Wapl repression by Pax5 promotes loop extrusion and V gene recombination at the Igh locus
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA591597 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10529600_1.fastq.gz | Fastqsanger.gz | |||
| SRR10529600_2.fastq.gz | Fastqsanger.gz | |||
| SRR10529601_1.fastq.gz | Fastqsanger.gz | |||
| SRR10529601_2.fastq.gz | Fastqsanger.gz | |||
| SRR10529602_1.fastq.gz | Fastqsanger.gz |
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Project description:The generation of a diverse antibody repertoire is essential for humoral immunity and requires the participation of all V genes in V(D)J recombination, which depends on the Pax5-regulated contraction of the 2.8-Mb long immunoglobulin heavy-chain (Igh) locus. How Pax5 controls Igh contraction in pro-B cells is, however, not known. Here, we demonstrate that locus contraction is caused by cohesin-mediated chromatin loop extrusion across the entire Igh locus. Notably, expression of the cohesin-release factor Wapl is repressed by Pax5 specifically in pro-B and pre-B cells, which facilitates extended loop extrusion by increasing the residence time of cohesin on chromatin. Pax5 mediates the transcriptional repression of Wapl through a single Pax5-binding site by recruiting the Polycomb repressive complex 2 to induce bivalent chromatin at the Wapl promoter. Reduced Wapl expression causes global alterations of the three-dimensional chromatin architecture, indicating that the potential to recombine all V genes entails structural changes of the entire genome in pro-B cells.
2020-07-01 | GSE140975 | GEO
Project description:Nuclear processes, such as V(D)J recombination, are orchestrated by the three-dimensional organization of chromosomes at multiple levels, including compartments1 and topologically associated domains (TADs)2,3 consisting of chromatin loops4. TADs are formed by chromatin-loop extrusion5-7, which depends on the loop-extrusion function of the ring-shaped cohesin complex8-12. Conversely, the cohesin-release factor Wapl13,14 restricts loop extension10,15. The generation of a diverse antibody repertoire, providing humoral immunity to pathogens, requires the participation of all V genes in V(D)J recombination16, which depends on contraction of the 2.8-Mb-long immunoglobulin heavy chain (Igh) locus by Pax517,18. However, how Pax5 controls Igh contraction in pro-B cells remains unknown. Here we demonstrate that locus contraction is caused by loop extrusion across the entire Igh locus. Notably, the expression of Wapl is repressed by Pax5 specifically in pro-B and pre-B cells, facilitating extended loop extrusion by increasing the residence time of cohesin on chromatin. Pax5 mediates the transcriptional repression of Wapl through a single Pax5-binding site by recruiting the polycomb repressive complex 2 to induce bivalent chromatin at the Wapl promoter. Reduced Wapl expression causes global alterations in the chromosome architecture, indicating that the potential to recombine all V genes entails structural changes of the entire genome in pro-B cells.
| S-EPMC7116900 | biostudies-literature
Project description:Extended loop extrusion across the immunoglobulin heavy-chain (Igh) locus facilitates VH-DJH recombination in pro-B cells by aligning the VH and DJH segments for RAG-mediated cleavage. This cohesin-mediated process, resulting in global changes of the chromosome architecture in pro-B cells, depends on a 3-fold downregulation of the cohesin-release factor Wapl by Pax5-induced repression of the Wapl promoter. Here, we demonstrate that chromatin looping and VK-JK recombination at the Igk locus was insensitive to a 3-fold Wapl increase in pre-B cells. Given the equally low Wapl mRNA levels in pro-B and pre-B cells, the Wapl protein was unexpectedly expressed at a 2.2-fold higher level in pre-B cells compared to pro-B cells, which resulted in a distinct chromosomal architecture with normalized loop sizes in pre-B cells. High-resolution analysis of the Igk locus identified multiple internal loops, which likely juxtapose VK and JK elements to facilitate VK-JK recombination. The higher Wapl expression in Igm-transgenic pre-B cells prevented extended loop extrusion at the Igh locus, leading to recombination of only the 6 most 3’ proximal VH genes and to allelic exclusion of all other VH genes in pre-B cells. Hence, the different chromosomal architectures in pro-B and pre-B cells forced the Igh and Igk loci to assume distinct folding principles to undergo V gene recombination.
2023-01-15 | GSE210289 | 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
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: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: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 | GSE151908 | 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 | GSE151907 | GEO