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.

Three-dimensional (3D) chromatin organization plays a key role in regulating mammalian genome function; however, many of its physical features at the single-cell level remain underexplored. Here, we use live- and fixed-cell 3D super-resolution and scanning electron microscopy to analyze structural and functional nuclear organization in somatic cells. We identify chains of interlinked ~200- to 300-nm-wide chromatin domains (CDs) composed of aggregated nucleosomes that can overlap with individual topologically associating domains and are distinct from a surrounding RNA-populated interchromatin compartment. High-content mapping uncovers confinement of cohesin and active histone modifications to surfaces and enrichment of repressive modifications toward the core of CDs in both hetero- and euchromatic regions. This nanoscale functional topography is temporarily relaxed in postreplicative chromatin but remarkably persists after ablation of cohesin. Our findings establish CDs as physical and functional modules of mesoscale genome organization.

Three-dimensional (3D) chromatin organisation plays a key role in regulating genome function in higher eukaryotes. Despite recognition that the genome partitions into ~1Mb-sized topological associated domains (TADs) based on ensemble Hi-C measurements, many features of the physical organisation at the single cell level remain underexplored. Using 3D super-resolution microscopy, we reveal a sequential curvilinear arrangement of globular chromatin domains with viscoelastic properties ('blobs') juxtaposed to an RNA-populated interchromatin (IC) network. Quantitative mapping of genome function markers uncovers a zonal distribution, with RNA-binding factors concentrated in the IC, confinement of structural proteins and transcriptionally active/permissive marks to chromatin domain surfaces, and enrichment of repressive marks towards the interior. This correlation between nanoscale topology and genome function is relaxed in postreplicative chromatin, accentuated in replicative senescence, persists upon ATP depletion and hyperosmolarity induced chromatin condensation and, remarkably, after inactivation of cohesin. Our findings support a model of a higher-order chromatin architecture on the size level of TADs that creates and modulates distinct functional environments through a combination of biophysical parameters such as density and ATP-driven processes such as replication and transcription, but independent of cohesin.The genome in mammalian cell nuclei is hierarchically organised at various size scales that correlate with diverse genomic functions 1,2 . At the base pair to kilobase pair level, DNA is wrapped around core histones to create nucleosomes that form the building blocks of chromatin 3 . At the 100 Mb scale, whole chromosomes harbour distinct territories within the nucleus with transcriptionally active euchromatic and inactive heterochromatic segments tending to segregate into specific nuclear sub-regions 4 . The intermediate chromatin organization ranging from several kb to 100 Mb still remains poorly understood 5 . Advances in next-generation sequencing based chromosome conformation capturing methods (3C/Hi-C) have revealed a genomic landscape composed of ~400 kb to ~1 Mb-sized topologically associating domains (TADs) 6,7 . TADs are genomic regions which have elevated intra-TAD interaction relative to sequences outside TADs; deletion of TAD boundaries leads to aberrant reprogramming of the transcriptome 8 . Chromatin immunoprecipitation sequencing (ChIP-Seq) identified the CCCTC-binding factor (CTCF) as a key regulator in TAD organisation, binding to convergently oriented recognition sequences which flank TADs and thus define their boundaries at the linear scale 9 . Although the exact mechanisms of TAD formation and maintenance has yet to be elucidated, co-occurrence of the ring-shaped 3 cohesin complex at CTCF binding sites suggest a regulatory role of cohesin in shaping TAD structures through chromatin loop extrusion [10][11][12][13] . Accordingly, loss of cohesin function leads to an erasure of TAD signat...