Noncoding transcription within the Igh distal V(H) region at PAIR elements affects the 3D structure of the Igh locus in pro-B cells

Abstract

Noncoding sense and antisense germ-line transcription within the Ig 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 germ-line transcription throughout the Igh locus has been done. Therefore, we performed directional RNA-seq, 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 Pax5-activated intergenic repeat (PAIR) elements in the distal IghV region. Importantly, long-distance loops measured by chromosome conformation capture (3C) are observed between these two active PAIR promoters and Eμ, the start site of Iμ germ-line 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 V(H) 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 V(H) genes close to the DJ(H) rearrangement which is adjacent to Eμ. Therefore, we hypothesize that one key role of noncoding germ-line transcription is to facilitate locus compaction, allowing distal V(H) genes to undergo efficient rearrangement.

Fig. 1.

RNA-seq analysis of the Igh locus. (A) RNA-seq was performed on Rag1^−/− pro–B-cell RNA enriched on a custom Agilent array coated with the entire nonrepetitive DNA of the Igh locus. Antisense transcripts throughout the V_H region of Igh locus are shown. (B) Mapping of antisense transcription in distal half of the V_HJ558 region. The relative position of all 14 PAIR elements is shown.

Fig. 2.

Roles of YY1. (A) Antisense and sense transcription levels from RNA from freshly isolated pro-B cells from Rag^−/− and YY1^−/−Rag^−/− mice were measured by qPCR. Results are presented as the ratio of the amount of transcription in YY1^−/−Rag^−/− pro-B cells compared with Rag^−/− pro-B cells, and are the mean ± SEM (n = 4–5). (B) ChIP-seq analysis of YY1 binding throughout the Igh locus in Rag1^−/− pro-B cells. Below the ChIP-seq Genome Browser picture, the vertical lines indicate the positions of V, D, and J genes. Note that the Igh orientation is the opposite from Fig. 2C. (C) ChIP/qPCR of indicated regions for YY1 binding in Rag1^−/− pro-B cells. The schematic representation of the Igh locus showing the relative locations of regions tested in ChIP is presented in the panel directly above. Results are presented as mean ± SEM (n = 5). (D) RT-PCR analysis showing expression of Pax5 in Rag^−/− and YY1^−/−Rag^−/− pro-B cells. Results are presented as mean ± SEM from four to five independent experiments. (E) ChIP assay for enrichment of Pax5 at PAIR promoters. Results are presented as mean ± SEM from two independent experiments.

Fig. 3.

The 3C analysis of long-range chromosomal loops between PAIR promoters and Eμ. (A) Schematic diagram of Igh locus showing the regions tested for 3C interactions and their relative distances. Primer pair C binds at two locations, indicated as C₁ and C₂. (B) The 3C analysis showing relative cross-linking frequencies between Eμ anchor fragment and HindIII fragments within the Igh locus using an Eμ TaqMan probe. Data are presented as mean ± SEM (n = 2–3). (C) The 3C analysis in YY1^−/− Rag1^−/− pro-B cells and Rag1^−/− pro-B cells. Data are presented as mean ± SEM (n = 3). (D) The 3C analysis in Rag2^−/− A-MuLV-transformed pro-B cells transduced with control or CTCF shRNA retroviruses. Data are presented as mean ± SEM (n = 3). (E) Antisense germ-line transcription is Eμ-independent. RNA was made from freshly isolated CD19⁺ cells from C.Rag1^−/− and Eμ^−/− bone marrow, and were assayed for antisense transcription. The Igh^a locus of these 129 background mice does not amplify well with PAIR6, J558 3′Int primers, so only the PAIR4 and J558 5′Int data are displayed. Two RNA preparations of each genotype were assayed. Data are presented ± SEM.

Fig. 4.

Effect of transcription inhibition on long-range chromosomal loops in the Igh locus. Rag1^−/− pro-B cells were subjected to heat shock at 45 °C for 30 min, or incubated with 100 μM DRB for 3 h. Cells were immediately harvested for RNA or for 3C lysate. (A and B) Relative expression levels following heat shock (A) and DRB (B) treatment on the indicated genes. Data are presented as mean ± SEM (n = 3). (C and D) The 3C analysis shows the relative cross-linking frequencies between the Eμ anchor fragment and PAIR6 and PAIR4. Data are presented as mean ± SEM (n = 4).