Genome-wide mapping of i-Motifs reveals their association with transcription regulation in live human cells [CUT&Tag]

i-Motifs (iMs) are four-stranded DNA structures that form at cytosine (C)-rich sequences and in acidic conditions in vitro. However, their formation and distribution in cells is still under debate. Here we performed CUT&Tag sequencing using the anti-iM antibody iMab and showed that iMs form within the human genome in live cells. We mapped native iMs in two different human cell lines and recovered C-rich sequences that were confirmed to fold into iMs in vitro. We found that iMs in cells are mainly present at actively transcribing gene promoters and that their abundance and distribution are specific to each cell type. iMs with both long and short C-tracts were recovered, further extending the relevance of iMs throughout the genome. By simultaneously mapping G-quadruplexes (G4s), four-stranded structures that form at guanine-rich regions, and comparing the results with iMs, we proved that the two structures can form in independent genomic regions; however, when both iMs and G4s are present in the same genomic tract, their formation is favored. Our findings support the in vivo formation of iM structures and provide new insights into their interplay with G4s as new regulatory elements in the human genome. i-motifs and G-quadruplex analysis in HEK293T (ATCC #CRL-3216) and WDLPS (ATCC #CRL-3043) through CUT&Tag method

i-基序（i-Motifs，简称iMs）是一类在体外富胞嘧啶（C）序列及酸性条件下形成的四链DNA结构。然而，其在细胞内的形成与分布仍存在争议。本研究使用抗iM抗体iMab开展CUT&Tag测序，证实活体细胞的人类基因组中可形成iMs。我们在两种不同的人类细胞系中定位了天然iMs，并富集得到经体外实验确认可折叠为iMs的富C序列。研究发现，细胞内的iMs主要存在于活跃转录的基因启动子区域，且其丰度与分布具有细胞类型特异性。我们同时回收了包含长、短胞嘧啶链（C-tracts）的iMs，进一步拓展了iMs在全基因组范围内的相关性。通过同时定位鸟嘌呤富集区域形成的四链结构——G-四链体（G-quadruplexes，简称G4s），并将其结果与iMs进行比对，我们证实两类结构可在独立的基因组区域形成；但若同一基因组区段同时存在iMs与G4s，则二者的形成均会受到促进。本研究结果支持iM结构在活体内的形成，并为其与G4s作为人类基因组新型调控元件的相互作用提供了新见解。本数据集通过CUT&Tag方法，对HEK293T（ATCC编号CRL-3216）与WDLPS（ATCC编号CRL-3043）两种细胞系中的i-基序与G-四链体开展分析。