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.

KLF4 (GKLF/EZF) encodes a transcription factor that is associated with both tumour suppression and oncogenesis. We describe the identification of KLF4 in a functional genomic screen for genes that bypass RAS V12 -induced senescence. However, in untransformed cells, KLF4 acts as a potent inhibitor of proliferation. KLF4-induced arrest is bypassed by oncogenic RAS V12 or by the RAS target cyclin-D1. Remarkably, inactivation of the cyclin-D1 target and the cell-cycle inhibitor p21 CIP1 not only neutralizes the cytostatic action of KLF4, but also collaborates with KLF4 in oncogenic transformation. Conversely, KLF4 suppresses the expression of p53 by directly acting on its promoter, thereby allowing for RAS V12 -mediated transformation and causing resistance to DNA-damage-induced apoptosis. Consistently, KLF4 depletion from breast cancer cells restores p53 levels and causes p53-dependent apoptosis. These results unmask KLF4 as a regulator of p53 that oncogenically transforms cells as a function of p21 CIP1 status. Furthermore, they provide a mechanistic explanation for the context-dependent oncogenic or tumour-suppressor functions of KLF4.Krüppel-like factor 4 (KLF4/GKLF/EZF) 1,2 is a transcription factor that can both activate and repress genes that are involved in cell-cycle regulation and differentiation. Among the KLF4-regulated cell-cycle genes, many upregulated genes are inhibitors of proliferation, whereas genes that promote proliferation are repressed 3 . This indicates that KLF4 regulates the expression of a set of cell-cycle genes to coordinately inhibit cellular proliferation. In keeping with this is the notion that ectopic expression of KLF4 acts cytostatically 1,4,5 . Moreover, KLF4 levels rise following DNA damage, cell-cycle arrest in response to serum withdrawal and contact inhibition 1,6 . Similarly, KLF4 levels are increased in the post-mitotic compartment of the gut and skin 1,2 . In mice, ectopic expression of Klf4 accelerates terminal differentiation, leading to premature skin-barrier acquisition 7 , whereas Klf4 deficiency prevents the terminal differentiation of colonic goblet cells 8 and the skin epithelium 9 , which leads to neonatal death 9 . These observations establish KLF4 as a stress-and differentiation-associated inhibitor of proliferation 10 , raising the possibility that KLF4 may have tumour-suppressive functions 8,11,12 .Indeed, KLF4 expression is frequently lost in various human cancer types 11,[13][14][15][16][17][18][19] . Recently, KLF4 has been shown to undergo promoter methylation and loss of heterozygosity in gastrointestinal cancer 16,17 . Consistent with a tumour-suppressor function for KLF4, its overexpression reduces the tumorigenicity of colonic and gastric cancer cells in vivo 17,20 . These observations, taken together, indicate that KLF4 acts as a tumour suppressor. This notion is further supported by recent data showing that specific ablation of Klf4 in the gastric epithelium of mice results in premalignant changes, including polypoid lesions 18 .Conversely, elevated K...

Cohesin's Smc1, Smc3, and Scc1 subunits form a tripartite ring that entraps sister DNAs. Scc3, Pds5, and Rad61 (Wapl) are regulatory subunits that control this process. We describe here smc3, scc3, pds5, and rad61 mutations that permit yeast cell proliferation and entrapment of sister DNAs by cohesin rings in the absence of Eco1, an acetyl transferase normally essential for establishing sister chromatid cohesion. The smc3 mutations cluster around and include a highly conserved lysine (K113) close to Smc3's ATP-binding pocket, which, together with K112, is acetylated by Eco1. Lethality caused by mutating both residues to arginine is suppressed by the scc3, pds5, and rad61 mutants. Scc3, Pds5, and Rad61 form a complex and inhibit entrapment of sister DNAs by a process involving the "K112/K113" surface on Smc3's ATPase. According to this model, Eco1 promotes sister DNA entrapment partly by relieving an antiestablishment activity associated with Scc3, Pds5, and Rad61.

| Krüppel-like factors are transcriptional regulators that influence several cellular functions, including proliferation. Recent studies have shown that one family member, KLF4, can function both as a tumour suppressor and an oncogene. The ability of KLF4 to affect the levels of expression of the cell-cycle regulator p21 seems to be involved, in that this protein might function as a switch that determines the outcome of KLF4 signalling. Is this role of p21 restricted to KLF4, or does p21 represent a nodal point for signals from multiple other factors with opposing functions in cancer?

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.