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Series GSE130216 Query DataSets for GSE130216
Status Public on Aug 19, 2019
Title The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination [HTGTS V(D)J-seq]
Organism Mus musculus
Experiment type Genome variation profiling by high throughput sequencing
Summary 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.
Contributor(s) Zhang Y, Zhang X, Alt FW
Citation(s) 31511698
NIH grant(s)
Grant ID Grant title Affiliation Name
R01 AI020047 Mechanisms That Control Antigen Receptor Variable Region Exon Assembly CHILDREN'S HOSPITAL Frederick W. Alt
Submission date Apr 23, 2019
Last update date Oct 02, 2019
Contact name Frederick W Alt
Organization name Boston Children's Hospital
Department PCMM
Lab Alt
Street address 1 Blackfan Circle
City Boston
State/province MA
ZIP/Postal code 02115
Country USA
Platforms (1)
GPL16417 Illumina MiSeq (Mus musculus)
Samples (76)
GSM3733975 DHJH_JH1bait_set1_rep1
GSM3733976 DHJH_JH1bait_set1_rep2
GSM3733977 DHJH_JH1bait_set1_rep3
This SubSeries is part of SuperSeries:
GSE130224 The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination
BioProject PRJNA534316
SRA SRP193546

Download family Format
SOFT formatted family file(s) SOFTHelp
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Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE130216_RAW.tar 142.2 Mb (http)(custom) TAR (of TXT)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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