Deep sequencing of the murine Igh repertoire reveals complex regulation of non-random V gene rearrangement frequencies
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE47766 | GEO | 2013/08/05
SECONDARY ACCESSION(S): PRJNA207764
REPOSITORIES: GEO
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Project description:A diverse antibody repertoire is formed through the rearrangement of V, D, and J segments at the immunoglobulin heavy chain (Igh) loci. The C57BL/6 murine Igh locus has over 100 functional VH gene segments that can recombine to a rearranged DJH. While the non-random usage of VH genes is well documented, it is not clear what elements determine recombination frequency. To answer this question we conducted deep sequencing of 5M-bM-^@M-^Y-RACE products of the Igh repertoire in pro-B cells, amplified in an unbiased manner. ChIP-seq results for several histone modifications and RNA polymerase II binding, RNA-seq for sense and antisense non-coding germline transcripts, and proximity to CTCF and Rad21 sites were compared to the usage of individual V genes. Computational analyses assessed the relative importance of these various accessibility elements. These elements divide the Igh locus into four epigenetically and transcriptionally distinct domains, and our computational analyses reveal different regulatory mechanisms for each region. Proximal V genes are relatively devoid of active histone marks and non-coding RNA in general, but having a CTCF site near their RSS is critical, suggesting that position near the base of the chromatin loops is important for rearrangement. In contrast, distal V genes have high levels of histone marks and non-coding RNA, which may compensate for their poorer RSS and for being distant from CTCF sites. Thus, the Igh locus has evolved a complex system for the regulation of V(D)J rearrangement that is different for of each the four domains that comprise this locus. For the ChIP-seq, input and immunoprecipitated DNA was given to The Scripps DNA Array Facility, where it was prepared for massively parallel sequencing on Illumina HiSeq2000.
2013-08-05 | E-GEOD-47766 | biostudies-arrayexpress
Project description:A diverse antibody repertoire is formed through the rearrangement of V, D, and J segments at the immunoglobulin heavy chain (Igh) loci. The C57BL/6 murine Igh locus has over 100 functional VH gene segments that can recombine to a rearranged DJH. While the non-random usage of VH genes is well documented, it is not clear what elements determine recombination frequency. To answer this question we conducted deep sequencing of 5’-RACE products of the Igh repertoire in pro-B cells, amplified in an unbiased manner. ChIP-seq results for several histone modifications and RNA polymerase II binding, RNA-seq for sense and antisense non-coding germline transcripts, and proximity to CTCF and Rad21 sites were compared to the usage of individual V genes. Computational analyses assessed the relative importance of these various accessibility elements. These elements divide the Igh locus into four epigenetically and transcriptionally distinct domains, and our computational analyses reveal different regulatory mechanisms for each region. Proximal V genes are relatively devoid of active histone marks and non-coding RNA in general, but having a CTCF site near their RSS is critical, suggesting that position near the base of the chromatin loops is important for rearrangement. In contrast, distal V genes have high levels of histone marks and non-coding RNA, which may compensate for their poorer RSS and for being distant from CTCF sites. Thus, the Igh locus has evolved a complex system for the regulation of V(D)J rearrangement that is different for of each the four domains that comprise this locus.
2013-08-05 | GSE47885 | GEO
Project description:A diverse antibody repertoire is formed through the rearrangement of V, D, and J segments at the immunoglobulin heavy chain (Igh) loci. The C57BL/6 murine Igh locus has over 100 functional VH gene segments that can recombine to a rearranged DJH. While the non-random usage of VH genes is well documented, it is not clear what elements determine recombination frequency. To answer this question we conducted deep sequencing of 5M-bM-^@M-^Y-RACE products of the Igh repertoire in pro-B cells, amplified in an unbiased manner. ChIP-seq results for several histone modifications and RNA polymerase II binding, RNA-seq for sense and antisense non-coding germline transcripts, and proximity to CTCF and Rad21 sites were compared to the usage of individual V genes. Computational analyses assessed the relative importance of these various accessibility elements. These elements divide the Igh locus into four epigenetically and transcriptionally distinct domains, and our computational analyses reveal different regulatory mechanisms for each region. Proximal V genes are relatively devoid of active histone marks and non-coding RNA in general, but having a CTCF site near their RSS is critical, suggesting that position near the base of the chromatin loops is important for rearrangement. In contrast, distal V genes have high levels of histone marks and non-coding RNA, which may compensate for their poorer RSS and for being distant from CTCF sites. Thus, the Igh locus has evolved a complex system for the regulation of V(D)J rearrangement that is different for of each the four domains that comprise this locus. RNA was extracted from C57BL/6 RAG-/- pro-B cells using TrizolM-BM-. (Life Technologies Corp., Carlsbad CA) and genomic DNA was eliminated using the genomic DNA wipeout buffer in the QuantiTect Reverse transcription kit (QIAGEN). A final purification of the RNA was performed with the RNeasy kit from QIAGEN. For each sample, 100 ng of total RNA was used to make RNASeq libraries using the NuGEN Encore Complete DR kits following manufacturer's recommended protocols. Sequencing libraries were gel purified to ensure insert sizes were larger than 100 bp in length and sequenced on an Ilumina HiSeq2000 for 100 bases plus 7 bases for indexing.
2013-08-05 | E-GEOD-47885 | biostudies-arrayexpress
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: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:Non-coding sense and antisense germline transcription within the immunoglobulin heavy chain locus precedes V(D)J recombination and has been proposed to be associated with Igh locus accessibility, although its precise role remains elusive. However, no global analysis of germline transcription throughout the Igh locus has been done. Therefore, we performed directional RNAseq, demonstrating the locations and extent of both sense and antisense transcription throughout the Igh locus. Surprisingly, the majority of antisense transcripts are localized around two PAIR elements in the distal IghV region. Importantly, long-distance loops measured by 3C are observed between these two active PAIR promoters and EM-NM-<, the start site of IM-NM-< germline transcription, in a lineage- and stage-specific manner, even though this antisense transcription is EM-NM-<-independent. YY1-/- pro-B cells are greatly impaired in distal VH gene rearrangement and Igh locus compaction, and we demonstrate that YY1 deficiency greatly reduces antisense transcription and PAIR-EM-NM-< interactions. ChIP-seq shows high level YY1 binding only at EM-NM-<, but low levels near some antisense promoters. PAIR-EM-NM-< interactions are not disrupted by DRB, which blocks transcription elongation without disrupting transcription factories once they are established, but the looping is reduced after heat shock treatment, which disrupts transcription factories. We propose that transcription-mediated interactions, most likely at transcription factories, initially compact the Igh locus, bringing distal VH genes close to the DJH rearrangement, which is adjacent to EM-NM-<. Therefore, we hypothesize that one key role of non-coding germline transcription is to facilitate locus compaction, allowing distal VH genes to undergo efficient rearrangement. ChIP Seq YY1 vs. input control
2012-10-18 | E-GEOD-40822 | biostudies-arrayexpress
Project description:Non-coding sense and antisense germline transcription within the immunoglobulin heavy chain locus precedes V(D)J recombination and has been proposed to be associated with Igh locus accessibility, although its precise role remains elusive. However, no global analysis of germline transcription throughout the Igh locus has been done. Therefore, we performed directional RNAseq, demonstrating the locations and extent of both sense and antisense transcription throughout the Igh locus. Surprisingly, the majority of antisense transcripts are localized around two PAIR elements in the distal IghV region. Importantly, long-distance loops measured by 3C are observed between these two active PAIR promoters and Eμ, the start site of Iμ germline transcription, in a lineage- and stage-specific manner, even though this antisense transcription is Eμ-independent. YY1-/- pro-B cells are greatly impaired in distal VH gene rearrangement and Igh locus compaction, and we demonstrate that YY1 deficiency greatly reduces antisense transcription and PAIR-Eμ interactions. ChIP-seq shows high level YY1 binding only at Eμ, but low levels near some antisense promoters. PAIR-Eμ interactions are not disrupted by DRB, which blocks transcription elongation without disrupting transcription factories once they are established, but the looping is reduced after heat shock treatment, which disrupts transcription factories. We propose that transcription-mediated interactions, most likely at transcription factories, initially compact the Igh locus, bringing distal VH genes close to the DJH rearrangement, which is adjacent to Eμ. Therefore, we hypothesize that one key role of non-coding germline transcription is to facilitate locus compaction, allowing distal VH genes to undergo efficient rearrangement. In order to determine the amount and location of sense and antisense non-coding RNA in the Igh locus, we prepared total RNA from CD19+ RAG1-/- pro-B cells. Samples were either pre-enriched in custom Agilent arrays, or directly sequenced. Data from one sample for each condition is included as reflected in the publication.
2012-10-18 | E-GEOD-40984 | biostudies-arrayexpress
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