Deep sequencing of the murine Igh repertoire reveals complex regulation of non-random V gene rearrangement frequencies (RNA-seq)

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. RNA was extracted from C57BL/6 RAG-/- pro-B cells using Trizol® (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.免疫球蛋白重链（Igh）基因座上的V、D、J基因片段重排，可形成多样化的抗体库。C57BL/6小鼠的Igh基因座拥有超过100个功能性VH基因片段，可与已重排的DJH片段发生重组。尽管VH基因的非随机使用已有大量文献报道，但目前尚不清楚哪些元件决定了重组频率。为解答这一问题，我们对前B细胞中Igh抗体库的5’-末端快速扩增（5’-RACE）产物进行了深度测序，扩增过程采用无偏倚方式。我们将多种组蛋白修饰及RNA聚合酶II结合的染色质免疫共沉淀测序（ChIP-seq）结果、正义与反义非编码生殖系转录本的RNA测序（RNA-seq）结果，以及与CTCF、Rad21结合位点的邻近性数据，与单个V基因的使用情况进行了比对分析。通过计算分析，我们评估了上述各类可及性元件的相对重要性。这些元件将Igh基因座划分为四个表观遗传与转录特征均不同的结构域，我们的计算分析揭示了每个结构域各自独特的调控机制。一般而言，近端V基因几乎不带有活性组蛋白修饰标记及非编码RNA，但在其重组信号序列（RSS）附近存在CTCF结合位点至关重要，这表明染色质环基部附近的位置对重组过程具有重要意义。与之相反，远端V基因则带有高水平的组蛋白修饰标记与非编码RNA，这或许可以弥补其较弱的RSS以及与CTCF结合位点距离较远的缺陷。由此可见，Igh基因座演化出了一套复杂的V(D)J重组调控系统，且构成该基因座的四个结构域各自拥有独特的调控方式。我们使用TRIzol®试剂（Life Technologies Corp.，加利福尼亚州卡尔斯巴德）从C57BL/6重组激活基因敲除（RAG-/-）前B细胞中提取RNA，并通过QuantiTect反转录试剂盒（QIAGEN）中的基因组DNA清除缓冲液去除基因组DNA。随后使用QIAGEN的RNeasy试剂盒对RNA进行最终纯化。每份样本取100 ng总RNA，按照NuGEN Encore Complete DR试剂盒的官方推荐操作流程构建RNA测序文库。测序文库通过凝胶纯化以确保插入片段长度大于100 bp，随后在Illumina HiSeq2000平台上进行测序，测序读长为100个碱基，同时加入7个碱基用于索引标记。