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| Status |
Public on May 11, 2020 |
| Title |
CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning [ChIP-Seq] |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
|
| Summary |
RAG endonuclease initiates V(D)J recombination in progenitor (pro)-B cells. Upon binding a recombination center (RC)-based JH, RAG scans upstream chromatin via loop extrusion, potentially mediated by cohesin, to locate Ds and assemble a DJH-based RC. CTCF looping factor-bound elements (CBEs) within the IGCR1 element upstream of the Ds impede RAG-scanning; but their inactivation allows scanning to proximal VHs where additional CBEs activate rearrangement and impede scanning any further upstream. Distal VH utilization is thought to involve diffusional RC access following large-scale Igh locus contraction. Here, we test the potential of linear RAG-scanning to mediate distal VH usage in G1-arrested, v-Abl-pro-B cell lines, which undergo robust D-to-JH rearrangement, but little VH-to-DJH rearrangement, presumably due to lack of locus contraction. Through an auxin-induced approach, we degrade cohesin-component Rad21 or CTCF in these G1-arrested lines, which maintain substantial viability throughout four-day experiments. Rad21 degradation eliminated all V(D)J recombination and RAG-scanning-associated interactions, except RC-located DQ52-to-JH joining in which synapsis occurs by diffusion11. Remarkably, while CTCF degradation suppressed most CBE-based chromatin interactions, it promoted robust RC interactions with, and VH-to-DJH joining of, distal VHs, with patterns similar to those of "locus-contracted" primary pro-B cells. Thus, down-modulation of CTCF-bound scanning-impediment activity promotes cohesin-driven RAG-scanning across the 2.7Mb Igh locus.
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| Overall design |
We performed LAM-HTGTS V(D)J-Seq, 3C-HTGTS, GRO-Seq and ChIP-Seq in BM pro-B cells and v- Abl transformed pro-B cells and its various mutant derivatives to study roles of Rad21 and CTCF in long-range cohesin-driven V(D)J recombinational scanning
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| |
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| Contributor(s) |
Alt FW, Zhaoqing B, Jiangman L |
| Citation(s) |
32717742 |
| |
| Submission date |
Dec 31, 2019 |
| Last update date |
Oct 08, 2020 |
| Contact name |
Frederick W Alt |
| E-mail(s) |
jianqiao.hu@childrens.harvard.edu
|
| 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 |
| |
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| Platforms (1) |
|
| Samples (24)
|
| GSM4240113 |
Rad21ChIP_HCT116spikingin_Rad21degron_NT_rep1 |
| GSM4240114 |
Rad21ChIP_HCT116spikingin_Rad21degron_IAA_rep1 |
| GSM4240115 |
Rad21ChIP_HCT116spikingin_Rad21degron_NT_input_rep1 |
|
| This SubSeries is part of SuperSeries: |
| GSE142781 |
CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning |
|
| Relations |
| BioProject |
PRJNA598324 |
| SRA |
SRP239168 |
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