MCM complexes are barriers that restrict cohesin-mediated loop extrusion

Abstract

Eukaryotic genomes are compacted into loops and topologically associating domains (TADs)1–3, which contribute to transcription, recombination and genomic stability4,5. Cohesin extrudes DNA into loops that are thought to lengthen until CTCF boundaries are encountered6–12. Little is known about whether loop extrusion is impeded by DNA-bound machines. Here we show that the minichromosome maintenance (MCM) complex is a barrier that restricts loop extrusion in G1 phase. Single-nucleus Hi-C (high-resolution chromosome conformation capture) of mouse zygotes reveals that MCM loading reduces CTCF-anchored loops and decreases TAD boundary insulation, which suggests that loop extrusion is impeded before reaching CTCF. This effect extends to HCT116 cells, in which MCMs affect the number of CTCF-anchored loops and gene expression. Simulations suggest that MCMs are abundant, randomly positioned and partially permeable barriers. Single-molecule imaging shows that MCMs are physical barriers that frequently constrain cohesin translocation in vitro. Notably, chimeric yeast MCMs that contain a cohesin-interaction motif from human MCM3 induce cohesin pausing, indicating that MCMs are ‘active’ barriers with binding sites. These findings raise the possibility that cohesin can arrive by loop extrusion at MCMs, which determine the genomic sites at which sister chromatid cohesion is established. On the basis of in vivo, in silico and in vitro data, we conclude that distinct loop extrusion barriers shape the three-dimensional genome.

Transcription shapes 3D chromatin organization by interacting with loop extrusion

“…However, while boundaries such as CTCF sites are stationary (6, 8–11), RNAPs are mobile and can be dynamically controlled by transcriptional regulators. Together with emerging evidence that extrusion might also be obstructed by other mobile complexes such as replication machinery (24, 32), this suggests that in addition to structural functions, cohesin has important dynamic functions in the spatiotemporal organization of the genome.…”

“…Extrusion barriers can thus facilitate or suppress functional interactions, such as enhancer-promoter contacts, which can impact differentiation, disease, and other physiological processes (8,(17)(18)(19)(20)(21)(22)(23). Other factors that do not occupy specific genomic positions, such as the replicative helicase MCM, can also act as barriers to loop extrusion (24). These observations raise the question of how chromatin organization by loop-extruding cohesins is shaped by other chromatin-bound factors, some of which may themselves be mobile.…”

Context-dependent transcriptional remodeling of TADs during differentiation

“…However, CTCF occupancy is not identical in all cell types and in any case does not account for all TADs (Nora et al, 2017; Taylor et al, 2022). Recent studies have shown that non-encoded features as diverse as MCM complex binding (minichromosome maintenance; most famous for replication origin licensing) and DNA double-strand breaks can also be barriers to loop extrusion (Arnould et al, 2021; Dequeker et al, 2022). Thus small modifications to the basic “loop extrusion versus compartmentalization” model could just as easily explain tissue-specific TAD structures.…”

Mars, a molecule archive suite for reproducible analysis and reporting of single-molecule properties from bioimages

“…Single-molecule total internal reflection fluorescence (smTIRF) microscopy has become an indispensable tool to study bio-macromolecular structure and functionality allowing the observation of, for example, molecule position and dynamics. Examples of such studies include kinetic studies of DNA replication (Dequeker et al ., 2022; Ha et al, 2002; Lewis et al, 2020), studies of the polymerization of structural elements like actin (Amann and Pollard, 2001), the direct observation of flagellar motor rotation (Sowa et al, 2005), as well as tracking of processes in vivo (Vizcay-Barrena et al, 2011). To illustrate the use of Mars for the analysis of such datasets, this example shows a typical Mars workflow for smTIRF studies of the kinetics of a fluorescently-labeled RNA polymerase transcribing on an immobilized, promoter-containing, 21 kb DNA molecule (Scherr et al, 2022) ( Figure 3A ).…”