Project description:VH-DJH recombination of the immunoglobulin heavy-chain (Igh) locus is temporally and spatially controlled during early B-cell development, and yet no regulatory elements other than the VH gene promoters have been identified throughout the entire 2.5-Mb VH gene cluster. Here we discovered novel regulatory sequences that are interspersed in the distal VH gene region. These conserved repeat elements were characterized by the presence of Pax5-dependent active chromatin, the binding of Pax5, E2A, CTCF and Rad21 as well as by Pax5-dependent antisense transcription in pro-B cells. The Pax5-activated intergenic repeat (PAIR) elements were no longer bound by Pax5 in pre-B and B cells consistent with the loss of antisense transcription, whereas E2A and CTCF interacted with PAIR elements throughout early B-cell development. The pro-B-cell-specific and Pax5-dependent activity of the PAIR elements suggests that they are involved in the regulation of distal VH-DJH recombination at the Igh locus. Analysis of chromatin and TF binding in rag2-/- and pax5-/- rag2-/- pro-B cells. Chip-Chip with one experiment for each antibody, 12 samples.
Project description:VH-DJH recombination of the immunoglobulin heavy-chain (Igh) locus is temporally and spatially controlled during early B-cell development, and yet no regulatory elements other than the VH gene promoters have been identified throughout the entire 2.5-Mb VH gene cluster. Here we discovered novel regulatory sequences that are interspersed in the distal VH gene region. These conserved repeat elements were characterized by the presence of Pax5-dependent active chromatin, the binding of Pax5, E2A, CTCF and Rad21 as well as by Pax5-dependent antisense transcription in pro-B cells. The Pax5-activated intergenic repeat (PAIR) elements were no longer bound by Pax5 in pre-B and B cells consistent with the loss of antisense transcription, whereas E2A and CTCF interacted with PAIR elements throughout early B-cell development. The pro-B-cell-specific and Pax5-dependent activity of the PAIR elements suggests that they are involved in the regulation of distal VH-DJH recombination at the Igh locus. Analysis of chromatin and TF binding in rag2-/- and wt pro-B, DP T and Mature B cells. Chip-Seq of CTCF and Rad21. The provided data is in mm8 coordinates.
Project description:VH-DJH recombination of the immunoglobulin heavy-chain (Igh) locus is temporally and spatially controlled during early B-cell development, and yet no regulatory elements other than the VH gene promoters have been identified throughout the entire 2.5-Mb VH gene cluster. Here we discovered novel regulatory sequences that are interspersed in the distal VH gene region. These conserved repeat elements were characterized by the presence of Pax5-dependent active chromatin, the binding of Pax5, E2A, CTCF and Rad21 as well as by Pax5-dependent antisense transcription in pro-B cells. The Pax5-activated intergenic repeat (PAIR) elements were no longer bound by Pax5 in pre-B and B cells consistent with the loss of antisense transcription, whereas E2A and CTCF interacted with PAIR elements throughout early B-cell development. The pro-B-cell-specific and Pax5-dependent activity of the PAIR elements suggests that they are involved in the regulation of distal VH-DJH recombination at the Igh locus.
Project description:VH-DJH recombination of the immunoglobulin heavy-chain (Igh) locus is temporally and spatially controlled during early B-cell development, and yet no regulatory elements other than the VH gene promoters have been identified throughout the entire 2.5-Mb VH gene cluster. Here we discovered novel regulatory sequences that are interspersed in the distal VH gene region. These conserved repeat elements were characterized by the presence of Pax5-dependent active chromatin, the binding of Pax5, E2A, CTCF and Rad21 as well as by Pax5-dependent antisense transcription in pro-B cells. The Pax5-activated intergenic repeat (PAIR) elements were no longer bound by Pax5 in pre-B and B cells consistent with the loss of antisense transcription, whereas E2A and CTCF interacted with PAIR elements throughout early B-cell development. The pro-B-cell-specific and Pax5-dependent activity of the PAIR elements suggests that they are involved in the regulation of distal VH-DJH recombination at the Igh locus.
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
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.
Project description:While extended loop extrusion across the entire Igh locus controls VH-DJH recombination, local regulatory sequences, such as the PAIR elements, may also activate VH gene recombination in pro-B-cells. Here we show that PAIR-associated VH8 genes contain a conserved putative regulatory element (V8E) in their downstream sequences. To investigate the function of PAIR4 and its V8.7E, we deleted all 14 PAIRs in the Igh 5’-region, which reduced distal VH gene recombination over a 100-kb distance on either side of the 890-kb deletion. Reconstitution by insertion of PAIR4-V8.7E strongly activated distal VH gene recombination. PAIR4 alone resulted in lower induction of recombination, indicating that PAIR4 and V8.7E function as one regulatory unit. The pro-B-cell-specific activity of PAIR4 depends on CTCF, as mutation of its CTCF-binding site led to sustained PAIR4 activity in pre-B and immature B-cells and in PAIR4 activation in T-cells. Notably, insertion of V8.8E was sufficient to activate VH gene recombination. Hence, enhancers of the PAIR4-V8.7E module and V8.8E element activate distal VH gene recombination and thus contribute to the diversification of the BCR repertoire in addition to loop extrusion.
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.
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.