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

Chromatin is the physiological template for all biological processes in the eukaryotic cell nucleus. In addition, the structure of chromatin is intrinsically related to its function. Thus, studying the dynamics of three-dimensional (3D) chromatin structure is essential to understand these biological processes. Recent publications based on integrative analysis of multi-omics studies have provided comprehensive and multilevel insights into 3D genome organization emphasizing its role during transcriptional regulation. However, the function of nuclear microRNAs in 3D genome organization has remained elusive. Here we show that mature microRNA 9 (miR-9) is enriched at promoters and super-enhancers (SE) of genes that are inducible by tissue growth factor beta 1 (TGFB1) signaling. Further, we found that nuclear miR-9 is required for broad domains of the euchromatin histone mark H3K4me3 (histone 3 tri-methylated lysine 4), as well as the nucleic acid secondary structure G-quadruplexes (G4s), both are chromatin features related to increased transcriptional activity. 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 pathway that plays important role in hyperproliferative diseases, such as cancer and fibrosis. Mlg cells were transfected with Mir9 complementary probe and biotinalited beads were used to obtain the DNA associated to Mir9

染色质（Chromatin）是真核细胞核内所有生物学过程的生理性模板。此外，染色质的结构与其功能存在内在关联。因此，研究三维（3D）染色质结构的动态变化，对于理解这些生物学过程至关重要。近年来，基于多组学整合分析的研究成果，为三维基因组组织提供了全面且多维度的见解，并着重强调了其在转录调控中的作用。然而，细胞核内微RNA（microRNA，miRNA）在三维基因组组织中的功能仍难以阐释。
本研究发现，成熟微RNA9（miR-9）富集于可被转化生长因子β1（TGFB1）信号通路诱导的基因的启动子与超级增强子（SE）区域。进一步研究表明，细胞核内的miR-9对于常染色质组蛋白标记H3K4me3（组蛋白3赖氨酸4三甲基化）的宽泛结构域，以及核酸二级结构G-四链体（G4s）均为必需，而这二者均为与转录活性增强相关的染色质特征。此外，本研究证实细胞核内的miR-9对于启动子-超级增强子环化过程不可或缺。
本研究将细胞核内微RNA置于三维基因组组织以及转化生长因子β1信号通路诱导的转录激活过程中，明确其与G-四链体、启动子-增强子相互作用处于相同的结构与功能框架内。而该信号通路在癌症、纤维化等过度增殖性疾病中发挥重要作用。本研究使用Mir9互补探针转染Mlg细胞，并采用生物素标记磁珠获取与Mir9结合的DNA。