The structural basis for cohesin–CTCF-anchored loops

In the paper

“Similarly, titration of a SGO1 phospho-T346 peptide, previously reported to preclude WAPL binding2, also displaced SA2-SCC1 from GST-CTCF (Extended Data Fig. 2d).”

What the sources say

• Supported

  The citation is attached to the statement that the phospho-T346 SGO1 peptide precludes WAPL binding, not to the citing authors’ GST-CTCF displacement result. In Results, “Sgo1 competes with Wapl for cohesin binding,” the source reports dose-dependent reduction of SA2–Scc1 binding to GST-Wapl-M by the phosphorylated peptide. (LLM, conf=0.92)

  cites [2] Hara, K. et al. Structure of cohesin subcomplex pinpoints direct shugoshin–Wapl antagonism in centromeric cohesion. Nat. Struct. Mol. Biol. 21, 864–870 (2014). source

In the paper

“Stabilization of cohesin by engagement of the CTCF N-terminus may in fact explain why TAD boundaries arise preferentially when CTCF binding sites are convergently oriented9,13–16,38,39.”

What the sources say

• Not checked yetcites [9] Sanborn, A. L. et al. Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes. Proc. Natl Acad. Sci. USA 112, E6456–E6465 (2015).
• Partly supported

  The Results section, under “Directional CTCF Binding in Genome-wide DNA Looping Specificity” (page unavailable), reports preferential orientations of CTCF-site pairs at TAD boundaries and links CTCF/cohesin occupancy to directional looping. It does not propose that engagement of the CTCF N-terminus stabilizes cohesin or explains that orientation preference. (LLM, conf=0.86)

  cites [13] Guo, Y. et al. CRISPR inversion of CTCF sites alters genome topology and enhancer/promoter function. Cell 162, 900–910 (2015). sourceExperimental — accuracy not yet measured
• Not checked yetcites [14] de Wit, E. et al. CTCF binding polarity determines chromatin looping. Mol. Cell 60, 676–684 (2015). source
• Partly supported

  The Results report that 92% of examined loop-anchor CTCF motif pairs are convergent, and the Discussion says loops often demarcate domains. But the Discussion proposes CTCF dimerization as a possible mechanism; it does not attribute the orientation preference to stabilization of cohesin by the CTCF N-terminus. No page number is available in our copy. (LLM, conf=0.87)

  cites [15] Rao, S. S. et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159, 1665–1680 (2014). sourceExperimental — accuracy not yet measured
• Partly supported

  The Results (section “Conserved CTCF Sites Are Directional and Interact with Other Conserved Sites”; page unavailable) link CTCF motif orientation to directional contacts and report oppositely oriented sites anchoring domain-defining interactions. But the paper does not propose stabilization through the CTCF N-terminus; the Discussion instead says cohesin interacts with CTCF primarily through its C-terminal tail. (LLM, conf=0.88)

  cites [16] Vietri Rudan, M. et al. Comparative Hi-C reveals that CTCF underlies evolution of chromosomal domain architecture. Cell Rep. 10, 1297–1309 (2015). sourceExperimental — accuracy not yet measured
• Not checked yetcites [38] Sedeño Cacciatore, Á. & Rowland, B. D. Loop formation by SMC complexes: turning heads, bending elbows, and fixed anchors. Curr. Opin. Genet. Dev. 55, 11–18 (2019). source
• Partly supported

  Results II reports that most chromatin-contact-domain boundaries have inward-facing CTCF motifs and that cohesin co-occupies many CTCF interaction anchors. The Discussion links convergent CTCF loops to CCD/TAD boundaries. Neither passage attributes that preference to engagement of the CTCF N-terminus stabilizing cohesin. (LLM, conf=0.85)

  cites [39] Tang, Z. et al. CTCF-mediated human 3D genome architecture reveals chromatin topology for transcription. Cell 163, 1611–1627 (2015). sourceExperimental — accuracy not yet measured

In the paper

“F228 inserts into a pocket comprising amino acids from SCC1 (S334, I337, L341) and SA2 (Y297, W334) (Extended Data Fig. 1f).”

In the paper

“Mutagenesis of Y226A or F228A in CTCF abolished SA2-SCC1 binding in a GST pulldown assay (Fig. 1e).”

In the paper

“The CTCF peptide is bound to the convex surface of SA2 (Fig. 1a,b).”

In the paper

“A similar motif is present in a number of established and novel cohesin ligands, including the cohesin release factor WAPL2,3.”

What the sources say

• Not checked yetcites [2] Hara, K. et al. Structure of cohesin subcomplex pinpoints direct shugoshin–Wapl antagonism in centromeric cohesion. Nat. Struct. Mol. Biol. 21, 864–870 (2014). source
• Not checked yetcites [3] Shintomi, K. & Hirano, T. Releasing cohesin from chromosome arms in early mitosis: opposing actions of Wapl–Pds5 and Sgo1. Genes Dev. 23, 2224–2236 (2009). source

In the paper

“and contributes to the positioning of cohesin at CTCF binding sites”

• Also stated in: § CTCF interaction promotes cohesin localization to CTCF sites · plain

  “CTCF interaction promotes cohesin localization to CTCF sites”

In the paper

“The lead ‘anchoring’ amino acids of CTCF, which bury the largest solvent-accessible surface area upon binding, are Y226 and F228 (Fig. 1b).”

In the paper

“A 2.6Å crystal structure of SA2-SCC1 in complex with CTCF reveals the molecular basis of the interaction.”

• Also stated in: § Structure of the SA2-SCC1-CTCF complex · plain

  “we produced crystals of the SA2-SCC1 complex in the presence of a peptide comprising the CTCF binding motif and determined the structure by molecular replacement at a resolution of 2.6 Å”

In the paper

“The hydroxyl group of Y226 hydrogen bonds with D326 of SA2 in a deep hydrophobic pocket lined by L329, L366 and F367 (Fig. 1c,d).”

In the paper

“We demonstrate that this interaction is specifically required for CTCF-anchored loops”

• Also stated in: § CTCF-CES interaction is required for CTCF-anchored loops · plain

  “CTCF-CES interaction is required for CTCF-anchored loops”

In the paper

“CTCF fragments containing amino acids 227-235 generally retained SA2-SCC1 on GST-beads (Extended Data Fig. 1a,b).”

In the paper

“Mapping of sequence conservation onto the structure confirms that the CES is highly conserved (Fig. 1g, Extended Data Fig. 2a).”

In the paper

“Isothermal calorimetry experiments further showed that the interaction is largely driven by amino acids 222-231 of CTCF as it retained an equilibrium dissociation constant (Kd = 1.04 ± 0.20 μM) comparable to an extended CTCF construct (Kd = 0.62 ± 0.07μM; Extended Data Fig. 1c and Extended Data Table 1a).”

In the paper

“Here we show that a segment within the CTCF N-terminus interacts with the SA2-SCC1 subunits of cohesin.”

• Also stated in: § Introduction · plain

  “We have identified an N-terminal segment of CTCF that directly engages the SA2-SCC1 subcomplex of cohesin.”

In the paper

“To determine whether WAPL competes with CTCF for binding to the SA2-SCC1 CES, we performed GST-pulldown competition assays. Indeed, titration of WAPL residues 1-600 against a preformed complex of GST-CTCF and SA2-SCC1 depleted the latter from the beads (Fig. 2b).”

In the paper

“In contrast, cohesin levels at a nearby locus that did not contain a CTCF site were not affected (Fig. 4b, and Extended Data Fig. 5b).”

In the paper

“the CTCF Y226A F228A mutation indeed led to a loss of the vast majority of detectable loops across the genome (from 2756 in the wild-type to 98 in the mutant cells) (Fig. 3b).”

In the paper

“The peptide containing the CTCF CES motif therefore binds with higher affinity than do peptides containing the WAPL motif(s).”

In the paper

“In wild-type cells, we found that, over a period of 20 minutes a fraction of the fluorescent cohesin population did not recover.”

In the paper

“Hi-C matrices of CTCF Y226A F228A mutant cells revealed a robust ablation of CTCF-anchored loops (Fig. 3a).”

In the paper

“An aggregate peak analysis, which quantifies the contact frequency of all the loops identified in wild-type cells, likewise showed a dramatic loss of these contacts (Fig. 3c and Extended Data Fig. 4d).”

In the paper

“Mapping of mutation frequencies onto the structure shows that amino acids that are largely buried in the interface are hotspots in cancer (Extended Data Fig. 2b).”

In the paper

“While the CTCF Y226A F228A mutations reduced cohesin levels at CTCF sites, cohesin was to a significant degree still present at CTCF sites.”

In the paper

“Accordingly, impairment of the CTCF-CES interaction renders cohesin more dynamic (Fig. 2c,d).”

In the paper

“In SGO1, the reported CES-binding domain (amino acids 313- 353) contains a conserved FGF motif which strongly resembles that of the CTCF peptide.”

In the paper

“The ablation of this interaction results in a near-complete loss of CTCF-cohesin anchored loops.”

In the paper

“found that the CTCF Y226A F228A mutation reduced the abundance of cohesin at the majority of these loci.”

In the paper

“Our study reveals that CTCF binds to a conserved essential surface (CES) on the SA2-SCC1 cohesin subcomplex.”

In the paper

“Consistent with our pulldowns, the CTCF Y226A, F228A, and Y226A/F228A peptide variants failed to retain SA2-SCC1.”

In the paper

“Robust binding was observed for MCM3, a subunit of the replicative helicase, SYCP3, a component of the synaptonemal complex, ZGPAT, a transcriptional repressor, and CENPU, a subunit of the inner kinetochore.”

In the paper

“We observed that a peptide spanning the second and third FGF motif of WAPL (amino acids 423-463) bound to SA2-SCC1 with a Kd of ~32.8 μM (Extended Data Fig. 2c)”

In the paper

“Despite this effect on gene expression and the loss of virtually all CTCF-anchored loops, cells harbouring only this mutant form of CTCF are viable.”

In the paper

“CTCF binding to the corresponding CTCF sites was also largely unaffected (Extended Data Fig. 5c-e).”

In the paper

“whereas a peptide comprising only the third motif bound more weakly (Extended Data Table 1a).”

In the paper

“and completely lack CTCF loops at their corners (Extended Data Fig. 4b,e).”

In the paper

“The CTCF Y226A F228A mutation affected the expression of >2000 genes.”

In the paper

“The CTCF Y226A F228A mutation, like CTCF depletion, only slightly affects the contact probability profile.”

What the sources say

• Not checked yetcites [12] Wutz, G. et al. Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins. EMBO J. 36, 3573–3599 (2017). source

In the paper

“CTCF Y226A F228A mutation does not evidently affect overall CTCF and cohesin levels on chromatin.”

In the paper

“This non-diffusive fraction is not evidently affected by the CTCF Y226A F228A mutation.”

In the paper

“CTCF remains essential for viability in CTCF Y226A F228A cells.”

In the paper

“CTCF* | 222-231 | 1.04 ± 0.20 | -11.08 ± 0.70 | -2.92 ± 0.82 | -8.16 ± 0.09 | 0.93 ± 0.04”

In the paper

“Genome-wide comparison of compartment scores for wild type and CTCF Y226A F228A cells (Pearson correlation = 0.97).”

In the paper

“CTCF Y226A F228A mutation has little effect on CTCF levels at CTCF sites.”

In the paper

“Compartmentalization is largely maintained in cells containing the CTCF Y226A F228A mutation.”

In the paper

“A series of missense mutations are found in SA2, SCC1 and CTCF in various cancer types24.”

What the sources say

• Not checked yetcites [24] Forbes, S. A. et al. COSMIC: somatic cancer genetics at high-resolution. Nucleic Acids Res. 45, D777–D783 (2017). source

In the paper

“Previous data indicate that CTCF directly interacts with the SA2 subunit of the cohesin complex19,20.”

What the sources say

• Not checked yetcites [19] Rubio, E. D. et al. CTCF physically links cohesin to chromatin. Proc. Natl Acad. Sci. USA 105, 8309–8314 (2008). source
• Not checked yetcites [20] Xiao, T., Wallace, J. & Felsenfeld, G. Specific sites in the C terminus of CTCF interact with the SA2 subunit of the cohesin complex and are required for cohesin-dependent insulation activity. Mol. Cell. Biol. 31, 2174–2183 (2011). source

In the paper

“Our data suggest that CTCF enables chromatin loop formation by protecting cohesin against loop release.”

• Also stated in: § Discussion · plain

  “CTCF does not simply present a passive barrier to cohesin-mediated loop extrusion, but specifically interacts with the CES to stabilize cohesin at these loci, and to prevent loop disruption.”

In the paper

“Hence, the CES of SA2-SCC1 is a general interaction hub for multiple cohesin regulators (Extended Data Fig. 2e).”

In the paper

“Specifically, the 34 amino acid flexible linker connecting the YxF motif to the first DNA-binding zinc finger of human CTCF, is sufficiently long to allow SA2-SCC1 DNA binding towards the N-, but not the C-terminus of CTCF (Fig. 4g), thus confirming previous mapping studies38.”

What the sources say

• Not checked yetcites [38] Sedeño Cacciatore, Á. & Rowland, B. D. Loop formation by SMC complexes: turning heads, bending elbows, and fixed anchors. Curr. Opin. Genet. Dev. 55, 11–18 (2019). source

In the paper

“Thus, the Y226 F228 interface of CTCF, and by extension cohesin-CTCF anchored loops, are apparently key to proper expression of these genes.”

In the paper

“Thus, SA2-SCC1 CES potentially facilitates cohesin regulation for a number of functionally divergent chromosomal processes.”

In the paper

“Therefore, the dysregulation of chromatin looping may be causally related to carcinogenesis (Extended Data Fig. 7a) 31,32.”

What the sources say

• Not checked yetcites [31] Lawrence, M. S et al. Discovery and saturation analysis of cancer genes across 21 tumour types. Nature 505, 495–501 (2014). source
• Not checked yetcites [32] Flavahan, W. A. et al. Insulator dysfunction and oncogene activation in IDH mutant gliomas. Nature 529, 110–114 (2016). source

In the paper

“The CTCF-CES interaction therefore stabilizes a sub-population of cohesin on chromatin.”

In the paper

“Previous data indicate that the SA2-SCC1 complex interacts with multiple cohesin regulators2,23,25.”

What the sources say

• Not checked yetcites [2] Hara, K. et al. Structure of cohesin subcomplex pinpoints direct shugoshin–Wapl antagonism in centromeric cohesion. Nat. Struct. Mol. Biol. 21, 864–870 (2014). source
• Not checked yetcites [23] Roig, M. B. et al. Structure and function of cohesin’s Scc3/SA regulatory subunit. FEBS Lett. 588, 3692–3702 (2014). source
• Not checked yetcites [25] Beckouët, F. et al. Releasing activity disengages cohesin’s Smc3/Scc1 interface in a process blocked by acetylation. Mol. Cell 61, 563–574 (2016). source

In the paper

“Thus, CTCF has essential roles that are apparently independent of CES engagement and the formation of CTCF-anchored loops in these cells.”

In the paper

“Vertebrate WAPL also contains several FGF motifs in its N-terminal region which are potentially involved in cohesin regulation3,29.”

What the sources say

• Not checked yetcites [3] Shintomi, K. & Hirano, T. Releasing cohesin from chromosome arms in early mitosis: opposing actions of Wapl–Pds5 and Sgo1. Genes Dev. 23, 2224–2236 (2009). source
• Not checked yetcites [29] Ouyang, Z. et al. Structure of the human cohesin inhibitor Wapl. Proc. Natl Acad. Sci. USA 110, 11355–11360 (2013). source

In the paper

“Similarly, this interface is also conserved through Scc3 in lower Eukaryotes, despite the absence of CTCF in such organisms.”

In the paper

“We therefore predict that a number of proteins containing F/YxF motifs engage the CES and thereby modulate cohesin’s ability to catalyse genome folding in functionally divergent chromosomal processes.”

In the paper

“The observation that CTCF-CES interaction controls DNA looping indicates that this aspect of cohesin function can be regulated by F/YxF motif containing cohesin ligands.”

In the paper

“Loop formation by the related condensin complex appears to involve a DNA anchoring function of its HEAT-repeat subunit Ycg1, a paralogue of SA2/Scc340–42.”

What the sources say

• Not checked yetcites [40] Nagy, G. et al. Motif oriented high-resolution analysis of ChIP-seq data reveals the topological order of CTCF and cohesin proteins on DNA. BMC Genomics 17, 637 (2016). source
• Not checked yetcites [41] Kschonsak, M. et al. Structural basis for a safety-belt mechanism that anchors condensin to chromosomes. Cell 171, 588–600 (2017). source
• Not checked yetcites [42] Ganji, M. et al. Real-time imaging of DNA loop extrusion by condensin. Science 360, 102–105 (2018). source

In the paper

“These different complexes may therefore use a similar anchoring principle to build loops and provide structure to genomes.”

In the paper

“The CES therefore is likely to represent an ancient interaction hub on cohesin.”