Mus musculus
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA317332 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR3331550_1.fastq.gz | Fastqsanger.gz | |||
| SRR3331550_2.fastq.gz | Fastqsanger.gz | |||
| SRR3331560_1.fastq.gz | Fastqsanger.gz | |||
| SRR3331560_2.fastq.gz | Fastqsanger.gz | |||
| SRR3331551_1.fastq.gz | Fastqsanger.gz |
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Project description:T cell antigen receptor δ (Tcrd) variable region exons are assembled by RAG-initiated V(D)J recombination. Here, we employ a high throughput method to map hundreds of thousands of RAG-initiated Tcrd D segment (Trdd1 and Trdd2) rearrangements in developing thymocytes. We find that Trdd2 joins directly to Trdv, Trdd1, and Trdj segments, but Trdd1 joining is ordered with joining to Trdd2 a prerequisite for further rearrangement. We also find frequent, previously unappreciated Trdd1 and Trdd2 rearrangements that inactivate Tcrd. Moreover, we find numerous RAG off-targets that are generated via unidirectional RAG tracking across the loop-domain containing Trdd1, Trdd2 and Trdj. Correspondingly, disruption of the upstream domain boundary causes spreading of on- and off-target RAG activity to the proximal Trdv domain. RAG-initiatd Tcrd D segment rearrangements in developing thymocytes were generated by deep sequencing using illumine Miseq
2016-08-04 | E-GEOD-79892 | biostudies-arrayexpress
Project description:T cell antigen receptor δ (Tcrd) variable region exons are assembled by RAG-initiated V(D)J recombination. Here, we employ a high throughput method to map hundreds of thousands of RAG-initiated Tcrd D segment (Trdd1 and Trdd2) rearrangements in developing thymocytes. We find that Trdd2 joins directly to Trdv, Trdd1, and Trdj segments, but Trdd1 joining is ordered with joining to Trdd2 a prerequisite for further rearrangement. We also find frequent, previously unappreciated Trdd1 and Trdd2 rearrangements that inactivate Tcrd. Moreover, we find numerous RAG off-targets that are generated via unidirectional RAG tracking across the loop-domain containing Trdd1, Trdd2 and Trdj. Correspondingly, disruption of the upstream domain boundary causes spreading of on- and off-target RAG activity to the proximal Trdv domain.
2016-08-04 | GSE79892 | GEO
Project description:Chromatin looping mediated by the CCCTC binding factor CTCF regulates V(D)J recombination at antigen receptor loci. CTCF-mediated looping can influence recombination signal sequence accessibility by regulating enhancer activation of germline promoters. CTCF-mediated looping has also been shown to limit directional tracking of the RAG recombinase along chromatin, and to regulate through-space interactions between recombination signal sequences, independent of the RAG recombinase. However, in all prior instances in which CTCF-mediated looping was shown to influence V(D)J recombination, it was not possible to fully resolve the relative contributions to the V(D)J recombination phenotype of changes in accessibility, RAG-tracking, and RAG-independent long-distance interactions. Here, to assess mechanisms by which CTCF-mediated looping can impact V(D)J recombination, we introduced an ectopic CTCF binding element (CBE) immediately downstream of Eδ in the murine Tcra-Tcrd locus. The ectopic CBE impaired inversional rearrangement of Trdv5 in the absence of measurable effects on Trdv5 transcription and chromatin accessibility. Moreover, although the ectopic CBE limited directional RAG tracking from the Tcrd recombination center, such tracking cannot account for Trdv5-to-Trdd2 inversional rearrangement. Rather, the defect in Trdv5 rearrangement could only be attributed to a reconfigured chromatin loop organization that limited RAG-independent through-space interactions between the Trdv5 and Trdd2 RSSs. We conclude that CTCF can regulate V(D)J recombination by segregating RSSs into distinct loop domains and inhibiting RSS synapsis, independent of any effects on transcription, RSS accessibility and RAG tracking. RAG-initiatd Tcrd D segment rearrangements in developing thymocytes were generated by deep sequencing using illumine Miseq
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Project description:RAG initiates V(D)J recombination in developing lymphocytes by generating "on-target" DNA double-stranded breaks at bona fide recombination signal sequence (RSS) pairs. We now employ RAG-generated DNA breaks in endogenous or ectopically-inserted RSS pairs as bait to identify huge numbers of RAG "off-target" sites. These off-target DNA breaks occur across convergent CTCF-binding element (CBE)-flanked loop domains containing the bait RSS pairs. Such off-target cleavage occurs at the simple CAC motif that defines the RSS cleavage-site and shows orientation-dependence best explained by a two-dimensional tracking mechanism. Deletion of the CBE-based IGCR1 IgH regulatory element disrupts antibody IgH recombination domains and, correspondingly, alters distributions of RAG on- and off-targets across the IgH locus. RAG off-targets frequently involved in chromosomal translocations occur in convergent RSS pairs at enhancer regions within a loop. Our findings reveal how RAG is developmentally focused and re-focused in lymphocytes and implicate mechanisms by which chromatin domains harness biological processes within them. We performed high-throughput genome-wide translocation sequencing (HTGTS) in progenitor B cells or cell lines to study signatures of RAG on and off-targeting activity using RAG-generated breaks in endogenous or ectopically-inserted paired bona fide RSSs as bait. Sequenceing was dong by Illumina Miseq.
2015-09-15 | E-GEOD-73007 | biostudies-arrayexpress
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.
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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.
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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.
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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.
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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.
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