Transcription shapes 3D chromatin organization by interacting with loop extrusion

Abstract

Cohesin organizes mammalian interphase chromosomes by reeling chromatin fibers into dynamic loops (Banigan and Mirny, 2020; Davidson et al., 2019; Kim et al., 2019; Yatskevich et al., 2019). "Loop extrusion" is obstructed when cohesin encounters a properly oriented CTCF protein (Busslinger et al., 2017; de Wit et al., 2015; Fudenberg et al., 2016; Nora et al., 2017; Sanborn et al., 2015; Wutz et al., 2017), and recent work indicates that other factors, such as the replicative helicase MCM (Dequeker et al., 2020), can also act as barriers to loop extrusion. It has been proposed that transcription relocalizes (Busslinger et al., 2017; Glynn et al., 2004; Lengronne et al., 2004) or interferes with cohesin (Heinz et al., 2018; Jeppsson et al., 2020; Valton et al., 2021; S. Zhang et al., 2021), and that active transcription start sites function as cohesin loading sites (Busslinger et al., 2017; Kagey et al., 2010; Zhu et al., 2021; Zuin et al., 2014), 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 (Busslinger et al., 2017), 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 (Ciosk et al., 2000) co-localizes with cohesin, but, unlike in previous reports (Busslinger et al., 2017; Kagey et al., 2010; Zhu et al., 2021; Zuin et al., 2014), 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.

Temporal analysis suggests a reciprocal relationship between 3D chromatin structure and transcription

“…Moreover, the temporal patterns of loop loss and internal transcription were anti-correlated. These temporal analyses agree with previous work showing accumulation of cohesin at the 3’ ends of genes in a manner correlated with the amount of transcription and also sensitive to transcription inhibition 24,27,28 , and recent studies demonstrating that RNA polymerase may act as a “moving barrier” to loop extrusion 49 . Transcription may also shape chromatin independently of cohesin.…”

“…(D) Average log2 fold-change of differential loops (blue) and inward-and outward-facing genes (orange, yellow) with promoters overlapping those loop anchors. Taken together, this suggests that the causal arrow between looping and transcription might point both ways: DNA loop formation may contribute to increased transcription of target genes, but very high levels of transcription could weaken loops by antagonizing loop extrusion as previously observed 24,25,27,[48][49][50] . An example of these potential phenomena can be seen at the GBP locus (Fig 5A).…”

Generation of dynamic three-dimensional genome structure through phase separation of chromatin

“…We represented this effect with U cohesin by assuming that cohesin stochastically moves along the chain, extruding a loop of the chromatin chain. In type-A chains, various functional complexes should block the cohesin movement [30, 31] as found in the large NCI (Fig. 1), while the cohesin movement is less disturbed in type-B chains [32].…”

“…Because the typical size of the loop domain is ∼ 200 kb [5], the larger NCI in a type-A region implies the more frequent intra-domain contacts. These intra-domain contacts may arise from DNA-protein complexes organized for transcription or replication [28,29], and cohesin molecules off the CTCFbound sites can associate with these complexes [30,31] to reinforce contacts and enhance NCI. A type-u region represents either the region showing the intermediate feature between euchromatin and heterochromatin as was identified by clustering the Hi-C contact data [32] or the mosaic of type-A and B regions averaged over a 100-kb interval.…”

A de novo transcription-dependent TAD boundary underpins critical multiway interactions during antibody class switch recombination

“…This conclusion was based on ChIP data showing that the cohesin loader, Nipbl, was highly enriched at transcription start sites ( 3, 12, 48–50 ). However, as shown in a new study ( 2 ), the Nipbl antibody used in these studies was non-specific resulting in erroneous maps of Nipbl chromatin occupancy. Using a new epitope-and degron-tagging approach, Nipbl was detected only rarely at transcription start sites ( 2 ).…”