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Extruding transcription elongation loops observed in high-resolution single-cell 3D genomes [RNA-seq and Smart-seq3, topoisomerase inhibition and cohesin depletion, mESC]

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE243523 | GEO | 2026/02/18

REPOSITORIES: GEO

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			Action	DRS
		Other
	GSE243523_mesc_topo_inhibition_gene_counts.tsv.gz	Tabular
	GSE243523_ss3_rad21_degron_readcount.matrix.tsv.gz	Tabular
	GSE243523_ss3_rad21_degron_umicount.matrix.tsv.gz	Tabular

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Project description:Cohesin organizes mammalian interphase chromosomes by reeling chromatin fibers into dynamic loops. "Loop extrusion" is obstructed when cohesin encounters a properly oriented CTCF protein. It has been proposed that transcription relocalizes or interferes with cohesin, and that active transcription start sites function as cohesin loading sites, but how these effects, and transcription in general, shape chromatin is unknown. To determine whether transcription can modulate loop extrusion, we studied cells in which the primary extrusion barriers could be removed by CTCF depletion and cohesin’s residence time and abundance on chromatin could be increased by Wapl knockout. We found evidence that transcription directly interacts with loop extrusion through a novel "moving barrier" mechanism, but not by loading cohesin at active promoters. Hi-C experiments showed intricate, cohesin-dependent genomic contact patterns near actively transcribed genes, and in CTCF-Wapl double knockout (DKO) cells, genomic contacts were enriched between sites of transcription-driven cohesin localization ("cohesin islands"). Similar patterns also emerged in polymer simulations in which transcribing RNA polymerases (RNAPs) acted as "moving barriers" by impeding, slowing, or pushing loop-extruding cohesins. The model predicts that cohesin does not load preferentially at promoters and instead accumulates at TSSs due to the barrier function of RNAPs. We tested this prediction by new ChIP-seq experiments, which revealed that the "cohesin loader" Nipbl co-localizes with cohesin, but, unlike in previous reports, Nipbl did not accumulate at active promoters. We propose that RNAP acts as a new type of barrier to loop extrusion that, unlike CTCF, is not stationary in its precise genomic position, but is itself dynamically translocating and relocalizes cohesin along DNA. In this way, loop extrusion could enable translocating RNAPs to maintain contacts with distal regulatory elements, allowing transcriptional activity to shape genomic functional organization.

Dataset Information

Extruding transcription elongation loops observed in high-resolution single-cell 3D genomes [RNA-seq and Smart-seq3, topoisomerase inhibition and cohesin depletion, mESC]

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