Systematic mapping of RNA-chromatin interactions in vivo

RNA molecules can attach to chromatin and thus provide a type of epigenomic information. It remains difficult to know what RNAs are associated with chromatin and where are the genomic target loci of these RNAs. Here, we present MARGI (Mapping RNA-genome interactions), a technology to massively reveal native RNA-chromatin interactions from unperturbed cells. The gist of this technology is to ligate chromatin-associated RNA (caRNA) with their target genomic sequence by proximity ligation, forming RNA-DNA chimeric sequences, which are converted to sequencing library for paired-end sequencing. Using MARGI, we produced RNA-genome interaction maps for human embryonic stem (ES) cells and HEK cells. MARGI revealed hundreds of chromatin-associated RNAs (caRNA), including previously known XIST, SNHG1, NEAT1, and MALAT1, as well as each caRNA's genomic interaction loci. Using a cross-species experiment, we estimated that approximately 2.2% of MARGI identified interactions are false positives. MARGI data suggested that 80-95% of interactions are proximal, where a caRNA is connected to its nearby genomic regions, 1-2% are distal, and 5-15% are inter-chromosomal. The majority of TSSs are associated with distal or inter-chromosomal caRNAs. ChIP-seq reported H3K27ac and H3K4me3 levels are positively correlated while H3K9me is negatively correlated with MARGI reported RNA attachment levels. The MARGI technology should facilitate revealing novel RNA functions and epigenomic events. Overall design: Two human cell lines were analysed: H9 and HEK. For each, we used two protocols, pxMARGI and diMARGI, having two replicates for the former and only one for the latter. Two samples from each replicate were sequenced. Whereas pxMARGI is designed to not differentiate passive and direct interactions, diMARGI is aimed to reveal protein tethered interactions. For the former, this was achieved by a combination of formaldehyde crosslinking and complete genome fragmentation, by overnight HaeIII digestion to ensure all genomic regions including heterochromatin were fragmented before the subsequent DNA ligation step. For the second, after crosslinking with formaldehyde and DSG, the chromatin was fragmented by sonication, and only the soluble fraction was passed onto the subsequent ligation steps.

RNA分子可结合染色质，从而提供一类表观基因组信息。目前仍难以明确哪些RNA与染色质相关联，以及这些RNA的基因组靶位点位于何处。本文介绍了MARGI（Mapping RNA-genome interactions，即RNA-基因组相互作用定位技术）——一种可从未受扰动的细胞中大规模揭示天然RNA-染色质相互作用的技术。该技术的核心原理是通过邻近连接，将与染色质结合的RNA（chromatin-associated RNA, caRNA）与其靶基因组序列进行酶促连接，形成RNA-DNA嵌合序列，随后将其转化为测序文库以开展双端测序。我们利用MARGI技术，绘制了人类胚胎干细胞（ES细胞）与HEK细胞的RNA-基因组相互作用图谱。MARGI可检测到数百种与染色质结合的RNA（caRNA），包括此前已报道的XIST、SNHG1、NEAT1及MALAT1，同时可获取每种caRNA的基因组相互作用位点。通过跨物种实验，我们估算出MARGI鉴定的相互作用中约2.2%为假阳性。MARGI数据显示，80%~95%的相互作用为近端相互作用，即caRNA与其邻近的基因组区域相连；1%~2%为远端相互作用；5%~15%为染色质间相互作用。大多数转录起始位点（Transcription Start Site, TSS）与远端或染色质间的caRNA相关联。染色质免疫共沉淀测序（ChIP-seq）检测到的H3K27ac、H3K4me3水平与MARGI报道的RNA结合水平呈正相关，而H3K9me水平则呈负相关。MARGI技术将有助于揭示新型RNA功能与表观基因组事件。总体实验设计：分析了两种人类细胞系：H9细胞与HEK细胞。针对每种细胞系，我们采用了两种实验方案——pxMARGI与diMARGI：前者设置两个生物学重复，后者仅设置一个重复。每个重复设置两份样本进行测序。pxMARGI的设计初衷是不区分被动相互作用与直接相互作用，而diMARGI则旨在揭示蛋白质锚定的相互作用。对于pxMARGI方案，我们通过甲醛交联结合完整的基因组片段化实现该目标：采用HaeIII限制性内切酶酶切过夜，以确保包括异染色质在内的所有基因组区域在后续DNA连接步骤前均被片段化。对于diMARGI方案，在经甲醛与DSG交联后，我们通过超声破碎染色质，仅取可溶性组分用于后续连接步骤。