Detection of statistically robust interactions from diverse RNA-DNA ligation data [ATAC-seq]

Chromatin-localized RNAs play diverse roles in gene regulation and nuclear architecture. Mapping genome-wide RNA-DNA interactions is possible using a variety of molecular methods, including using bridging oligonucleotides to ligate RNA and DNA in proximity. While molecular methods have progressed, a robust computational method for calling biologically meaningful RNA-DNA interactions from these data is lacking. Herein, we present RADIAnT, a reads-to-interactions pipeline for analyzing RNA-DNA ligation data. RADIAnT calls interactions against a dataset-specific, unified background which considers RNA binding site-TSS distance and genomic region bias. By scaling the background with RNA abundance, RADIAnT is sensitive enough to detect specific interactions of lowly expressed transcripts, while remaining specific enough to discount false positive interactions of highly abundant RNAs. RADIAnT outperforms previously proposed methods in the accurate recall of genome-wide Malat1-DNA interactions, and in a use-case, was utilized to identify dynamic chromatin-associated RNAs in the physiologically- and pathlologically-relevant process of endothelial-to-mesenchymal transition. Overall design: ATAC-sequencing was performed on 3 HUVEC replicates, once in wildtype (endothelial) phenotype (CTL), once after IL-1ß + TGF-ß induced phenotypic switch to mesenchymal-like cells (EMT).

定位于染色质的RNA在基因调控与核结构维持中发挥多种功能。全基因组RNA-DNA互作的图谱绘制可通过多种分子生物学方法实现，其中包括利用桥接寡核苷酸将邻近状态下的RNA与DNA进行连接。尽管分子实验方法已取得进展，但目前仍缺乏可从此类数据中识别具有生物学意义的RNA-DNA互作的可靠计算方法。本研究推出RADIAnT工具，这是一款用于分析RNA-DNA连接测序数据的读段-互作分析流程。RADIAnT会基于数据集专属的统一背景模型进行互作识别，该模型整合了RNA结合位点与转录起始位点（Transcription Start Site, TSS）的距离以及基因组区域偏好性。通过结合RNA表达量对背景模型进行校准，RADIAnT既能具备足够灵敏度以检测低表达转录本的特异性互作，又能保持足够特异性以排除高丰度RNA带来的假阳性互作。在全基因组Malat1-DNA互作的准确召回任务中，RADIAnT的表现优于此前已报道的方法；在一项应用案例中，该工具被用于鉴定内皮细胞向间充质细胞转化（Endothelial-to-Mesenchymal Transition, EMT）这一兼具生理与病理意义的过程中动态变化的染色质相关RNA。实验整体设计：对3组人脐静脉内皮细胞（Human Umbilical Vein Endothelial Cells, HUVEC）生物学重复样本进行转座酶可及性测序（ATAC-sequencing），分别在野生型（内皮细胞）表型（CTL）以及经IL-1β与TGF-β诱导转化为间充质样细胞的表型（EMT组）下开展检测。