Crystal structure of the cohesin loader Scc2 and insightinto cohesinopathySotaro 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 TexasSouthwestern 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-somes and regulates chromosome segregation, transcription, andDNA repair. The cohesin core consists of the structural maintenance ofchromosomes 1 and 3 (Smc1–Smc3) heterodimeric ATPase, the kleisinsubunit sister chromatid cohesion 1 (Scc1) that links the two ATPaseheads, and the Scc1-bound adaptor protein Scc3. The sister chromatidcohesion 2 and 4 (Scc2–Scc4) complex loads cohesin onto chromo-somes. Mutations of cohesin and its regulators, including Scc2, causehuman developmental diseases termed cohesinopathy. Here, we re-port the crystal structure of Chaetomium thermophilum (Ct) Scc2 andexamine its interaction with cohesin. Similar to Scc3 and another Scc1-interacting cohesin regulator, precocious dissociation of sisters 5 (Pds5),Scc2 consists mostly of helical repeats that fold into a hook-shapedstructure. Scc2 binds to Scc1 through an N-terminal region of Scc1 thatoverlaps with its Pds5-binding region. Many cohesinopathy mutationstarget conserved residues in Scc2 and diminish Ct Scc2 binding to CtScc1. Pds5 binding to Scc1 weakens the Scc2–Scc1 interaction. Ourstudy defines a functionally important interaction between the kleisinsubunit of cohesin and the hook of Scc2. Through competingwith Scc2for Scc1 binding, Pds5 might contribute to the release of Scc2 fromloaded cohesin, freeing Scc2 for additional rounds of loading.

transcription | cohesinopathy | X-ray crystallography | cohesin loading |HEAT repeat

Cohesin consists of four core subunits: structural mainte-nance of chromosomes 1 and 3 (Smc1 and Smc3), and sister

chromatid cohesion 1 and 3 (Scc1 and Scc3). (1–5). Smc1 and Smc3are related ATPases that heterodimerize through their hinge do-mains. The C-terminal winged helix domain and the N-terminalhelical domain (NHD) of Scc1 bind to the Smc1 ATPase head and acoiled-coil segment adjacent to the Smc3 ATPase head, respectively,forming a tripartite ring (6, 7). Scc3 contains Huntingtin-elongationfactor 3-protein phosphatase 2A-TOR1 (HEAT) repeats, binds tothe middle region of Scc1, and provides a landing pad for severalcohesin regulators (8, 9). Cohesin can topologically trap DNA insideits ring (10), thereby regulating many facets of chromosome biology.During interphase, cohesin mediates the formation of chromosomeloops that impact transcription (11). During S phase, cohesin physi-cally links replicated chromosomes to establish sister-chromatidcohesion (12, 13), which is a prerequisite for faithful chromosomesegregation during mitosis. Finally, cohesin contributes to homology-directed repair of DNA breaks, in part, through holding thesister chromatid in proximity of the breaks and presenting it asthe repair template (14).For cohesin to accomplish these diverse tasks, its association

with chromosomes has to be tightly controlled both spatially andtemporally. Cohesin dynamics on chromosomes are regulatedby a set of accessory proteins, including the Scc2–Scc4 loadingcomplex and the releasing complex comprising Pds5 and wingedapart-like protein (Wapl) (2, 4, 5). Scc2–Scc4 promotes theloading of cohesin onto chromosomes in telophase and G1 (15).Pds5–Wapl can release cohesin from chromosomes by openingthe Smc3–Scc1 interface (6, 7, 16, 17). Both cohesin loading andrelease by these complexes require the ATPase activity of cohesin(17–21). Therefore, the Scc2–Scc4 and Pds5–Wapl complexesharness the energy of ATP hydrolysis to open the cohesin ring,

allowing cohesin to entrap DNA dynamically. The acetyltransfer-ase Eco1 (Esco1/2 in vertebrates) acetylates the Smc3 ATPase headand stabilizes cohesin on chromosomes (22–25), possibly throughattenuating the ATPase activity of cohesin (19). In vertebrates,Smc3 acetylation enables the binding of sororin to cohesin and Pds5(26). Sororin alters the molecular interactions among cohesin, Pds5,and Wapl, and inhibits the releasing activity of the Pds5–Waplcomplex (26, 27).Mutations of cohesin subunits and accessory proteins cause

human developmental diseases termed cohesinopathy, includingCornelia de Lange syndrome (CdLS) (28, 29). About 60% ofCdLS cases involve mutations of Scc2 [also called Nipped B-likeprotein (NIPBL)] (30–32). Thus, Scc2 is crucial for normal hu-man development. Although both Scc2 and Scc4 are required forcohesin loading in vivo, Scc2 alone has been reported to enhancethe topological loading of recombinant fission yeast cohesin ontocircular DNA in vitro (18). The crystal structure of Scc4 bound tothe N-terminal segment of Scc2 has been determined (33, 34).Low-resolution structures of the Scc2–Scc4 complex have alsobeen determined by EM (33, 34). Despite the progress, themechanism by which the Scc2–Scc4 complex promotes cohesinloading is not understood. In particular, the Scc2–Scc4 complexhas been reported to interact with multiple subunits of cohesin(18), but which interaction is functionally important is unclear.To gain more insight into Scc2 function, we have determined the

crystal structure of Scc2 from the thermophilic fungus, Chaetomiumthermophilum (Ct). Scc2 consists almost entirely of helical repeatsthat fold into a molecular hook. The overall structure of Scc2 issimilar to two other Scc1-binding HEAT repeat proteins, Scc3 andPds5, which also adopt highly bent structures (8, 9, 27, 35, 36). LikeScc3 and Pds5, Scc2 interacts with Scc1. Mutation of conserved Scc2

Significance

The ring-shaped cohesin traps chromosomes inside its ringand regulates chromosome segregation during mitosis andtranscription during interphase. The sister chromatid cohesion 2protein (Scc2) opens the cohesin ring and loads it onto chromo-somes. Mutations of cohesin subunits and regulators perturbtranscription and cause human developmental diseases calledcohesinopathy. Scc2 is the most frequently mutated cohesin reg-ulator in cohesinopathy. In this study, we report the crystal struc-ture of a fungal Scc2 protein, which represents a high-resolutionsnapshot of the cohesin loader. We have identified a set of Scc2mutations in cohesinopathy that disrupt the binding of Scc2 to thekleisin subunit of cohesin. Our results provide critical insight intocohesin loading and cohesinopathy.

Author contributions: S.K. and H.Y. designed research; S.K., D.M.B., Z.O., and D.R.T. per-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.,and H.Y. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org (PDB ID code 5T8V).1To whom correspondence should be addressed. Email: hongtao.yu@utsouthwestern.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1611333113/-/DCSupplemental.

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residues targeted by missense cohesinopathy mutations diminishesScc1 binding, establishing the functional relevance of the observedScc2–Scc1 interaction. Pds5 competes with Scc2 for binding to Scc1.Through competing with Scc2 for Scc1 binding, Pds5 may help torelease Scc2 from the loaded and acetylated cohesin, allowing Scc2to perform additional rounds of loading.

ResultsCrystal Structure of Ct Scc2. Scc2 contains an N-terminal disor-dered region that binds to Scc4 and a C-terminal HEAT repeatdomain (Fig. 1A). Structures of Scc4 in complex with Scc2Nreveal that Scc2N wraps around the tetratricopeptide repeat(TPR) domain of Scc4, forming extensive interactions (33, 34).Scc4 is required for cohesin loading in vivo likely through stabi-lizing Scc2 and through targeting Scc2 to defined chromosomeloci (33), but is dispensable for cohesin loading in an in vitroreconstituted assay (18). Thus, the C-terminal HEAT repeatdomain of Scc2 is critical for cohesin loading.

We coexpressed Ct Scc2 and Ct Scc4 in insect cells with thebaculoviral expression system and obtained the full-length Scc2–Scc4 complex. Consistent with previous reports (33, 34), Scc4 sta-bilized the full-length Scc2 protein. In the absence of Scc4, Scc2underwent extensive proteolysis presumably at its N-terminal region.Because we could not crystalize the Scc2–Scc4 complex, we made aseries of truncation mutants of Scc2 and obtained diffracting crystalsof Scc2385–1,840, which contained the entire HEAT repeat domain ofScc2. The crystal structure of Scc2 was then determined to 2.8-Åresolution with the single-wavelength anomalous diffraction (SAD)method (Table S1).Scc2 consists of 24 HEAT repeats and a helical insert domain

located between repeats H7 and H8 (Fig. 1B and Fig. S1). Scc2 isshaped like a handled hook, with H1–H7 forming the handle andH8–H24 forming the hook. This highly bent architecture is alsoobserved in the low-resolution EM structures of the budding andfission yeast Scc2 proteins (33, 34). Thus, the curvature of Scc2 isa defined, conserved feature of the cohesin loader.

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 twoorientations. The helical insert domain (HID) is colored gray. The rest of the protein is colored blue. The positions of the HEAT repeats are indicated. Allstructure 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 thesame 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 ofLachancea thermotolerans (Lt) Pds5 with the bound Scc1 shown in sticks, in the same orientation and scale as Scc2 on the left in B.

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The highly bent structure of Scc2 is reminiscent of the structures ofScc3 [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 serveridentified the structure of SA2 bound to Scc1 to be most closelyrelated to the structure of SA2 bound to Scc2. Both Scc3 and Pds5have highly bent structures, which are shaped like a dragon and aplier lever, respectively. In the cases of Scc2 and Scc3, the curvaturesin their structures are formed by highly distorted HEAT repeatswhose 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 middleregion of Scc1, whereas Pds5 binds to a region immediately adjacentto the NHD. The structure of the human SA2–Scc1 complex revealsthat a 70-residue fragment of Scc1 binds across a large segment ofSA2, forming extensive interactions (8) (Fig. 1C). Mutagenesis re-sults have shown that the interfaces between the three C-terminalhelices of Scc1 (αB–αD) and the bottom of the U-shaped body of theSA2 dragon contribute much of the binding energy to the SA2–Scc1interaction (8). Likewise, the structures of the fungal Pds5–Scc1complexes reveal that Scc1 binds at the C-terminal jaw of the Pds5plier lever (35, 36) (Fig. 1D). The fact that Scc2 shares a similar,curved shape with Scc3 and Pds5 raises the intriguing possibility thatScc2 might use its C-terminal hook to interact with Scc1.

Scc1 Binding by Scc2. To identify with which cohesin subunit Scc2interacts, we obtained 35S-labeled Smc1–Smc3 heterodimer, Smc1,Smc3, Scc3, and Scc1 through in vitro translation, and tested theirbinding to GST-Scc2 (Fig. 2A). GST-Scc2 only bound to Scc1, butnot to Smc1, Smc3, or Scc3. Deletion mutagenesis mapped theScc2-binding region of Scc1 to an N-terminal region encompassingresidues 126–230 (Fig. 2B and Fig. S2). As determined by iso-thermal titration calorimetry, purified recombinant Scc2385–1,840and Scc1126–230 bound to each other with a dissociation constant(Kd) of 20.4 nM (Fig. 2 C and D). Thus, Scc2 can directly bind tothe N-terminal region of Scc1 with high affinity. Ct Scc1 fragmentsthat contain the NHD cannot be expressed in bacteria or insectcells, preventing us from quantitatively measuring a potential con-tribution of that domain to Scc2 binding.Our Scc1-binding results are consistent with an earlier report that

identified residues 145–152 in the fission yeast Rad21 (Scc1 ortholog)as a binding element of Scc2–Scc4 in binding reactions on peptidearrays (18). The same study also reported numerous interactionsbetween Scc2–Scc4 and peptides from other cohesin subunits, in-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.It is possible that some isolated peptides used in the previous studyare not properly folded and can develop nonphysiological interactionswith Scc2–Scc4. In any case, our binding assays clearly establish aninteraction between Scc2 and the N-terminal region of Scc1. Westress that our binding assays do not rule out possible, functionalinteractions between Scc2 and other cohesin subunits.

Cohesinopathy Mutations Diminish the Scc2–Scc1 Interaction. Themajority of CdLS cases are caused by mutations in one of the twoalleles of NIPBL (encoding human Scc2), including missense,nonsense, and deletions, suggesting that these Scc2 mutationsare loss-of-function mutations and Scc2 haploinsufficiency is theunderlying cause for disease (32). Human Scc2 is a much largerprotein, with its C-terminal HEAT repeat domain sharing highsequence similarity with Scc2 proteins from other species, in-cluding Ct Scc2 (Fig. S1). Mapping of the Scc2 residues con-served from yeast to man onto the structure of Ct Scc2 revealsthat these conserved residues cluster around two patches (I andII) in the hook of Scc2 (Fig. 3A). Interestingly, many of theseconserved residues are targeted by missense CdLS mutations inhuman Scc2 (Fig. 3 B and C and Fig. S3), suggesting that theseresidues are functionally important. We note that not all residuescorresponding to those residues mutated in CdLS are surface-

exposed. Mutations of buried residues may disrupt the foldingand stability of Scc2 locally or globally.We then created a panel of Ct Scc2 mutants targeting residues in

patches I and II, based on the corresponding CdLS missense muta-tions in human Scc2, and expressed them as GST fusion proteins.Strikingly, the vast majority (16 of 19) of these GST-Ct Scc2 mutantswere deficient in binding to the N-terminal fragment of Ct Scc1, albeitto varying degrees (Fig. 4 A and B). Our mutagenesis results establishthe specificity of the Scc2–Scc1 interaction, and suggest that the hookof Scc2 might contact the N-terminal region of Scc1. The Scc2–Scc1interaction is likely conserved in humans and other species. A deficientScc2–Scc1 interaction might be an underlying cause of cohesinopathy,although this hypothesis remains to be formally tested.We again emphasize that not all mutated residues are likely to

make direct contact with Scc1. For example, A1367 and L1373are buried in a hydrophobic core of Scc2 and cannot form directcontact with Scc1. Instead, the A1367F and L1373P mutationsmay perturb the conformation of this segment of Scc2, indirectlyaffecting the binding of Scc1. Likewise, R1053 and R1081 formfavorable electrostatic interactions with D1084, whereas R1090and R1120 contact D1123, along the spine of Scc2. These residuesmay not form direct contact with Scc1 either. Their mutations mayalter the local conformation of HEAT repeats H9, H10, andH11, thus impacting Scc1 binding.

Fig. 2. Ct Scc2 binds to the N-terminal region of Ct Scc1. (A) GST or GST-Scc2385–1,840 proteins were immobilized on Glutathione Sepharose beads.Beads were incubated with the indicated 35S-labeled cohesin subunits. Theinput and bound proteins were separated by SDS/PAGE gels, which werestained with Coomassie (Bottom) and analyzed with a phosphorimager(Top). (B) Domains and motifs of Ct Scc1. SCS, separase cleavage site; WHD,winged helix domain. (C) Coomassie-stained gel of purified recombinantScc2385–1,840 and Scc1126–230. (D) Isothermal titration calorimetry analysis ofthe binding between Scc2385–1,840 and Scc1126–230.

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The fission yeast Scc2 has DNA-binding activity, and the Scc2–Scc4 complex stimulates the ATPase activity of the fission yeastcohesin only in the presence of DNA (18). An inspection of theelectrostatic potential of Ct Scc2 reveals no large, contiguous,positively charged surfaces that could serve as DNA-binding sites(Fig. 4C). The largest positively charged patch is the conservedpatch I in the middle region of Scc2. We have shown, however,that mutations of several basic residues in this patch, includingR1081, R1090, K1091, R1092, and R1120, diminish Scc1 binding.Because the N-terminal Scc2-binding region of Scc1 (residues 126–230) is highly negatively charged and has a pI of 3.73, this positivelycharged site is likely involved in Scc1 binding, as opposed to DNAbinding. The C-terminal region of Ct Scc2 (residues 1,888–1,946;not included in our crystallization construct) contains a long stretchof basic residues. It will be interesting to test whether thisC-terminal basic region of Scc2 contributes to DNA binding byScc2, although this region is poorly conserved.

Pds5 Competes with Scc2 for Binding to Scc1. Because Pds5 andScc2 bind to overlapping N-terminal regions in Scc1 (Fig. 2B), wetested whether they competed for binding to a fragment of Scc1(residues 51–230) that encompasses both binding regions. Asexpected, GST-Scc2 bound efficiently to this larger Scc1 frag-ment (Fig. 5A). Addition of increasing amounts of recombinantCt 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. Theycannot 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 assayin vitro (19). Our demonstration of a direct competition betweenPds5 and Scc2 for Scc1 binding provides a straightforward expla-nation for the inhibitory effect of Pds5 in cohesin loading. Takentogether, these findings support a role of the Scc2–Scc1 interactionin cohesin loading. Unfortunately, we cannot test this possibility di-rectly, because we have so far failed to reconstitute Scc2-dependenttopological loading of recombinant Ct cohesin onto DNA.

DiscussionMechanism of Scc2-Dependent Cohesin Loading. The cohesin ringcan topologically entrap DNA. For DNA to enter this ring, oneor more of the three interfaces or gates—the hinge interface, theSmc3–Scc1N interface, and the Smc1–Scc1C interface—need tobe opened (38) (Fig. 5B). Artificial tethering of the Smc1 and Smc3hinges prevents cohesin loading in the budding yeast, whereasfusing Smc3 to the N terminus of Scc1 and tethering of theSmc1–Scc1C interface still allows functional cohesin loading (39).These results suggest that cohesin loading involves the opening ofthe hinge interface. Cohesin loading requires the ATPase activityof cohesin. It is unclear how ATP hydrolysis by the ATPase headscan open the hinge interface that is tens of nanometers away. An

alternative model posits that Scc2 and DNA form a transientcomplex with cohesin and stimulate ATP hydrolysis by cohesin,opening the Smc3–Scc1N interface to allow DNA entry (19).Because the Smc3–Scc1N interface is adjacent to the ATPaseheads, it is easier to envision how ATP hydrolysis might open thisinterface. On the other hand, this model cannot easily explain the

Fig. 3. Cohesinopathy mutations target conserved residues in Scc2. (A) Surface drawing of Ct Scc2 with conserved residues colored red and nonconservedresidues 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 colorscheme is the same as in A.

Fig. 4. Cohesinopathy mutations in Scc2 diminish Scc1 binding. (A) GST-Scc2385–1,840 wild type (WT) and the indicated mutant proteins were immo-bilized on Glutathione Sepharose beads. Beads were incubated with 35S-labeledScc1126–230. The bound proteins were separated by SDS/PAGE gels, which werestained with Coomassie (Bottom) and analyzed with a phosphorimager (Top).(B) Quantification of the relative Scc1 intensities in A. Mean ± SD, n = 3 in-dependent experiments. Mutants with less than 50% of the WT activity areshown as red bars. (C) Surface drawing of Scc2 colored by the electrostaticpotential (blue, positive; red, negative; white, neutral) in two orientations.

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finding that the Smc3–Scc1 fusion protein supports functionalcohesion. Thus, the identity of the DNA entry gate(s) of cohesinremains to be established.In this study, we have shown that Scc2 interacts with the

N-terminal region of Scc1, but not other cohesin subunits. Sev-eral lines of evidence suggest that the Scc2–Scc1 interaction isspecific and might be functionally important for cohesin loading.First, Scc2 has an overall fold and shape that are similar to twoother Scc1-binding proteins, Scc3 and Pds5. Second, cohesin-opathy mutations targeting conserved residues in Scc2 disruptthe Scc2–Scc1 interaction. Finally, Pds5, which is known to blockScc2-dependent cohesin loading in vitro, blocks the Scc2–Scc1interaction. We speculate that this Scc2–Scc1 interaction, alongwith DNA-bridged interactions between Scc2 and other cohesinsubunits, might stabilize the ATP-bound, heads-engaged confor-mation of cohesin, thereby promoting ATP hydrolysis and cohesinloading through the yet-to-be-determined entry gate(s) (Fig. 5B).Alternatively, Scc2 rigidifies the flexible segments of Scc1 andallows the ATPase-driven head disengagement to disrupt the

Smc3–Scc1N or Smc1–Scc1C interface. We do not know thebiochemical activity of the handle of Scc2, which harbors a highlyconserved motif (residues 656–671 in Ct Scc2), but suspect thatthis handle might contact subunit interfaces in intact cohesin topromote 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 isregulated by the ATPase activity of cohesin (40, 41). Althoughbinding of Pds5 to cohesin does not require Smc3 acetylation inhuman cells, the Pds5-dependent binding of sororin to cohesinrequires Smc3 acetylation (27). In the budding yeast, Pds5protects 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 thatorganism. Thus, Smc3 acetylation and subsequent binding ofPds5 (Pds5–sororin interaction in vertebrates) might activelypromote the release of Scc2 from loaded cohesin, freeing it toperform 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 thebudding yeast and by Hdac8 in human cells (43, 44). Inactiva-tion of Hdac8 in human cells traps cohesin in the acetylated andsororin-bound form (44), which likely also contains Pds5. Inacti-vation of Hos1 in the budding yeast also prevents Smc3 deacety-lation during mitosis and proper cohesion in the ensuing S phase(43). In these situations, persistent Pds5 binding to acetylatedcohesin is expected to prevent Scc2-dependent cohesin loading,contributing to the observed phenotypes. We speculate that cohesindeacetylation might be critical for releasing Pds5 (or Pds5–sororininteraction) from cohesin, enabling a fresh cycle of Scc2-dependentloading onto chromosomes.

Implications for Cohesinopathy. Mutations of one of the two Scc2alleles account for the majority of known CdLS cases (32). Theexpression of the remaining wild-type Scc2 allele is variable in thecells of patients who have CdLS, and is inversely correlated withthe severity of disease phenotypes (45). Patients with higher Scc2expression show milder phenotypes. This correlation is ob-served even in patients with CdLS who have mutations in othercohesin subunits. Thus, proper levels of Scc2 are particularlyimportant for human development. Because the cells of pa-tients with CdLS have no gross defects in cohesin loading orsister-chromatid cohesion, the prevailing model is that Scc2mutations cause cohesinopathy by perturbing the transcriptionof developmental genes (28, 29). Although it is possible thatlow levels of Scc2 impair cohesin loading at specific genes, it isequally possible that the cohesin–Scc2 complex is the functionalentity that regulates transcription. In support of this hypothesis,in mouse embryonic stem (ES) cells, cohesin, Scc2, and themediator co-occupy the enhancer and promoter regions of ac-tively transcribed genes and activate transcription through theformation of enhancer-promoter DNA loops (46). We haveshown that many Scc2 missense mutations in CdLS specificallydisrupt the Scc2–Scc1 interaction. In the future, it will be in-teresting to test whether these Scc2 mutations disrupt thecohesin–Scc2 mediator complex and the formation of theenhancer-promoter loops in human ES cells or induced plu-ripotent stem cells. These experiments will provide fresh in-sight into the molecular basis of cohesinopathy.

Materials and MethodsProtein Expression and Purification. The cDNAs of Ct Scc2 and Pds5 weresynthesized and subcloned into the pFastBacHT vector. Ct Scc2 (native andselenomethionine-labeled) and Pds5 proteins were then expressed in insectcells and purified with standard procedures. Details are provided in SI Ma-terials and Methods.

Crystallization of Ct Scc2. Ct Scc2385–1,840 crystals grew within a few days af-ter the mixing of the protein solution (5 mg/mL) with the reservoir solu-tion containing 150 mM sodium citrate tribasic dihydrate (pH 5.2), 5 mM

Fig. 5. Pds5 competes with Scc2 for Scc1 binding. (A) GST or GST-Scc2385–1,840proteins were immobilized on Glutathione Sepharose beads. Beads were incu-batedwith 35S-labeled Scc151–230 in the absence or presence of increasing amountsof Pds5. Input and bound proteins were separated by SDS/PAGE gels, which werestained with Coomassie (Bottom) and analyzed with a phosphorimager (Top). Therelative binding intensities of Scc1 are quantified and indicated below the auto-radiograph. (B) Model for Scc2-dependent cohesin loading onto DNA, highlight-ing the importance of the Scc2–Scc1 interaction. The Scc2–cohesin complex(mediated, in part, by the Scc2–Scc1 interaction) might be the functional entitythat promotes transcription. The competition between Pds5 and Scc2 for Scc1binding might trigger the release of Scc2 from the loaded and acetylated cohesin.

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tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), and 9% (wt/vol) PEG6000. Details are provided in SI Materials and Methods.

Data Collection and Structure Determination. X-ray diffraction data were col-lectedat anAdvancedPhotonSourcebeamline.HKL-3000wasused toprocessboththe L-selenomethionine and native diffraction datasets (47). Phases were obtainedthrough a SAD experiment. Details are provided in SI Materials andMethods. Dataprocessing, phasing, and model refinement statistics are provided in Table S1.

Protein-Binding Assays. GST pull-down assays and isothermal titration calo-rimetry were used to analyze the binding between Scc2 and cohesin subunits.Details are provided in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank Ningyan Cheng, Zhuqing Ouyang, andZhonghui Lin for their participation in the project. They performed exper-iments that yielded negative results. We thank Shih-Chia Tso for assistancewith isothermal titration calorimetry and Kim Nasmyth for communicatingresults prior to publication and helpful discussions. Use of Argonne Na-tional Laboratory Structural Biology Center beamlines at the AdvancedPhoton Source was supported by the US Department of Energy under Con-tract DE-AC02-06CH11357. This study is supported by grants from the Can-cer Prevention and Research Institute of Texas (Grants RP110465-P3 andRP120717-P2 to H.Y.), the Welch Foundation (Grant I-1441 to H.Y.), andthe National Institutes of Health (Grants R01GM053163 and R01GM117080to Z.O.). H.Y. is an Investigator with the Howard Hughes Medical Institute.

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