCR1), the DHs, and a RAG-bound recombination center (RC). The RC comprises JH-proximal D (DQ52), 4 JHs, and the intronic enhancer (“iEmu”). Robust RAG cleavage is restricted to paired V(D)J segments flanked by complementary recombination signal sequences (12RSSs and 23RSSs). Ds are flanked downstream and upstream by 12RSSs that, respectively, mediate deletional joining with convergently-oriented JH-23RSSs and VH-23RSSs. Despite 12/23 compatibility, inversional D to JH joining via upstream D-12RSSs is rare. Plasmid-based assays attributed lack of inversional D to JH joining to sequence-based preference for downstream D-12RSSs, as opposed to putative linear scanning mechanisms. Given recent findings that RAG linearly scans convergent CBE-anchored chromatin loops, potentially formed by cohesin-mediated loop extrusion, we revisited a scanning role. Here, we report that JH-23RSS chromosomal orientation programs RC-bound RAG to linearly scan upstream chromatin in the 3’Igh sub-domain for convergently-oriented D-12RSSs and, thereby, to mediate deletional joining of all Ds, except RC-based DQ52 that joins by a diffusion-related mechanism. In a DQ52-based RC, formed in the absence of JHs, RAG bound by the downstream DQ52-RSS scans the downstream constant region exon-containing 3’Igh sub-domain in which scanning can be impeded by targeted nuclease-dead Cas9 (dCas9) binding, by transcription through repetitive Igh switch sequences, and by the 3’Igh CBE-based loop anchor. Notably, each scanning impediment focally increases RAG activity on potential substrate sequences within the impeded region. High resolution mapping of RC chromatin interactions reveals that such focal RAG targeting is associated with corresponding impediments to the loop extrusion process that drives chromatin past RC-bound RAG. Overall design: We performed 3C-HTGTS using IgH intronic enhancer as bait in v-Abl transformed pro-B cells to study roles of chromatin loop extrusion-mediated RAG scanning in IgH V(D)J recombination.
2019-08-19 | GSE130214 | GEO
Project description:RAG endonuclease initiates IgH locus (Igh) V(D)J assembly in progenitor (pro)-B cells by joining Ds to JHs, before joining upstream VHs to DJH intermediates. In mouse pro-B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain at the 3’end of Igh contains an internal sub-domain spanning the 5’CBE anchor (IGCR1), the DHs, and a RAG-bound recombination center (RC). The RC comprises JH-proximal D (DQ52), 4 JHs, and the intronic enhancer (“iEmu”). Robust RAG cleavage is restricted to paired V(D)J segments flanked by complementary recombination signal sequences (12RSSs and 23RSSs). Ds are flanked downstream and upstream by 12RSSs that, respectively, mediate deletional joining with convergently-oriented JH-23RSSs and VH-23RSSs. Despite 12/23 compatibility, inversional D to JH joining via upstream D-12RSSs is rare. Plasmid-based assays attributed lack of inversional D to JH joining to sequence-based preference for downstream D-12RSSs, as opposed to putative linear scanning mechanisms. Given recent findings that RAG linearly scans convergent CBE-anchored chromatin loops, potentially formed by cohesin-mediated loop extrusion, we revisited a scanning role. Here, we report that JH-23RSS chromosomal orientation programs RC-bound RAG to linearly scan upstream chromatin in the 3’Igh sub-domain for convergently-oriented D-12RSSs and, thereby, to mediate deletional joining of all Ds, except RC-based DQ52 that joins by a diffusion-related mechanism. In a DQ52-based RC, formed in the absence of JHs, RAG bound by the downstream DQ52-RSS scans the downstream constant region exon-containing 3’Igh sub-domain in which scanning can be impeded by targeted nuclease-dead Cas9 (dCas9) binding, by transcription through repetitive Igh switch sequences, and by the 3’Igh CBE-based loop anchor. Notably, each scanning impediment focally increases RAG activity on potential substrate sequences within the impeded region. High resolution mapping of RC chromatin interactions reveals that such focal RAG targeting is associated with corresponding impediments to the loop extrusion process that drives chromatin past RC-bound RAG. Overall design: We performed GRO-Seq in v-Abl transformed pro-B cells to study roles of chromatin loop extrusion-mediated RAG scanning in IgH V(D)J recombination.
2019-08-19 | GSE130215 | GEO
Project description:RAG endonuclease initiates IgH locus (Igh) V(D)J assembly in progenitor (pro)-B cells by joining Ds to JHs, before joining upstream VHs to DJH intermediates. In mouse pro-B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain at the 3’end of Igh contains an internal sub-domain spanning the 5’CBE anchor (IGCR1), the DHs, and a RAG-bound recombination center (RC). The RC comprises JH-proximal D (DQ52), 4 JHs, and the intronic enhancer (“iEmu”). Robust RAG cleavage is restricted to paired V(D)J segments flanked by complementary recombination signal sequences (12RSSs and 23RSSs). Ds are flanked downstream and upstream by 12RSSs that, respectively, mediate deletional joining with convergently-oriented JH-23RSSs and VH-23RSSs. Despite 12/23 compatibility, inversional D to JH joining via upstream D-12RSSs is rare. Plasmid-based assays attributed lack of inversional D to JH joining to sequence-based preference for downstream D-12RSSs, as opposed to putative linear scanning mechanisms. Given recent findings that RAG linearly scans convergent CBE-anchored chromatin loops, potentially formed by cohesin-mediated loop extrusion, we revisited a scanning role. Here, we report that JH-23RSS chromosomal orientation programs RC-bound RAG to linearly scan upstream chromatin in the 3’Igh sub-domain for convergently-oriented D-12RSSs and, thereby, to mediate deletional joining of all Ds, except RC-based DQ52 that joins by a diffusion-related mechanism. In a DQ52-based RC, formed in the absence of JHs, RAG bound by the downstream DQ52-RSS scans the downstream constant region exon-containing 3’Igh sub-domain in which scanning can be impeded by targeted nuclease-dead Cas9 (dCas9) binding, by transcription through repetitive Igh switch sequences, and by the 3’Igh CBE-based loop anchor. Notably, each scanning impediment focally increases RAG activity on potential substrate sequences within the impeded region. High resolution mapping of RC chromatin interactions reveals that such focal RAG targeting is associated with corresponding impediments to the loop extrusion process that drives chromatin past RC-bound RAG. Overall design: We performed HTGTS V(D)J-seq in v-Abl transformed pro-B cells to study roles of chromatin loop extrusion-mediated RAG scanning in IgH V(D)J recombination.
2019-08-19 | GSE130216 | GEO
Project description:RAG endonuclease initiates IgH locus (Igh) V(D)J assembly in progenitor (pro)-B cells by joining Ds to JHs, before joining upstream VHs to DJH intermediates. In mouse pro-B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain at the 3’end of Igh contains an internal sub-domain spanning the 5’CBE anchor (IGCR1), the DHs, and a RAG-bound recombination center (RC). The RC comprises JH-proximal D (DQ52), 4 JHs, and the intronic enhancer (“iEmu”). Robust RAG cleavage is restricted to paired V(D)J segments flanked by complementary recombination signal sequences (12RSSs and 23RSSs). Ds are flanked downstream and upstream by 12RSSs that, respectively, mediate deletional joining with convergently-oriented JH-23RSSs and VH-23RSSs. Despite 12/23 compatibility, inversional D to JH joining via upstream D-12RSSs is rare. Plasmid-based assays attributed lack of inversional D to JH joining to sequence-based preference for downstream D-12RSSs, as opposed to putative linear scanning mechanisms. Given recent findings that RAG linearly scans convergent CBE-anchored chromatin loops, potentially formed by cohesin-mediated loop extrusion, we revisited a scanning role. Here, we report that JH-23RSS chromosomal orientation programs RC-bound RAG to linearly scan upstream chromatin in the 3’Igh sub-domain for convergently-oriented D-12RSSs and, thereby, to mediate deletional joining of all Ds, except RC-based DQ52 that joins by a diffusion-related mechanism. In a DQ52-based RC, formed in the absence of JHs, RAG bound by the downstream DQ52-RSS scans the downstream constant region exon-containing 3’Igh sub-domain in which scanning can be impeded by targeted nuclease-dead Cas9 (dCas9) binding, by transcription through repetitive Igh switch sequences, and by the 3’Igh CBE-based loop anchor. Notably, each scanning impediment focally increases RAG activity on potential substrate sequences within the impeded region. High resolution mapping of RC chromatin interactions reveals that such focal RAG targeting is associated with corresponding impediments to the loop extrusion process that drives chromatin past RC-bound RAG. Overall design: We performed Hi-C in v-Abl transformed pro-B cells to study roles of chromatin loop extrusion-mediated RAG scanning in IgH V(D)J recombination.
2019-08-19 | GSE134543 | GEO
Project description:RAG endonuclease initiates IgH locus (Igh) V(D)J assembly in progenitor (pro)-B cells by joining Ds to JHs, before joining upstream VHs to DJH intermediates. In mouse pro-B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain at the 3’end of Igh contains an internal sub-domain spanning the 5’CBE anchor (IGCR1), the DHs, and a RAG-bound recombination center (RC). The RC comprises JH-proximal D (DQ52), 4 JHs, and the intronic enhancer (“iEmu”). Robust RAG cleavage is restricted to paired V(D)J segments flanked by complementary recombination signal sequences (12RSSs and 23RSSs). Ds are flanked downstream and upstream by 12RSSs that, respectively, mediate deletional joining with convergently-oriented JH-23RSSs and VH-23RSSs. Despite 12/23 compatibility, inversional D to JH joining via upstream D-12RSSs is rare. Plasmid-based assays attributed lack of inversional D to JH joining to sequence-based preference for downstream D-12RSSs, as opposed to putative linear scanning mechanisms. Given recent findings that RAG linearly scans convergent CBE-anchored chromatin loops, potentially formed by cohesin-mediated loop extrusion, we revisited a scanning role. Here, we report that JH-23RSS chromosomal orientation programs RC-bound RAG to linearly scan upstream chromatin in the 3’Igh sub-domain for convergently-oriented D-12RSSs and, thereby, to mediate deletional joining of all Ds, except RC-based DQ52 that joins by a diffusion-related mechanism. In a DQ52-based RC, formed in the absence of JHs, RAG bound by the downstream DQ52-RSS scans the downstream constant region exon-containing 3’Igh sub-domain in which scanning can be impeded by targeted nuclease-dead Cas9 (dCas9) binding, by transcription through repetitive Igh switch sequences, and by the 3’Igh CBE-based loop anchor. Notably, each scanning impediment focally increases RAG activity on potential substrate sequences within the impeded region. High resolution mapping of RC chromatin interactions reveals that such focal RAG targeting is associated with corresponding impediments to the loop extrusion process that drives chromatin past RC-bound RAG. Overall design: We performed RAD21 and NIPBL ChIP-Seq in v-Abl transformed pro-B cells to study roles of chromatin loop extrusion mediated-RAG scanning in IgH V(D)J recombination.
2019-08-19 | GSE130213 | GEO
Project description:RAG endonuclease initiates IgH locus (Igh) V(D)J assembly in progenitor (pro)-B cells by joining Ds to JHs, before joining upstream VHs to DJH intermediates. In mouse pro-B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain at the 3’end of Igh contains an internal sub-domain spanning the 5’CBE anchor (IGCR1), the DHs, and a RAG-bound recombination center (RC). The RC comprises JH-proximal D (DQ52), 4 JHs, and the intronic enhancer (“iEmu”). Robust RAG cleavage is restricted to paired V(D)J segments flanked by complementary recombination signal sequences (12RSSs and 23RSSs). Ds are flanked downstream and upstream by 12RSSs that, respectively, mediate deletional joining with convergently-oriented JH-23RSSs and VH-23RSSs. Despite 12/23 compatibility, inversional D to JH joining via upstream D-12RSSs is rare. Plasmid-based assays attributed lack of inversional D to JH joining to sequence-based preference for downstream D-12RSSs, as opposed to putative linear scanning mechanisms. Given recent findings that RAG linearly scans convergent CBE-anchored chromatin loops, potentially formed by cohesin-mediated loop extrusion, we revisited a scanning role. Here, we report that JH-23RSS chromosomal orientation programs RC-bound RAG to linearly scan upstream chromatin in the 3’Igh sub-domain for convergently-oriented D-12RSSs and, thereby, to mediate deletional joining of all Ds, except RC-based DQ52 that joins by a diffusion-related mechanism. In a DQ52-based RC, formed in the absence of JHs, RAG bound by the downstream DQ52-RSS scans the downstream constant region exon-containing 3’Igh sub-domain in which scanning can be impeded by targeted nuclease-dead Cas9 (dCas9) binding, by transcription through repetitive Igh switch sequences, and by the 3’Igh CBE-based loop anchor. Notably, each scanning impediment focally increases RAG activity on potential substrate sequences within the impeded region. High resolution mapping of RC chromatin interactions reveals that such focal RAG targeting is associated with corresponding impediments to the loop extrusion process that drives chromatin past RC-bound RAG. Overall design: We performed HTGTS V(D)J-seq, 3C-HTGTS, GRO-Seq, ChIP-Seq and Hi-C in v-Abl transformed pro-B cells to study roles of chromatin loop extrusion-mediated RAG scanning in IgH V(D)J recombination.
2019-08-19 | GSE130224 | GEO
Project description:V(D)J recombination at the immunoglobulin heavy chain (IgH) locus follows the 12/23 rule to ensure the correct assembly of the variable region gene segments. Here, we report characterization of an in vivo model that allowed us to study recombination violating the 12/23 rule, namely a mouse strain lacking canonical D elements in its IgH locus. We demonstrate that VH to JH joining can support the generation of all B cell subsets. However, the process is inefficient in that B cells and antibodies derived from the DH-less allele are not detectable if the latter is combined with a wild-type IgH allele. There is no preferential usage of any particular VH gene family or JH element in VHJH junctions, indicating that 23/23-guided recombination is possible, but is a low frequency event at the IgH locus in vivo.
| S-EPMC1237122 | BioStudies
Project description:AIMS:To demonstrate the usefulness of polymerase chain reaction (PCR) methodology with both the FR2A/LJH/VLJH and the FR1c/LJH/VLJH primer sets for detecting monoclonal immunoglobulin heavy chain (IgH) gene rearrangement in B cell non-Hodgkin lymphomas (B-NHLs). METHODS:Eighty three patients with B-NHL were enrolled in this study. DNA was isolated from paraffin wax embedded sections and amplified by PCR to determine the sequences of the rearranged IgH gene. Each PCR product was subcloned. Cycle sequences and sequence analyses were done to determine the clone specific IgH variable region (VH) sequences. RESULTS:Clonal IgH gene rearrangements were detected in 45 cases with FR2a/JH/VLJH and in 14 of the remaining cases with FR1c/JH/VLJH. Most of the cases detectable with FR2a/JH/VLJH were derived from VH3 and VH4 families. Five of six cases in the VH1 family and one in the VH7 family were amplified with the FR1c/JH/VLJH primer set only. CONCLUSION:The detection rate of IgH rearrangement in B-NHLs can be increased by using both FR2A/LJH/VLJH and FR1c/LJH/VLJH, and these two primer sets are suitable for routine PCR methodology. Moreover, each primer set appears to be closely related to VH family specificity.
2003-01-01 | S-EPMC1770088 | BioStudies
Project description:The formation of nontemplated (N) regions during Ig gene rearrangement is a major contributor to Ab diversity. To gain insights into the mechanisms behind this, we studied the nucleotide composition of N regions within 29,962 unique human VHDJH rearrangements and 8728 unique human DJH rearrangements containing exactly one identifiable D gene segment and thus two N regions, N1 and N2. We found a distinct decreasing content of cytosine (C) and increasing content of guanine (G) across each N region, suggesting that N regions are typically generated by concatenation of two 3' overhangs synthesized by addition of nucleoside triphosphates with a preference for dCTP. This challenges the general assumption that the terminal deoxynucleotidyl transferase favors dGTP in vivo. Furthermore, we found that the G and C gradients depended strongly on whether the germline gene segments were trimmed or not. Our data show that C-enriched N addition preferentially happens at trimmed 3' ends of VH, D, and JH gene segments, indicating a dependency of the transferase mechanism upon the nuclease mechanism.
| S-EPMC6121214 | BioStudies