G4access reveals a role for G-quadruplexes in promoter opening and control of imprinting regions

Metazoan promoters are highly enriched in secondary structure forming motifs, among which G-quadruplexes (G4s). We describe G4access, a novel approach to isolate and sequence G4s when associated to open chromatin, based on their resistance to Micrococcal nuclease. G4access is an antibody- and crosslink-independent procedure that allows for high enrichment of predicted G4s (PG4s) motifs, most of which can be confirmed in vitro. Using this technique in several human and mouse cell lines, we were able to show a cell-specific enrichment that both relates to apparent nucleosome depletion and transcription at promoters. G4access allows scoring for PG4 pattern variation linked to nucleosome positioning changes that occur following treatment with a G4 ligand. It also reveals an unexpected role of G4s in hybrid mouse ES cells in the context of imprinting control regions, in which we propose that they will hallmark allelic active loci. Finally, we extended our procedure to non-mammalian species showing less annotated G4s in their genome. We found a decreased but still substantial G4 enrichment and confirmed their association to open and transcriptionally active regions of the yeast genome. Overall, our study not only provides a novel tool for studying G4 forming sequences in the cellular context but also indicates their essential roles as promoter elements, in chromatin opening and nucleosome positioning.后生动物启动子（Metazoan promoters）高度富集可形成二级结构的基序，其中G-四链体（G-quadruplexes, G4s）是最为典型的一类。本研究介绍了G4access这一全新方法：该方法基于G4对微球菌核酸酶（Micrococcal nuclease）的抗性，可分离并测序与开放染色质结合的G4序列。G4access属于无抗体且无交联的实验流程，能够高效富集预测性G-四链体（predicted G4s, PG4s）基序，其中绝大多数均可在体外得到验证。我们在多株人源与小鼠细胞系中应用该技术，证实其可实现细胞特异性富集，且该富集现象与启动子区域的核小体缺失及转录活性紧密相关。G4access可用于评估经G4配体（G4 ligand）处理后，伴随核小体定位改变而产生的预测性G4基序模式变化。此外，该技术还揭示了G4在杂交小鼠胚胎干细胞（mouse embryonic stem cells, ES cells）的印记调控区（imprinting control regions）中存在未被预期的功能：我们推测G4可作为等位基因活性位点的标志性序列。最后，我们将该实验流程拓展至基因组中注释G4序列较少的非哺乳动物物种，发现其G4富集程度虽有所下降但仍较为显著，并证实G4与酵母基因组的开放染色质及转录活跃区域存在关联。总体而言，本研究不仅为在细胞环境中研究可形成G4结构的序列提供了全新工具，同时也明确了G4作为启动子元件、参与染色质开放及核小体定位的核心功能。