SummaryThe spatial organization of chromosomes influences many nuclear processes including gene expression. The cohesin complex shapes the 3D genome by looping together CTCF sites along chromosomes. We show here that chromatin loop size can be increased and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. Cohesin’s DNA release factor WAPL restricts this loop extension and also prevents looping between incorrectly oriented CTCF sites. We reveal that the SCC2/SCC4 complex promotes the extension of chromatin loops and the formation of topologically associated domains (TADs). Our data support the model that cohesin structures chromosomes through the processive enlargement of loops and that TADs reflect polyclonal collections of loops in the making. Finally, we find that whereas cohesin promotes chromosomal looping, it rather limits nuclear compartmentalization. We conclude that the balanced activity of SCC2/SCC4 and WAPL enables cohesin to correctly structure chromosomes.

Cohesin catalyzes folding of the genome into loops that are anchored by CTCF 1. The molecular mechanism of how cohesin and CTCF structure the 3D genome has remained unclear. Here we show that a segment within the CTCF N-terminus interacts with the SA2-SCC1 subunits of cohesin. A 2.6Å crystal structure of SA2-SCC1 in complex with CTCF reveals the molecular basis of the interaction. We demonstrate that this interaction is specifically required for CTCF-anchored loops and contributes to the positioning of cohesin at CTCF binding sites. A similar motif is present in a number of established and novel cohesin ligands, including the cohesin release factor WAPL 2,3. Our data suggest that CTCF enables chromatin loop formation by protecting cohesin against loop release. These results provide fundamental insights into the molecular mechanism that enables dynamic regulation of chromatin folding by cohesin and CTCF.

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Chromatin folding contributes to the regulation of genomic processes such as gene activity. Existing conformation capture methods characterize genome topology through analysis of pairwise chromatin contacts in populations of cells but cannot discern whether individual interactions occur simultaneously or competitively. Here we present multi-contact 4C (MC-4C), which applies Nanopore sequencing to study multi-way DNA conformations of individual alleles. MC-4C distinguishes cooperative from random and competing interactions and identifies previously missed structures in subpopulations of cells. We show that individual elements of the β-globin superenhancer can aggregate into an enhancer hub that can simultaneously accommodate two genes. Neighboring chromatin domain loops can form rosette-like structures through collision of their CTCF-bound anchors, as seen most prominently in cells lacking the cohesin-unloading factor WAPL. Here, massive collision of CTCF-anchored chromatin loops is believed to reflect 'cohesin traffic jams'. Single-allele topology studies thus help us understand the mechanisms underlying genome folding and functioning.

The X shape of chromosomes is one of the iconic images in biology. Cohesin actually connects the sister chromatids along their entire length, from S phase until mitosis. Then, cohesin's antagonist Wapl allows the separation of chromosome arms by opening a DNA exit gate in cohesin rings. Centromeres are protected against this removal activity, resulting in the X shape of mitotic chromosomes. The destruction of the remaining centromeric cohesin by Separase triggers chromosome segregation. We review the two-phase regulation of cohesin removal and discuss how this affects chromosome alignment and decatenation in mitosis and cohesin reloading in the next cell cycle.

SummaryCohesin stably holds together the sister chromatids from S phase until mitosis. To do so, cohesin must be protected against its cellular antagonist Wapl. Eco1 acetylates cohesin’s Smc3 subunit, which locks together the sister DNAs. We used yeast genetics to dissect how Wapl drives cohesin from chromatin and identified mutants of cohesin that are impaired in ATPase activity but remarkably confer robust cohesion that bypasses the need for the cohesin protectors Eco1 in yeast and Sororin in human cells. We uncover a functional asymmetry within the heart of cohesin’s highly conserved ABC-like ATPase machinery and find that both ATPase sites contribute to DNA loading, whereas DNA release is controlled specifically by one site. We propose that Smc3 acetylation locks cohesin rings around the sister chromatids by counteracting an activity associated with one of cohesin’s two ATPase sites.

The classical X shape of mitotic human chromosomes is the consequence of two distinct waves of cohesin removal. First, during prophase and prometaphase, the bulk of cohesin is driven from chromosome arms by the cohesin antagonist WAPL. This arm-specific cohesin removal is referred to as the prophase pathway [1-4]. The subsequent cleavage of the remaining centromeric cohesin by Separase is known to be the trigger for anaphase onset [5-7]. Remarkably the biological purpose of the prophase pathway is unknown. We find that this pathway is essential for two key mitotic processes. First, it is important to focus Aurora B at centromeres to allow efficient correction of erroneous microtubule-kinetochore attachments. In addition, it is required to facilitate the timely decatenation of sister chromatids. As a consequence, WAPL-depleted cells undergo anaphase with segregation errors, including both lagging chromosomes and catenanes, resulting in micronuclei and DNA damage. Stable WAPL depletion arrests cells in a p53-dependent manner but causes p53-deficient cells to become highly aneuploid. Our data show that the WAPL-dependent prophase pathway is essential for proper chromosome segregation and is crucial to maintain genomic integrity.

Conformation capture-approaches like Hi-C can elucidate chromosome structure at a genome-wide scale. Hi-C datasets are large and require specialised software. Here, we present GENOVA: a user-friendly software package to analyse and visualise chromosome conformation capture (3C) data. GENOVA is an R-package that includes the most common Hi-C analyses, such as compartment and insulation score analysis. It can create annotated heatmaps to visualise the contact frequency at a specific locus and aggregate Hi-C signal over user-specified genomic regions such as ChIP-seq data. Finally, our package supports output from the major mapping-pipelines. We demonstrate the capabilities of GENOVA by analysing Hi-C data from HAP1 cell lines in which the cohesin-subunits SA1 and SA2 were knocked out. We find that ΔSA1 cells gain intra-TAD interactions and increase compartmentalisation. ΔSA2 cells have longer loops and a less compartmentalised genome. These results suggest that cohesinSA1 forms longer loops, while cohesinSA2 plays a role in forming and maintaining intra-TAD interactions. Our data supports the model that the genome is provided structure in 3D by the counter-balancing of loop formation on one hand, and compartmentalization on the other hand. By differentially controlling loops, cohesinSA1 and cohesinSA2 therefore also affect nuclear compartmentalization. We show that GENOVA is an easy to use R-package, that allows researchers to explore Hi-C data in great detail.