The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination [GRO-Seq]
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA534315 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8946860_1.fastq.gz | Fastqsanger.gz | |||
| SRR8946860_2.fastq.gz | Fastqsanger.gz | |||
| SRR8946870_1.fastq.gz | Fastqsanger.gz | |||
| SRR8946870_2.fastq.gz | Fastqsanger.gz | |||
| SRR8946861_1.fastq.gz | Fastqsanger.gz |
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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.
2019-08-19 | GSE130215 | GEO
Project description:The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination
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Project description:Loop Extrusion Mediates Physiological Locus Contraction for V(D)J Recombination (GRO-Seq)
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Project description:The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination [Hi-C]
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Project description:The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination [ChIP-Seq]
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Project description:The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination [3C-HTGTS]
| PRJNA534314 | ENA
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.
2019-08-19 | GSE130224 | GEO
Project description:Chromatin Loop Extrusion Plays a Fundamental Mechanistic Role in Antibody Class Switching [GRO-Seq]
| PRJNA534493 | ENA
Project description:Immunoglobulin heavy chain locus (Igh) VH, D, and JH gene segments are developmentally assembled into V(D)J exons. RAG endonuclease initiates V(D)J recombination by binding a JH-recombination signal sequence (RSS) within a chromatin-based recombination center (RC) and then, in an orientation-dependent process, scans upstream D-containing chromatin presented by cohesin-mediated loop extrusion for convergent D-RSSs to initiate DJH-RC formation. In primary pro-B cells, 100s of upstream VH-associated RSSs, embedded in convergent orientation to the DJH-RC-RSS, gain proximity to the DJH-RC for VH-to-DJH joining via a mechanistically-undefined VH-locus contraction process. Here, we report that a 2.4 mega-base VH locus inversion in primary pro-B cells nearly abrogates rearrangements of normally convergent VH-RSSs and cryptic RSSs, even though locus contraction per se is maintained. Moreover, this inversion activated rearrangement of both cryptic VH-locus RSSs normally in the opposite orientation and, unexpectedly, of normally-oriented cryptic RSSs within multiple, sequential upstream convergent-CBE domains. Primary pro-B cells had significantly reduced transcription of Wapl, a cohesin-unloading factor, versus levels in v-Abl pro-B lines that lack marked locus contraction or distal VH rearrangements. Correspondingly, Wapl depletion in v-Abl lines activated VH-locus contraction and orientation-specific RAG-scanning across the VH-locus. Our findings indicate that locus contraction and physiological VH-to-DJH joining both are regulated via circumvention of CBE scanning impediments.
2020-11-08 | GSE151907 | 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.
2019-08-19 | GSE134543 | GEO