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

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 germline 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 E mu, the start site of E mu germ-line transcription, in a lineage-and stage-specific manner, even though this antisense transcription is E mu-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-E mu interactions. ChIP-seq shows high level YY1 binding only at E mu, but low levels near some antisense promoters. PAIR-E mu 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 DJ(H) rearrangement which is adjacent to E mu. 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.