Crystal Structure of the Cohesin Loader Scc2 and Insight Into Cohesinopathy

Crystal structure of the cohesin loader Scc2 and insight into cohesinopathy

Sotaro Kikuchia,b, Dominika M. Borekc, Zbyszek Otwinowskic, Diana R. Tomchickc, and Hongtao Yua,b,1

aDepartment of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390; bHoward Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390; and cDepartment of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390

Edited by Brenda A. Schulman, St. Jude Children’s Research Hospital, Memphis, TN, and approved September 20, 2016 (received for review July 12, 2016)

The ring-shaped cohesin complex topologically entraps chromo- allowing cohesin to entrap DNA dynamically. The acetyltransfer- somes and regulates chromosome segregation, transcription, and ase Eco1 (Esco1/2 in vertebrates) acetylates the Smc3 ATPase head DNA repair. The cohesin core consists of the structural maintenance of and stabilizes cohesin on chromosomes (22–25), possibly through chromosomes 1 and 3 (Smc1–Smc3) heterodimeric ATPase, the kleisin attenuating the ATPase activity of cohesin (19). In vertebrates, subunit sister chromatid cohesion 1 (Scc1) that links the two ATPase Smc3 acetylation enables the binding of sororin to cohesin and Pds5 heads, and the Scc1-bound adaptor protein Scc3. The sister chromatid (26). Sororin alters the molecular interactions among cohesin, Pds5, – cohesion 2 and 4 (Scc2–Scc4) complex loads cohesin onto chromo- and Wapl, and inhibits the releasing activity of the Pds5 Wapl somes. Mutations of cohesin and its regulators, including Scc2, cause complex (26, 27). human developmental diseases termed cohesinopathy. Here, we re- Mutations of cohesin subunits and accessory proteins cause port the crystal structure of Chaetomium thermophilum (Ct) Scc2 and human developmental diseases termed cohesinopathy, including examine its interaction with cohesin. Similar to Scc3 and another Scc1- Cornelia de Lange syndrome (CdLS) (28, 29). About 60% of CdLS cases involve mutations of Scc2 [also called Nipped B-like interacting cohesin regulator, precocious dissociation of sisters 5 (Pds5), protein (NIPBL)] (30–32). Thus, Scc2 is crucial for normal hu- Scc2 consists mostly of helical repeats that fold into a hook-shaped man development. Although both Scc2 and Scc4 are required for structure. Scc2 binds to Scc1 through an N-terminal region of Scc1 that cohesin loading in vivo, Scc2 alone has been reported to enhance overlaps with its Pds5-binding region. Many cohesinopathy mutations the topological loading of recombinant fission yeast cohesin onto target conserved residues in Scc2 and diminish Ct Scc2 binding to Ct circular DNA in vitro (18). The crystal structure of Scc4 bound to – Scc1. Pds5 binding to Scc1 weakens the Scc2 Scc1 interaction. Our the N-terminal segment of Scc2 has been determined (33, 34). study defines a functionally important interaction between the kleisin Low-resolution structures of the Scc2–Scc4 complex have also subunit of cohesin and the hook of Scc2. Through competing with Scc2 been determined by EM (33, 34). Despite the progress, the for Scc1 binding, Pds5 might contribute to the release of Scc2 from mechanism by which the Scc2–Scc4 complex promotes cohesin loaded cohesin, freeing Scc2 for additional rounds of loading. loading is not understood. In particular, the Scc2–Scc4 complex has been reported to interact with multiple subunits of cohesin transcription | cohesinopathy | X-ray crystallography | cohesin loading | (18), but which interaction is functionally important is unclear. HEAT repeat To gain more insight into Scc2 function, we have determined the crystal structure of Scc2 from the thermophilic fungus, Chaetomium thermophilum ohesin consists of four core subunits: structural mainte- (Ct). Scc2 consists almost entirely of helical repeats Cnance of chromosomes 1 and 3 (Smc1 and Smc3), and sister that fold into a molecular hook. The overall structure of Scc2 is chromatid cohesion 1 and 3 (Scc1 and Scc3). (1–5). Smc1 and Smc3 similar to two other Scc1-binding HEAT repeat proteins, Scc3 and are related ATPases that heterodimerize through their hinge do- Pds5, which also adopt highly bent structures (8, 9, 27, 35, 36). Like Scc3 and Pds5, Scc2 interacts with Scc1. Mutation of conserved Scc2 mains. The C-terminal winged helix domain and the N-terminal helical domain (NHD) of Scc1 bind to the Smc1 ATPase head and a coiled-coil segment adjacent to the Smc3 ATPase head, respectively, Significance forming a tripartite ring (6, 7). Scc3 contains Huntingtin-elongation factor 3-protein phosphatase 2A-TOR1 (HEAT) repeats, binds to The ring-shaped cohesin traps chromosomes inside its ring the middle region of Scc1, and provides a landing pad for several and regulates chromosome segregation during mitosis and cohesin regulators (8, 9). Cohesin can topologically trap DNA inside transcription during interphase. The sister chromatid cohesion 2 its ring (10), thereby regulating many facets of chromosome biology. protein (Scc2) opens the cohesin ring and loads it onto chromo- During interphase, cohesin mediates the formation of chromosome somes. Mutations of cohesin subunits and regulators perturb loops that impact transcription (11). During S phase, cohesin physi- transcription and cause human developmental diseases called cally links replicated chromosomes to establish sister-chromatid cohesinopathy. Scc2 is the most frequently mutated cohesin reg- cohesion (12, 13), which is a prerequisite for faithful chromosome ulator in cohesinopathy. In this study, we report the crystal struc- segregation during mitosis. Finally, cohesin contributes to homology- ture of a fungal Scc2 protein, which represents a high-resolution directed repair of DNA breaks, in part, through holding the snapshot of the cohesin loader. We have identified a set of Scc2 sister chromatid in proximity of the breaks and presenting it as mutations in cohesinopathy that disrupt the binding of Scc2 to the the repair template (14). kleisin subunit of cohesin. Our results provide critical insight into For cohesin to accomplish these diverse tasks, its association cohesin loading and cohesinopathy. with chromosomes has to be tightly controlled both spatially and temporally. Cohesin dynamics on chromosomes are regulated Author contributions: S.K. and H.Y. designed research; S.K., D.M.B., Z.O., and D.R.T. per- by a set of accessory proteins, including the Scc2–Scc4 loading formed research; S.K., D.M.B., Z.O., D.R.T., and H.Y. analyzed data; and S.K., D.M.B., D.R.T., complex and the releasing complex comprising Pds5 and winged and H.Y. wrote the paper. apart-like protein (Wapl) (2, 4, 5). Scc2–Scc4 promotes the The authors declare no conflict of interest. loading of cohesin onto chromosomes in telophase and G1 (15). This article is a PNAS Direct Submission. Pds5–Wapl can release cohesin from chromosomes by opening Data deposition: The atomic coordinates and structure factors have been deposited in the the Smc3–Scc1 interface (6, 7, 16, 17). Both cohesin loading and Protein Data Bank, www.pdb.org (PDB ID code 5T8V). release by these complexes require the ATPase activity of cohesin 1To whom correspondence should be addressed. Email: [email protected]. (17–21). Therefore, the Scc2–Scc4 and Pds5–Wapl complexes This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. harness the energy of ATP hydrolysis to open the cohesin ring, 1073/pnas.1611333113/-/DCSupplemental.

12444–12449 | PNAS | November 1, 2016 | vol. 113 | no. 44 www.pnas.org/cgi/doi/10.1073/pnas.1611333113 Downloaded by guest on September 30, 2021 residues targeted by missense cohesinopathy mutations diminishes We coexpressed Ct Scc2 and Ct Scc4 in insect cells with the Scc1 binding, establishing the functional relevance of the observed baculoviral expression system and obtained the full-length Scc2– Scc2–Scc1 interaction. Pds5 competes with Scc2 for binding to Scc1. Scc4 complex. Consistent with previous reports (33, 34), Scc4 sta- Through competing with Scc2 for Scc1 binding, Pds5 may help to bilized the full-length Scc2 protein. In the absence of Scc4, Scc2 release Scc2 from the loaded and acetylated cohesin, allowing Scc2 underwent extensive proteolysis presumably at its N-terminal region. to perform additional rounds of loading. Because we could not crystalize the Scc2–Scc4 complex, we made a series of truncation mutants of Scc2 and obtained diffracting crystals Results of Scc2385–1,840, which contained the entire HEAT repeat domain of Crystal Structure of Ct Scc2. Scc2 contains an N-terminal disor- Scc2. The crystal structure of Scc2 was then determined to 2.8-Å dered region that binds to Scc4 and a C-terminal HEAT repeat resolution with the single-wavelength anomalous diffraction (SAD) domain (Fig. 1A). Structures of Scc4 in complex with Scc2N method (Table S1). reveal that Scc2N wraps around the tetratricopeptide repeat Scc2 consists of 24 HEAT repeats and a helical insert domain (TPR) domain of Scc4, forming extensive interactions (33, 34). located between repeats H7 and H8 (Fig. 1B and Fig. S1). Scc2 is Scc4 is required for cohesin loading in vivo likely through stabi- shaped like a handled hook, with H1–H7 forming the handle and lizing Scc2 and through targeting Scc2 to defined chromosome H8–H24 forming the hook. This highly bent architecture is also loci (33), but is dispensable for cohesin loading in an in vitro observed in the low-resolution EM structures of the budding and reconstituted assay (18). Thus, the C-terminal HEAT repeat fission yeast Scc2 proteins (33, 34). Thus, the curvature of Scc2 is domain of Scc2 is critical for cohesin loading. a defined, conserved feature of the cohesin loader. BIOCHEMISTRY

Fig. 1. Crystal structure of Ct Scc2. (A) Schematic drawing of the Ct Scc2–Scc4 complex. (B) Cartoon drawing of the crystal structure of Ct Scc2385–1,840 in two orientations. The helical insert domain (HID) is colored gray. The rest of the protein is colored blue. The positions of the HEAT repeats are indicated.All structure figures are made with PyMOL (www.pymol.org). (C) Cartoon drawing of the crystal structure of the human [Homo sapiens (Hs)] SA2–Scc1 complex, in the same orientation and scale as Scc2 on the left in B. SA2 and Scc1 are colored blue and magenta, respectively. (D) Cartoon drawing of the crystal structure of Lachancea thermotolerans (Lt) Pds5 with the bound Scc1 shown in sticks, in the same orientation and scale as Scc2 on the left in B.

Kikuchi et al. PNAS | November 1, 2016 | vol. 113 | no. 44 | 12445 Downloaded by guest on September 30, 2021 The highly bent structure of Scc2 is reminiscent of the structures of Scc3 [stromal antigen 1 or 2 (SA1/2) in vertebrates] and Pds5 (8, 9, 27, 35, 36) (Fig. 1 C and D). In fact, a search through the Dali server identified the structure of SA2 bound to Scc1 to be most closely related to the structure of SA2 bound to Scc2. Both Scc3 and Pds5 have highly bent structures, which are shaped like a dragon and a plier lever, respectively. In the cases of Scc2 and Scc3, the curvatures in their structures are formed by highly distorted HEAT repeats whose two helices deviate substantially from their antiparallel ar- rangement. In the case of human Pds5, the curvature is further sta- bilized by a tightly bound cofactor, inositol hexakisphosphate (27). Both Scc3 and Pds5 are Scc1-binding proteins, and they bind to different regions of Scc1 (8, 9, 27, 35–37). Scc3 binds to the middle region of Scc1, whereas Pds5 binds to a region immediately adjacent to the NHD. The structure of the human SA2–Scc1 complex reveals that a 70-residue fragment of Scc1 binds across a large segment of SA2, forming extensive interactions (8) (Fig. 1C). Mutagenesis results have shown that the interfaces between the three C-terminal helices of Scc1 (αB–αD) and the bottom of the U-shaped body of the SA2 dragon contribute much of the binding energy to the SA2–Scc1 interaction (8). Likewise, the structures of the fungal Pds5–Scc1 complexes reveal that Scc1 binds at the C-terminal jaw of the Pds5 plier lever (35, 36) (Fig. 1D).ThefactthatScc2sharesasimilar, curved shape with Scc3 and Pds5 raises the intriguing possibility that Scc2 might use its C-terminal hook to interact with Scc1.

Scc1 Binding by Scc2. To identify with which cohesin subunit Scc2 interacts, we obtained 35S-labeled Smc1–Smc3 heterodimer, Smc1, Smc3, Scc3, and Scc1 through in vitro translation, and tested their binding to GST-Scc2 (Fig. 2A). GST-Scc2 only bound to Scc1, but not to Smc1, Smc3, or Scc3. Deletion mutagenesis mapped the Scc2-binding region of Scc1 to an N-terminal region encompassing residues 126–230 (Fig. 2B and Fig. S2). As determined by isothermal titration calorimetry, purified recombinant Scc2385–1,840 and Scc1126–230 boundtoeachotherwithadissociationconstant (Kd) of 20.4 nM (Fig. 2 C and D). Thus, Scc2 can directly bind to the N-terminal region of Scc1 with high affinity. Ct Scc1 fragments that contain the NHD cannot be expressed in bacteria or insect Fig. 2. Ct Scc2 binds to the N-terminal region of Ct Scc1. (A) GST or GST- cells, preventing us from quantitatively measuring a potential con- Scc2385–1,840 proteins were immobilized on Glutathione Sepharose beads. Beads were incubated with the indicated 35S-labeled cohesin subunits. The tributionofthatdomaintoScc2binding. input and bound proteins were separated by SDS/PAGE gels, which were Our Scc1-binding results are consistent with an earlier report that stained with Coomassie (Bottom) and analyzed with a phosphorimager identified residues 145–152 in the fission yeast Rad21 (Scc1 ortholog) – (Top). (B) Domains and motifs of Ct Scc1. SCS, separase cleavage site; WHD, as a binding element of Scc2 Scc4 in binding reactions on peptide winged helix domain. (C) Coomassie-stained gel of purified recombinant arrays (18). The same study also reported numerous interactions Scc2 – and Scc1 – .(D) Isothermal titration calorimetry analysis of – 385 1,840 126 230 between Scc2 Scc4 and peptides from other cohesin subunits, in- the binding between Scc2385–1,840 and Scc1126–230. cluding Smc1, Smc3, and Scc3. In contrast, we did not observe de- tectable interactions between Scc2 and these other cohesin subunits. The underlying reasons for this discrepancy are unknown at present. exposed. Mutations of buried residues may disrupt the folding It is possible that some isolated peptides used in the previous study and stability of Scc2 locally or globally. are not properly folded and can develop nonphysiological interactions We then created a panel of Ct Scc2 mutants targeting residues in with Scc2–Scc4. In any case, our binding assays clearly establish an patches I and II, based on the corresponding CdLS missense muta- interaction between Scc2 and the N-terminal region of Scc1. We tions in human Scc2, and expressed them as GST fusion proteins. stress that our binding assays do not rule out possible, functional Strikingly, the vast majority (16 of 19) of these GST-Ct Scc2 mutants interactions between Scc2 and other cohesin subunits. were deficient in binding to the N-terminal fragment of Ct Scc1, albeit to varying degrees (Fig. 4 A and B). Our mutagenesis results establish Cohesinopathy Mutations Diminish the Scc2–Scc1 Interaction. The the specificity of the Scc2–Scc1 interaction, and suggest that the hook majority of CdLS cases are caused by mutations in one of the two of Scc2 might contact the N-terminal region of Scc1. The Scc2–Scc1 alleles of NIPBL (encoding human Scc2), including missense, interaction is likely conserved in humans and other species. A deficient nonsense, and deletions, suggesting that these Scc2 mutations Scc2–Scc1 interaction might be an underlying cause of cohesinopathy, are loss-of-function mutations and Scc2 haploinsufficiency is the although this hypothesis remains to be formally tested. underlying cause for disease (32). Human Scc2 is a much larger We again emphasize that not all mutated residues are likely to protein, with its C-terminal HEAT repeat domain sharing high make direct contact with Scc1. For example, A1367 and L1373 sequence similarity with Scc2 proteins from other species, in- are buried in a hydrophobic core of Scc2 and cannot form direct cluding Ct Scc2 (Fig. S1). Mapping of the Scc2 residues con- contact with Scc1. Instead, the A1367F and L1373P mutations served from yeast to man onto the structure of Ct Scc2 reveals may perturb the conformation of this segment of Scc2, indirectly that these conserved residues cluster around two patches (I and affecting the binding of Scc1. Likewise, R1053 and R1081 form II) in the hook of Scc2 (Fig. 3A). Interestingly, many of these favorable electrostatic interactions with D1084, whereas R1090 conserved residues are targeted by missense CdLS mutations in and R1120 contact D1123, along the spine of Scc2. These residues human Scc2 (Fig. 3 B and C and Fig. S3), suggesting that these may not form direct contact with Scc1 either. Their mutations may residues are functionally important. We note that not all residues alter the local conformation of HEAT repeats H9, H10, and corresponding to those residues mutated in CdLS are surface- H11, thus impacting Scc1 binding.

12446 | www.pnas.org/cgi/doi/10.1073/pnas.1611333113 Kikuchi et al. Downloaded by guest on September 30, 2021 Fig. 3. Cohesinopathy mutations target conserved residues in Scc2. (A) Surface drawing of Ct Scc2 with conserved residues colored red and nonconserved residues mutated in CdLS colored yellow. Zoomed-in views of the cartoon diagram of Scc2, with residues in patches I (B) and II (C) shown in sticks. The color scheme is the same as in A.

The fission yeast Scc2 has DNA-binding activity, and the Scc2– alternative model posits that Scc2 and DNA form a transient Scc4 complex stimulates the ATPase activity of the fission yeast complex with cohesin and stimulate ATP hydrolysis by cohesin, cohesin only in the presence of DNA (18). An inspection of the opening the Smc3–Scc1N interface to allow DNA entry (19). electrostatic potential of Ct Scc2 reveals no large, contiguous, Because the Smc3–Scc1N interface is adjacent to the ATPase positively charged surfaces that could serve as DNA-binding sites heads, it is easier to envision how ATP hydrolysis might open this (Fig. 4C). The largest positively charged patch is the conserved interface. On the other hand, this model cannot easily explain the patch I in the middle region of Scc2. We have shown, however, that mutations of several basic residues in this patch, including

R1081, R1090, K1091, R1092, and R1120, diminish Scc1 binding. BIOCHEMISTRY Because the N-terminal Scc2-binding region of Scc1 (residues 126– 230) is highly negatively charged and has a pI of 3.73, this positively chargedsiteislikelyinvolvedinScc1binding,asopposedtoDNA binding. The C-terminal region of Ct Scc2 (residues 1,888–1,946; not included in our crystallization construct) contains a long stretch of basic residues. It will be interesting to test whether this C-terminal basic region of Scc2 contributes to DNA binding by Scc2, although this region is poorly conserved.

Pds5 Competes with Scc2 for Binding to Scc1. Because Pds5 and Scc2 bind to overlapping N-terminal regions in Scc1 (Fig. 2B), we tested whether they competed for binding to a fragment of Scc1 (residues 51–230) that encompasses both binding regions. As expected, GST-Scc2 bound efficiently to this larger Scc1 fragment (Fig. 5A). Addition of increasing amounts of recombinant Ct Pds5 diminished the binding of Scc1 to GST-Scc2 in a dose- dependent manner. Ct Pds5 itself did not bind to GST-Scc2 beads. Thus, Pds5 and Scc2 indeed compete for binding to Scc1. They cannot simultaneously bind to Scc1 to form a ternary complex. Pds5 inhibits Scc2-dependent topological loading of the fission yeast cohesin onto DNA in a reconstituted cohesin-loading assay in vitro (19). Our demonstration of a direct competition between Pds5 and Scc2 for Scc1 binding provides a straightforward expla- nation for the inhibitory effect of Pds5 in cohesin loading. Taken together, these findings support a role of the Scc2–Scc1 interaction in cohesin loading. Unfortunately, we cannot test this possibility directly, because we have so far failed to reconstitute Scc2-dependent topological loading of recombinant Ct cohesin onto DNA. Discussion Mechanism of Scc2-Dependent Cohesin Loading. The cohesin ring can topologically entrap DNA. For DNA to enter this ring, one or more of the three interfaces or gates—the hinge interface, the Smc3–Scc1N interface, and the Smc1–Scc1C interface—need to be opened (38) (Fig. 5B). Artificial tethering of the Smc1 and Smc3 Fig. 4. Cohesinopathy mutations in Scc2 diminish Scc1 binding. (A) GST- Scc2385–1,840 wild type (WT) and the indicated mutant proteins were immo- hinges prevents cohesin loading in the budding yeast, whereas bilized on Glutathione Sepharose beads. Beads were incubated with 35S-labeled fusing Smc3 to the N terminus of Scc1 and tethering of the – Scc1126–230. The bound proteins were separated by SDS/PAGE gels, which were Smc1 Scc1C interface still allows functional cohesin loading (39). stained with Coomassie (Bottom) and analyzed with a phosphorimager (Top). These results suggest that cohesin loading involves the opening of (B) Quantification of the relative Scc1 intensities in A. Mean ± SD, n = 3in- the hinge interface. Cohesin loading requires the ATPase activity dependent experiments. Mutants with less than 50% of the WT activity are of cohesin. It is unclear how ATP hydrolysis by the ATPase heads shown as red bars. (C) Surface drawing of Scc2 colored by the electrostatic can open the hinge interface that is tens of nanometers away. An potential (blue, positive; red, negative; white, neutral) in two orientations.

Kikuchi et al. PNAS | November 1, 2016 | vol. 113 | no. 44 | 12447 Downloaded by guest on September 30, 2021 Smc3–Scc1N or Smc1–Scc1C interface. We do not know the biochemical activity of the handle of Scc2, which harbors a highly conserved motif (residues 656–671 in Ct Scc2), but suspect that this handle might contact subunit interfaces in intact cohesin to promote the opening of the cohesin ring. The competition between Pds5 and Scc2 for binding to Scc1 is intriguing and possibly relevant to cohesin regulation in vivo. Smc3 acetylation can occur at low levels outside S phase and is regulated by the ATPase activity of cohesin (40, 41). Although binding of Pds5 to cohesin does not require Smc3 acetylation in human cells, the Pds5-dependent binding of sororin to cohesin requires Smc3 acetylation (27). In the budding yeast, Pds5 protects cohesin from deacetylation and turns over more rap- idly on pericentric chromatin in cells lacking Eco1 (37, 42), suggesting that Pds5 might interact with acetylated Smc3 in that organism. Thus, Smc3 acetylation and subsequent binding of Pds5 (Pds5–sororin interaction in vertebrates) might actively promote the release of Scc2 from loaded cohesin, freeing it to perform additional rounds of cohesin loading (Fig. 5B). Con- versely, the soluble cohesin released from chromosomes by Pds5– Wapl interaction or separase is deacetylated by Hos1 in the budding yeast and by Hdac8 in human cells (43, 44). Inactiva- tion of Hdac8 in human cells traps cohesin in the acetylated and sororin-bound form (44), which likely also contains Pds5. Inacti- vation of Hos1 in the budding yeast also prevents Smc3 deacetylation during mitosis and proper cohesion in the ensuing S phase (43). In these situations, persistent Pds5 binding to acetylated cohesin is expected to prevent Scc2-dependent cohesin loading, contributing to the observed phenotypes. We speculate that cohesin deacetylation might be critical for releasing Pds5 (or Pds5–sororin interaction) from cohesin, enabling a fresh cycle of Scc2-dependent loading onto chromosomes.

Implications for Cohesinopathy. Mutations of one of the two Scc2 alleles account for the majority of known CdLS cases (32). The expression of the remaining wild-type Scc2 allele is variable in the cells of patients who have CdLS, and is inversely correlated with the severity of disease phenotypes (45). Patients with higher Scc2 expression show milder phenotypes. This correlation is observed even in patients with CdLS who have mutations in other Fig. 5. Pds5 competes with Scc2 for Scc1 binding. (A) GST or GST-Scc2385–1,840 cohesin subunits. Thus, proper levels of Scc2 are particularly proteins were immobilized on Glutathione Sepharose beads. Beads were incu- 35 important for human development. Because the cells of pa- bated with S-labeled Scc151–230 in the absence or presence of increasing amounts of Pds5. Input and bound proteins were separated by SDS/PAGE gels, which were tients with CdLS have no gross defects in cohesin loading or stained with Coomassie (Bottom) and analyzed with a phosphorimager (Top). The sister-chromatid cohesion, the prevailing model is that Scc2 relative binding intensities of Scc1 are quantified and indicated below the auto- mutations cause cohesinopathy by perturbing the transcription radiograph. (B) Model for Scc2-dependent cohesin loading onto DNA, highlight- of developmental genes (28, 29). Although it is possible that ing the importance of the Scc2–Scc1 interaction. The Scc2–cohesin complex low levels of Scc2 impair cohesin loading at specific genes, it is (mediated, in part, by the Scc2–Scc1 interaction) might be the functional entity equally possible that the cohesin–Scc2 complex is the functional that promotes transcription. The competition between Pds5 and Scc2 for Scc1 entity that regulates transcription. In support of this hypothesis, binding might trigger the release of Scc2 from the loaded and acetylated cohesin. in mouse embryonic stem (ES) cells, cohesin, Scc2, and the mediator co-occupy the enhancer and promoter regions of actively transcribed genes and activate transcription through the finding that the Smc3–Scc1 fusion protein supports functional formation of enhancer-promoter DNA loops (46). We have cohesion. Thus, the identity of the DNA entry gate(s) of cohesin shown that many Scc2 missense mutations in CdLS specifically remains to be established. disrupt the Scc2–Scc1 interaction. In the future, it will be in- In this study, we have shown that Scc2 interacts with the teresting to test whether these Scc2 mutations disrupt the N-terminal region of Scc1, but not other cohesin subunits. Sev- cohesin–Scc2 mediator complex and the formation of the eral lines of evidence suggest that the Scc2–Scc1 interaction is enhancer-promoter loops in human ES cells or induced plu- specific and might be functionally important for cohesin loading. ripotent stem cells. These experiments will provide fresh in- First, Scc2 has an overall fold and shape that are similar to two sight into the molecular basis of cohesinopathy. other Scc1-binding proteins, Scc3 and Pds5. Second, cohesinopathy mutations targeting conserved residues in Scc2 disrupt Materials and Methods – the Scc2 Scc1 interaction. Finally, Pds5, which is known to block Protein Expression and Purification. The cDNAs of Ct Scc2 and Pds5 were – Scc2-dependent cohesin loading in vitro, blocks the Scc2 Scc1 synthesized and subcloned into the pFastBacHT vector. Ct Scc2 (native and interaction. We speculate that this Scc2–Scc1 interaction, along selenomethionine-labeled) and Pds5 proteins were then expressed in insect with DNA-bridged interactions between Scc2 and other cohesin cells and purified with standard procedures. Details are provided in SI Ma- subunits, might stabilize the ATP-bound, heads-engaged confor- terials and Methods. mation of cohesin, thereby promoting ATP hydrolysis and cohesin B loading through the yet-to-be-determined entry gate(s) (Fig. 5 ). Crystallization of Ct Scc2. Ct Scc2385–1,840 crystals grew within a few days af- Alternatively, Scc2 rigidifies the flexible segments of Scc1 and ter the mixing of the protein solution (5 mg/mL) with the reservoir solu- allows the ATPase-driven head disengagement to disrupt the tion containing 150 mM sodium citrate tribasic dihydrate (pH 5.2), 5 mM

12448 | www.pnas.org/cgi/doi/10.1073/pnas.1611333113 Kikuchi et al. Downloaded by guest on September 30, 2021 tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), and 9% (wt/vol) PEG ACKNOWLEDGMENTS. We thank Ningyan Cheng, Zhuqing Ouyang, and 6000. Details are provided in SI Materials and Methods. Zhonghui Lin for their participation in the project. They performed experiments that yielded negative results. We thank Shih-Chia Tso for assistance Data Collection and Structure Determination. X-ray diffraction data were col- with isothermal titration calorimetry and Kim Nasmyth for communicating lected at an Advanced Photon Source beamline. HKL-3000 was used to process both results prior to publication and helpful discussions. Use of Argonne Na- the L-selenomethionine and native diffraction datasets (47). Phases were obtained tional Laboratory Structural Biology Center beamlines at the Advanced through a SAD experiment. Details are provided in SI Materials and Methods.Data Photon Source was supported by the US Department of Energy under Con- processing, phasing, and model refinement statistics are provided in Table S1. tract DE-AC02-06CH11357. This study is supported by grants from the Can- cer Prevention and Research Institute of Texas (Grants RP110465-P3 and Protein-Binding Assays. GST pull-down assays and isothermal titration calo- RP120717-P2 to H.Y.), the Welch Foundation (Grant I-1441 to H.Y.), and rimetry were used to analyze the binding between Scc2 and cohesin subunits. the National Institutes of Health (Grants R01GM053163 and R01GM117080 Details are provided in SI Materials and Methods. to Z.O.). H.Y. is an Investigator with the Howard Hughes Medical Institute.

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