3D genome organization during TGFB-induced transcription requires nuclear microRNA and G-quadruplexes [Cut&Tag]

Studying the dynamics of three-dimensional (3D) chromatin structure is essential to the understanding of biological processes in the nucleus. Integrative analysis of multi-omics data in recent publications have provided comprehensive and multilevel insight into 3D genome organization emphasizing its role for transcriptional regulation. While enhancers are regulatory elements that play a central role in the spatiotemporal control of gene expression, chromatin looping has been broadly accepted as a means for enhancer-promoter interactions yielding cell-type-specific gene expression signatures. On the other hand, G-quadruplexes (G4s) are non-canonical DNA secondary structures that are enriched at promoters and related to increased gene expression. However, the role of G4s in promoter-distal regulatory elements, such as super-enhancers (SE), as well as in 3D genome organization and chromatin looping mediating long-range enhancer-promoter interactions has remained elusive. Here we show that mature microRNA 9 (miR-9) is enriched at promoters and SE of genes that are inducible by tissue growth factor beta 1 (TGFB1) signaling. Further, we find that nuclear miR-9 is required for chromatin features related to increased transcriptional activity, such as broad domains of the euchromatin histone mark H3K4me3 (histone 3 tri-methylated lysine 4) and G4s. Moreover, we show that nuclear miR-9 is required for promoter-super-enhancer looping. Our study places a nuclear microRNA in the same structural and functional context with G4s and promoter-enhancer interactions during 3D genome organization and transcriptional activation induced by TGFB1 signaling, a critical regulator of proliferation programs in cancer and fibrosis. Overall design: MLg or MLE12 cells were transfected with antagomiR probes (Ambion) using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions and after 48 h cells were harvested. Followed Cut&Tag protocol was performed to obtained DNA associated to the Histone mark or G-quadruplexes structure

研究三维染色质结构（three-dimensional chromatin structure，3D）的动态变化，对于解析细胞核内的生物学过程具有关键意义。近期研究中对多组学数据（multi-omics data）的整合分析，为三维基因组组织（three-dimensional genome organization，3D genome organization）提供了全面且多层次的认知，并强调了其在转录调控中的核心作用。增强子（enhancer）是一类在基因表达的时空调控中发挥核心作用的调控元件，而染色质环（chromatin looping）已被广泛认为是介导增强子-启动子（enhancer-promoter）相互作用、进而产生细胞类型特异性基因表达特征的重要方式。另一方面，G四链体（G-quadruplexes，G4s）属于非经典DNA二级结构，在启动子区域富集且与基因表达上调相关。然而，G4s在启动子远端调控元件（如超级增强子（super-enhancers，SE））中的作用，以及其在三维基因组组织和介导远程增强子-启动子相互作用的染色质环中的功能，至今仍未明确。本研究发现，成熟微小RNA9（microRNA 9，miR-9）在受转化生长因子β1（tissue growth factor beta 1，TGFB1）信号通路诱导的基因的启动子及超级增强子区域富集。进一步研究表明，细胞核内的miR-9对于与转录活性上调相关的染色质特征至关重要，例如常染色质标志性组蛋白修饰H3K4me3（histone 3 tri-methylated lysine 4，组蛋白3三甲基化赖氨酸4）的宽结构域以及G4s。此外，本研究证实，细胞核内的miR-9对于启动子-超级增强子环的形成不可或缺。本研究将微小RNA置于转化生长因子β1信号通路诱导的三维基因组组织与转录激活过程中，明确了其与G4s及启动子-增强子相互作用共享结构与功能层面的关联——而该信号通路是癌症与纤维化中增殖程序的关键调控因子。实验设计概述：使用脂质体2000（Lipofectamine 2000，Invitrogen）按照厂商说明书，将反义miRNA探针（antagomiR probes，Ambion）转染至MLg或MLE12细胞中，于转染48小时后收集细胞；随后采用Cut&Tag实验流程，获取与组蛋白修饰或G四链体结构相结合的DNA片段。