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

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 Eu, the start site of Iu germline transcription, in a lineage- and stage-specific manner, even though this antisense transcription is Eu-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-EÎŒ interactions. ChIP-seq shows high level YY1 binding only at Eu, but low levels near some antisense promoters. PAIR-Eu 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 Eu. 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.免疫球蛋白重链基因座（immunoglobulin heavy chain locus）内的非编码有义与反义生殖系转录发生于V(D)J重组（V(D)J recombination）之前，且已被提出与Igh基因座的染色质可及性相关，但其确切功能仍尚不明确。目前尚未开展针对整个Igh基因座生殖系转录的全局分析。为此，我们开展了定向RNA测序（directional RNA-seq），明确了整个Igh基因座范围内有义与反义转录的位置与覆盖范围。令人意外的是，绝大多数反义转录本定位于Igh基因座远端V区的两个PAIR元件（PAIR elements）附近。值得注意的是，尽管这类反义转录不依赖于Eu（Iu生殖系转录的起始位点），但通过染色体构象捕获（3C，Chromosome Conformation Capture）检测到的远距离环化相互作用，仍以谱系和阶段特异性的方式存在于这两个活跃的PAIR启动子与Eu之间。阴阳因子1（YY1，Yin Yang 1）缺陷的前B细胞（pro-B cells）在远端VH基因重排与Igh基因座染色质压缩过程中存在严重缺陷，我们的研究证实，YY1缺失会显著降低反义转录水平与PAIR-Eu相互作用强度。染色质免疫共沉淀测序（ChIP-seq，Chromatin Immunoprecipitation sequencing）结果显示，YY1仅在Eu位点存在高水平结合，而在部分反义启动子附近仅呈现低水平结合。PAIR-Eu相互作用不会被DRB阻断：DRB可在转录工厂（transcription factories）建立后阻断转录延伸，但不会破坏已形成的转录工厂；而热休克处理会破坏转录工厂，此时环化相互作用会显著减弱。我们提出，介导转录的相互作用（最有可能发生在转录工厂中）最初会使Igh基因座发生染色质压缩，使远端VH基因靠近紧邻Eu的DJH重排（DJH rearrangement）位点。因此，我们推测非编码生殖系转录的关键功能之一是促进基因座压缩，从而使远端VH基因能够进行高效重排。为明确Igh基因座内有义与反义非编码RNA的含量与位置，我们从CD19+ RAG1-/-前B细胞中提取了总RNA。部分样本通过定制安捷伦芯片（Agilent arrays）进行预富集，其余样本直接进行测序。本研究包含每种实验条件下的一个样本数据，与已发表文献中的描述一致。