Extruding transcription elongation loops observed in high-resolution single-cell 3D genomes [RNA-seq and Smart-seq3, topoisomerase inhibition and cohesin depletion, mESC]
Inside human nuclei, genes are transcribed within a highly packed genome, whose organization is facilitated by cohesin-mediated loop extrusion. However, how cohesin folds transcribed genes and affects transcription remain unclear. Here we report that highly expressed long genes form a “stripe-like” structure termed transcription elongation loop (TEL), which aligns between the transcription start site (TSS) and the transcription termination site (TTS). We proved that TELs formation results from the joint interactions between cohesin-mediated loop extrusion, RNA polymerase II (RNAPII) and topoisomerases. By improving the spatial resolution of single-cell 3D genome mapping to 5 kb with micrococcal nuclease (MNase) in our new single-cell Micro-C (scMicro-C) method, we directly observed the loop expansion of TELs. Furthermore, we demonstrated that TEL-associated genes have higher transcriptional burst frequency and cohesin modulates transcriptional bursting by facilitating the release of RNAPII pausing. Thus, our results demonstrated the fundamental role of cohesin in transcription regulation. Overall design: Smart-seq3 was performed on single mouse embryonic stem cells with cohesin depleted for 0h, 3h, and 6h. Bulk RNA-seq was performed on mouse embryonic stem cells treated with DMSO, ETO and TPT, repectively (two replicates per condition).