Altered 3D chromatin structure permits inversional recombination at the IgH locus (Capture Hi-C)
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE145472 | GEO | 2020/07/01
REPOSITORIES: GEO
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Project description:We report that in the absence of intergenic control region 1 (IGCR1), a regulatory sequence that specifies the RAG scanning domain on WT IgH alleles, VH and DH gene segments can recombine with each other by both deletion and inversion of intervening DNA.
2020-06-18 | GSE133185 | GEO
Project description:We report that in the absence of intergenic control region 1 (IGCR1), a regulatory sequence that specifies the RAG scanning domain on WT IgH alleles, VH and DH gene segments can recombine with each other by both deletion and inversion of intervening DNA.
2020-07-02 | GSE135763 | 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
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: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: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:RAG endonuclease initiates antibody heavy chain variable region exon assembly from V, D, and J segments within a chromosomal V(D)J recombination center (RC) by cleaving between paired gene segments and flanking recombination signal sequences (RSSs). The IGCR1 control region promotes DJH intermediate formation by isolating Ds, JHs, and RC from upstream VHs in a chromatin loop anchored by CTCF-binding elements ("CBEs"). How VHs access the DJHRC for VH to DJH rearrangement was unknown. We report that CBEs immediately downstream of frequently rearranged VH-RSSs increase recombination potential of their associated VH far beyond that provided by RSSs alone. This CBE activity becomes particularly striking upon IGCR1 inactivation, which allows RAG, likely via loop extrusion, to linearly scan chromatin far upstream. VH-associated CBEs stabilize interactions of D-proximal VHs first encountered by the DJHRC during linear RAG scanning and, thereby, promote dominant rearrangement of these VHs by an unanticipated chromatin accessibility-enhancing CBE function.
2018-07-03 | GSE112822 | GEO
Project description:RAG endonuclease initiates antibody heavy chain variable region exon assembly from V, D, and J segments within a chromosomal V(D)J recombination center (RC) by cleaving between paired gene segments and flanking recombination signal sequences (RSSs). The IGCR1 control region promotes DJH intermediate formation by isolating Ds, JHs, and RC from upstream VHs in a chromatin loop anchored by CTCF-binding elements ("CBEs"). How VHs access the DJHRC for VH to DJH rearrangement was unknown. We report that CBEs immediately downstream of frequently rearranged VH-RSSs increase recombination potential of their associated VH far beyond that provided by RSSs alone. This CBE activity becomes particularly striking upon IGCR1 inactivation, which allows RAG, likely via loop extrusion, to linearly scan chromatin far upstream. VH-associated CBEs stabilize interactions of D-proximal VHs first encountered by the DJHRC during linear RAG scanning and, thereby, promote dominant rearrangement of these VHs by an unanticipated chromatin accessibility-enhancing CBE function.
2018-07-03 | GSE112781 | GEO
Project description:RAG endonuclease initiates antibody heavy chain variable region exon assembly from V, D, and J segments within a chromosomal V(D)J recombination center (RC) by cleaving between paired gene segments and flanking recombination signal sequences (RSSs). The IGCR1 control region promotes DJH intermediate formation by isolating Ds, JHs, and RC from upstream VHs in a chromatin loop anchored by CTCF-binding elements ("CBEs"). How VHs access the DJHRC for VH to DJH rearrangement was unknown. We report that CBEs immediately downstream of frequently rearranged VH-RSSs increase recombination potential of their associated VH far beyond that provided by RSSs alone. This CBE activity becomes particularly striking upon IGCR1 inactivation, which allows RAG, likely via loop extrusion, to linearly scan chromatin far upstream. VH-associated CBEs stabilize interactions of D-proximal VHs first encountered by the DJHRC during linear RAG scanning and, thereby, promote dominant rearrangement of these VHs by an unanticipated chromatin accessibility-enhancing CBE function.
2018-07-03 | GSE113022 | GEO