GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Series GSE263123 Query DataSets for GSE263123
Status Public on Apr 08, 2024
Title Molecular Basis for Differential Igk Versus Igh V(D)J Joining Mechanisms [3C-HTGTS data]
Organism Mus musculus
Experiment type Other
Summary In developing B lymphocytes, V(D)J recombination assembles IgH and Igk variable region exons from hundreds of gene segments clustered across mega-base Igh and Igk loci. V, D, and J segments are flanked by conserved recombination signal sequences (RSSs) that target RAG endonuclease. RAG orchestrates Igh V(D)J recombination upon capturing a JH-RSS within the JH-RSS-based recombination center (RC). JH-RSS orientation programs RAG to scan upstream D- and VH-containing chromatin linearly presented by cohesin-mediated loop extrusion. During Igh scanning, RAG robustly utilizes only D- or VH-RSSs in convergent ("deletional") orientation with JH-RSSs. However, for Vk-to-Jk joining, RAG utilizes Vk-RSSs from deletional- and inversional-oriented clusters, inconsistent with linear scanning. Here, we elucidate the Vk-to-Jk joining mechanism. Igk undergoes robust primary and secondary rearrangements, which confounds scanning assays. Thus, we engineered cells to undergo only primary Vk-to-Jk rearrangements and found that RAG-scanning from the primary Jk-RC terminates just 8kb upstream within the CTCF-Site-based Sis element. While Sis and the Jk-RC barely interacted with the Vk locus, the CTCF-site-based Cer element, 4kb upstream of Sis, interacted with various loop-extrusion impediments across the locus. Like VH-locus inversion, DJH inversion abrogated VH-to-DJH joining; yet Vk-locus or Jk inversion allowed robust Vk-to-Jk joining. Together, these experiments implicated loop extrusion in bringing Vks near Cer for short-range diffusion-mediated capture by RC-based RAG. To elucidate key mechanistic elements for diffusional V(D)J recombination in Igk versus Igh, we assayed Vk-to-JH and D-to-Jk rearrangements in hybrid Igh-Igk loci generated by targeted chromosomal translocations, and pinpointed remarkably strong Vk and Jk RSSs. Indeed, RSS replacements in hybrid or normal Igk and Igh loci confirmed ability of Igk versus Igh RSSs to promote robust diffusional joining. We propose that Igk evolved strong RSSs to mediate diffusional Vk-to-Jk joining; while Igh evolved weaker RSSs requisite for modulating VH joining by RAG scanning impediments.
Overall design We performed 3C-HTGTS in BM pre-B cells and v- Abl transformed pro-B cells and its various mutant derivatives to study the differential molecular mechanisms of Igk versus Igh V(D)J recombination.
Contributor(s) Alt FW, Hu H, Zhang Y, Li X
Citation(s) 38811728
Submission date Apr 03, 2024
Last update date Jun 14, 2024
Contact name Frederick Alt
Organization name Boston Children's Hospital / Harvard Medical School
Department Program in Cellular and Molecular Medicine
Lab Frederick Alt
Street address 1 Blackfan Circle
City Boston
State/province MA
ZIP/Postal code 02115
Country USA
Platforms (1)
GPL21626 NextSeq 550 (Mus musculus)
Samples (16)
GSM8186161 v-Abl_iEk_rep1
GSM8186162 v-Abl_iEk_rep2
GSM8186163 v-Abl_SisCBE2_rep1
This SubSeries is part of SuperSeries:
GSE263124 Molecular Basis for Differential Igk Versus Igh V(D)J Joining Mechanisms
BioProject PRJNA1095894

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

Supplementary file Size Download File type/resource
GSE263123_RAW.tar 51.4 Mb (http)(custom) TAR (of BEDGRAPH, TLX)
SRA Run SelectorHelp
Raw data are available in SRA

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap