Long-Range IgH V(D)J Recombination [3C-HTGTS]
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE295768 | GEO | 2026/04/22
REPOSITORIES: GEO
Similar Datasets
Project description:Cohesin-mediated loop extrusion plays a crucial role in IgH V(D)J recombination, driving D-to-JH rearrangements followed by VH-to-DJH rearrangements.
2026-04-22 | GSE295770 | GEO
Project description:Cohesin-mediated loop extrusion plays a crucial role in IgH V(D)J recombination, driving D-to-JH rearrangements followed by VH-to-DJH rearrangements.
2026-04-22 | GSE295769 | GEO
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 | GSE151909 | GEO
Project description: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.
2024-04-08 | GSE263123 | GEO
Project description: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.
2020-05-11 | GSE142778 | GEO
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 | GSE151906 | GEO
Project description: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.
2024-04-08 | GSE254039 | GEO
Project description:Immunoglobulin heavy chain variable region exons are assembled in progenitor-B cells, from VH, D, and JH gene segments located in separate clusters across the Igh locus. RAG endonuclease orchestrates V(D)J recombination from a JH-based recombination center (RC). Cohesin-mediated extrusion of upstream chromatin past RC-bound RAG presents Ds for joining to JHs to form a DJHRC, from which RAG scans upstream for VHs to assemble VHDJH exons in an ordered recombination process. Cohesin-mediated chromatin loop extrusion is impeded by CTCF-bound elements (CBEs), with extrusion between two CBEs, particularly if convergently-oriented, forming chromatin-contact loops genome-wide. Igh has provocative CBE organization, with two divergently-oriented CBEs (CBE1 and CBE2) in the IGCR1 element between the VH and D/JH domains, dozens of VH domain CBEs convergent to CBE1, and 10 3'Igh-CBEs convergent to CBE2. IGCR1 CBEs segregate D/JH and VH domains by impeding RAG-scanning. After DJH formation, down-regulation of WAPL, a cohesin unloader, neutralizes IGCR1 and VH-domain CBEs, allowing RAG to scan the VH domain. To elucidate potential mechanisms underlying the developmental transition from D-to-JH to VH-to-DJH recombination and potential roles of IGCR1-based CBEs and 3'Igh-CBEs in regulating RAG-scanning, we tested effects of deleting or inverting IGCR1 or 3'Igh-CBEs in mice and/or progenitor-B cell lines. These studies revealed that normal IGCR1 CBE orientation augments its RAG-scanning impediment activity and that the 3'Igh-CBEs reinforce RC activity as a dynamic loop extrusion impediment, thereby, increasing its ability to support V(D)J recombination. Finally, our findings implicate a mechanism by which developmental WAPL down-regulation contributes to ordered V(D)J recombination.
2023-05-30 | GSE230604 | GEO
Project description:Immunoglobulin heavy chain variable region exons are assembled in progenitor-B cells, from VH, D, and JH gene segments located in separate clusters across the Igh locus. RAG endonuclease orchestrates V(D)J recombination from a JH-based recombination center (RC). Cohesin-mediated extrusion of upstream chromatin past RC-bound RAG presents Ds for joining to JHs to form a DJHRC, from which RAG scans upstream for VHs to assemble VHDJH exons in an ordered recombination process. Cohesin-mediated chromatin loop extrusion is impeded by CTCF-bound elements (CBEs), with extrusion between two CBEs, particularly if convergently-oriented, forming chromatin-contact loops genome-wide. Igh has provocative CBE organization, with two divergently-oriented CBEs (CBE1 and CBE2) in the IGCR1 element between the VH and D/JH domains, dozens of VH domain CBEs convergent to CBE1, and 10 3'Igh-CBEs convergent to CBE2. IGCR1 CBEs segregate D/JH and VH domains by impeding RAG-scanning. After DJH formation, down-regulation of WAPL, a cohesin unloader, neutralizes IGCR1 and VH-domain CBEs, allowing RAG to scan the VH domain. To elucidate potential mechanisms underlying the developmental transition from D-to-JH to VH-to-DJH recombination and potential roles of IGCR1-based CBEs and 3'Igh-CBEs in regulating RAG-scanning, we tested effects of deleting or inverting IGCR1 or 3'Igh-CBEs in mice and/or progenitor-B cell lines. These studies revealed that normal IGCR1 CBE orientation augments its RAG-scanning impediment activity and that the 3'Igh-CBEs reinforce RC activity as a dynamic loop extrusion impediment, thereby, increasing its ability to support V(D)J recombination. Finally, our findings implicate a mechanism by which developmental WAPL down-regulation contributes to ordered V(D)J recombination.
2023-05-30 | GSE230251 | GEO
Project description:Immunoglobulin heavy chain variable region exons are assembled in progenitor-B cells, from VH, D, and JH gene segments located in separate clusters across the Igh locus. RAG endonuclease orchestrates V(D)J recombination from a JH-based recombination center (RC). Cohesin-mediated extrusion of upstream chromatin past RC-bound RAG presents Ds for joining to JHs to form a DJHRC, from which RAG scans upstream for VHs to assemble VHDJH exons in an ordered recombination process. Cohesin-mediated chromatin loop extrusion is impeded by CTCF-bound elements (CBEs), with extrusion between two CBEs, particularly if convergently-oriented, forming chromatin-contact loops genome-wide. Igh has provocative CBE organization, with two divergently-oriented CBEs (CBE1 and CBE2) in the IGCR1 element between the VH and D/JH domains, dozens of VH domain CBEs convergent to CBE1, and 10 3'Igh-CBEs convergent to CBE2. IGCR1 CBEs segregate D/JH and VH domains by impeding RAG-scanning. After DJH formation, down-regulation of WAPL, a cohesin unloader, neutralizes IGCR1 and VH-domain CBEs, allowing RAG to scan the VH domain. To elucidate potential mechanisms underlying the developmental transition from D-to-JH to VH-to-DJH recombination and potential roles of IGCR1-based CBEs and 3'Igh-CBEs in regulating RAG-scanning, we tested effects of deleting or inverting IGCR1 or 3'Igh-CBEs in mice and/or progenitor-B cell lines. These studies revealed that normal IGCR1 CBE orientation augments its RAG-scanning impediment activity and that the 3'Igh-CBEs reinforce RC activity as a dynamic loop extrusion impediment, thereby, increasing its ability to support V(D)J recombination. Finally, our findings implicate a mechanism by which developmental WAPL down-regulation contributes to ordered V(D)J recombination.
2023-05-30 | GSE230249 | GEO