The FACT Spt16 ‘‘peptidase’’ domain is a –H4 binding module

Tobias Stuwe, Michael Hothorn*, Erwan Lejeune, Vladimir Rybin, Miriam Bortfeld, Klaus Scheffzek, and Andreas G. Ladurner†

European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany

Edited by Alan R. Fersht, University of Cambridge, Cambridge, United Kingdom, and approved March 27, 2008 (received for review December 28, 2007) The FACT complex is a conserved cofactor for RNA polymerase II scription and replication by removing one H2A–H2B dimer from elongation through . FACT bears histone nucleosomes, thus relieving the barrier to polymerase progres- activity and contributes to integrity. However, the sion. After Pol II passage, FACT may restore the proper molecular mechanisms behind FACT function remain elusive. Here chromatin state (14, 20). we report biochemical, structural, and mutational analyses that There is little mechanistic insight into how FACT may be able identify the peptidase homology domain of the Schizosaccharo- to perform its chaperoning functions. In particular, it is unclear myces pombe FACT large subunit Spt16 (Spt16-N) as a binding how it interacts with histones. To start dissecting the structure module for histones H3 and H4. The 2.1-Å crystal structure of and function of the essential Spt16 subunit of FACT, we sought Spt16-N reveals an aminopeptidase P fold whose enzymatic activ- to identify the molecular functions inherent to this Ͼ100-kDa ity has been lost. Instead, the highly conserved fold directly binds multidomain protein. Structural information on FACT exists for histones H3–H4 through a tight interaction with their globular core the HMG-like module Nhp6A from S. cerevisiae (23), which domains, as well as with their N-terminal tails. Mutations within a binds DNA, and for the middle domain of S. cerevisiae Pob3 (24), conserved surface pocket in Spt16-N or posttranslational modifi- which resembles a double PH-like fold and interacts with cation of the tail reduce interaction in vitro, whereas the replication protein A. The highly conserved Spt16 consists of globular domains of H3–H4 and the H3 tail bind distinct Spt16-N three domains: an acidic segment at the C terminus (10) that is surfaces. Our analysis suggests that the N-terminal domain of required for binding histones H2A–H2B in vitro (20) and re- Spt16 may add to the known H2A–H2B chaperone activity of FACT sembles the acidic domains of Nap1, nucleolin, and Asf1 (25–27); by including a H3–H4 tail and H3–H4 core binding function medi- two central domains, one of which interacts with Pob3 (21, 24); ated by the N terminus of Spt16. We suggest that these interactions and an N-terminal region of Ϸ450 residues (Spt16-N) showing may aid FACT-mediated reorganization events. homology with aminopeptidases (28). We were intrigued by the presence of a peptidase-like domain within this essential and histone chaperone ͉ histone modifications ͉ protein evolution ͉ conserved histone chaperone [supporting information (SI) Fig. site-directed mutagenesis ͉ transcription S1]. We have characterized biochemical functions of Spt16-N by combining structural approaches with quantitative binding stud- ucleosomes create a natural barrier to RNA polymerase II ies and site-directed mutagenesis. N(Pol II) progression. Transcription of histone-wrapped DNA thus requires factors that promote nucleosome remodel- Results ing, such as the histone chaperone FACT (facilitates chromatin A Catalytically Inactive Enzyme Fold Interacts with Histones H3–H4. transcription), which in human cells purifies as a heterodimer of We expressed and crystallized the N-terminal ‘‘peptidase’’ mod- Spt16 and SSRP1 (1, 2). ule of the S. pombe FACT complex Spt16. The structure of the FACT is a highly conserved complex (2–5). In fungi, SSRP1 domain (residues 1–442, Spt16-N) was solved and refined to is largely encoded by POB3, whereas NHP6 encodes the arche- 2.1-Å resolution (Table S1 and Materials and Methods). Our typal HMG-box of SSRP1. The gene for the Spt16 subunit is structure is highly similar to the recently published structure of essential in Saccharomyces cerevisiae and Schizosaccharomyces the related domain from S. cerevisiae (29), revealing the con- pombe, probably reflecting important chromatin-related func- served pita-bread fold (C-terminal lobe; residues 178–442) of tions in transcription, replication, and DNA repair (4, 6–9). aminopeptidases (30), preceded by a smaller domain (N- Genetic screens identified Spt16 as a factor whose mutation terminal lobe; residues 1–174) (Fig. 1A). Structural homology restores the expression of a Ty1 transposon-silenced reporter searches return a bacterial prolidase and creatinase [Protein gene by promoting its cryptic transcription, known as a suppres- Data Bank ID codes 1CHM and 1PV9 (31, 32); DALI (55) sor of Ty1 phenotype, [SptϪ] (4, 7, 10, 11). Several POB3 alleles genes display [SptϪ] phenotypes (12), indicating that the main- tenance of correct chromatin structure involves Pol II cofactors Author contributions: T.S. and M.H. contributed equally to this work; T.S., M.H., E.L., and such as FACT (13). Indeed, FACT acts as a coactivator of A.G.L. designed research; T.S., M.H., and M.B. purified proteins and carried out biochemical assays, T.S. and M.H. crystallized the proteins and collected diffraction data, M.H. phased transcriptional initiation and elongation (14), and many Spt16 and refined the structures, and V.R. conducted ultracentrifugation assay; T.S., M.B., and alleles display genetic interactions with basal transcription fac- A.G.L. contributed new reagents/analytic tools; T.S., M.H., E.L., V.R., K.S., and A.G.L. tors. Furthermore, FACT subunits biochemically interact with analyzed data; and T.S., M.H., E.L., K.S., and A.G.L. wrote the paper. the Pol II elongation complex Paf1 (15), bind the coding region The authors declare no conflict of interest. of transcribed Pol II genes, and are recruited to inducible genes This article is a PNAS Direct Submission. upon activation (16–19). Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, FACT’s biological roles in transcription (and replication) may www.pdb.org (PDB ID codes 3CB5 and 3CB6). stem from its histone chaperone activity (20, 21). Histone *Present address: The Salk Institute, Plant Biology Laboratory, 10010 North Torrey Pines chaperones stimulate reactions involving the transfer of histones Road, La Jolla, CA 92037. (22), thereby mediating chromatin reorganization. At the mo- †To whom correspondence should be addressed. E-mail: [email protected]. lecular level, FACT binds nucleosomes and destabilizes inter- This article contains supporting information online at www.pnas.org/cgi/content/full/ actions between H2A–H2B dimers and (H3–H4)2 tetramers 0712293105/DCSupplemental. (20). Mechanistically this suggests that FACT may help tran- © 2008 by The National Academy of Sciences of the USA

8884–8889 ͉ PNAS ͉ July 1, 2008 ͉ vol. 105 ͉ no. 26 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712293105 Downloaded by guest on October 2, 2021 the conserved Spt16-N domain may bind histones using GST A pull-down assays between Spt16-N and recombinant core his- tones H2A–H2B or H3–H4, as well as native octam- ers. Surprisingly, the Spt16-N peptidase module directly interacts with both recombinant and native histones H3–H4 (Fig. 2 A and B). Stable H3–H4 association occurs even under high ionic

N N-terminal lobe strength, in contrast to histones H2A–H2B, which do not bind N Spt16-N specifically (Fig. 2A). Isothermal titration calorimetry 90º (ITC) assays confirm binding of recombinant H3–H4 to Spt16-N under physiological salt concentrations (Fig. S3A). To test whether the interacting proteins form a stable complex, we measured the interaction between the proteins by comigration on size-exclusion chromatography. Spt16-N, together with re- folded –H2B dimers, shows no shift in its elution volume (Fig. S2C). Thus, H2A–H2B do not bind Spt16-N under these conditions, consistent with Spt16 requiring its acidic C terminus for H2A–H2B interaction (20). In contrast, we observe C-terminal lobe comigration of Spt16-N with H3–H4, as indicated by the shift to C C higher molecular masses of Spt16-N when bound to histones H3–H4 (Fig. 2C). There is an equilibrium between H3–H4 B dimers and tetramers that depends on pH and protein and salt concentration (33). Based on column calibration using molecular mass markers, the two shifted peaks correspond approximately to a dimer of H3–H4 bound to one Spt16-N domain, as well as two Spt16-N domains bound to a H3–H4 tetramer. To resolve the stoichiometry of the complex extrapolated from our size-exclusion chromatography, we have performed analyt-

90º ical ultracentrifugation. The analysis shows that Spt16-N forms BIOCHEMISTRY a 1:1 complex with H3–H4 dimers and, to a lesser extent, a peak that fits the molecular masses of two Spt16-N modules with four H3–H4 histones (Fig. 3A). Taken together, our structural and biochemical data reveal that Spt16-N uses an ancient enzymatic fold to mediate molecular interactions with histones H3–H4 in vitro.

Spt16-N Interacts with the Globular Domains of Histone H3 and H4. To determine whether Spt16-N binds histones H3–H4 through their globular core domains or through their N-terminal tails, we used 0% 100% gel-filtration and GST pull-down assays to measure the binding Sequence conservation of Spt16-N to the globular domains of tailless H3 and H4 Fig. 1. Structure of the aminopeptidase domain of Spt16. (A) View of the complexes (Fig. 3B and Fig. S3B). The assays reveal that Spt16-N N-terminal domain (residues 1–442) of S. pombe FACT subunit Spt16 in two makes direct interactions with the globular domains of histone orientations. The module consists of an N-terminal lobe (Upper) and a larger H3–H4. Furthermore, the interaction with H3–H4 cores is C-terminal lobe (Lower) that is structurally related to aminopeptidases. (B) The equally stable to that of full-length H3–H4 even at high salt Spt16-N domain is conserved across eukaryotes. Surfaces are colored according to concentrations (Figs. S2A and S3B). This suggests that the sequence conservation, ranging from dark orange for invariant residues to white globular domains are a major contributor for Spt16-N binding in for variable residues, identifying a conserved pocket in the C-terminal lobe (the vitro. Mutagenesis of either of the two conserved surface regions pita-bread fold) of Spt16, which corresponds to the catalytic site in active ami- in Spt16-N (Fig. 1B) does not disrupt binding to H3–H4 globular nopeptidase enzymes, and a second conserved pocket in the N-terminal lobe. The domains (Fig. S3B), indicating that the H3–H4 interaction is relative orientation of the two structures is the same as in A. mediated by a distinct region on the extended surface of the Spt16-N module. Z-scores 30.5 and 30.3, respectively]. Their catalytic domains closely align with the corresponding segment in Spt16 (rmsd Spt16-N Interacts with Histone H3 and H4 Tails. Many chromatin factors recognize native or posttranslationally modified residues between Ϸ300 C␣ atoms is Ͻ2.5 Å). Importantly, neither resi- dues required for divalent cation coordination in metal- on histones (34–36), usually through conserved ligand-binding cavities. Because Spt16-N bears two conserved surface pockets, dependent aminopeptidases (30) nor the catalytic histidine in we determined whether Spt16-N might recognize H3 or H4 creatinase (32) are conserved in Spt16 (Fig. S2 A and B). Spt16-N N-terminal tails. We quantitated binding of Spt16-N to tail may thus have evolutionary diverged from aminopeptidases. peptides using ITC. Our in-solution assays reveal a high affinity The Spt16-N module harbors two surface patches that are of Spt16-N for the N-terminal tails of histone H3 (KD ϭ 11 ␮M) strictly conserved among its orthologues (Fig. 1B and Fig. S1). and H4 (KD ϭ 3 ␮M) (Fig. 4 A–C). The interaction between One maps to the center of the ‘‘pita-bread core,’’ and a second histone tails and Spt16-N is strongly salt-dependent (data not is found on the N-terminal lobe (Fig. 1B). The presence of shown; histone tail binding modules typically bind their ligands conserved surface-exposed residues in this peptidase domain only at low salt in vitro), in contrast to interaction with the prompted us to test whether Spt16-N may bind proteins. Inferred globular domain. Consistent with our GST pull-down and gel- mechanisms for FACT function in chromatin reorganization filtration assays (Fig. 2 A and C), Spt16-N does not detectably involve a chaperone activity for H2A–H2B histone dimers, likely interact with H2B tails (Fig. 4A). Furthermore, the calorimetry mediated by the C terminus of Spt16 (20, 22). We tested whether shows that S. pombe Spt16-N binds H3 and H4 tails with 1:1

Stuwe et al. PNAS ͉ July 1, 2008 ͉ vol. 105 ͉ no. 26 ͉ 8885 Downloaded by guest on October 2, 2021 0.2 A H2A-H2B H3-H4 A 0.2 x2 0.0030.003 0.0025 x3

[NaCl] mM 0.15 150 250 350 450 550 150 250 350 450 550 0.15 x1 0.0020.002 70 GST-Spt16-N 0.0015

0.001 55 0.10.1 0.001 40 0.0005 C (M)

35 0 0 120 140 160 180 200 220 25 0.050.05 120140160 108 0 02 220

0 15 00050 5010 1000 10 1505 20 2000 25052 300 300 H3 Molecular weight (kDa) kDa H2A-H2B H4 B 200150 29 kDa B Native histone octamers 0.06 Spt16-N.H3-H4 glob [NaCl] mM Input 250 350 450 550 0.04 Spt16-N GST-Spt16-N 70 0.02

Abs ( λ = 280 nm) 0 kDa 9210 11 131415171116 10291 28 Elution volume (ml) 25 45 Spt16-N H3 25 15 H2A-H2B H4 15 H3 globular C 200 150 29 kDa kDa H4 globular

0.06 Spt16-N.H3-H4 Fig. 3. Spt16-N recognizes the globular core of tailless histones H3–H4. (A) H3-H4 Analytical ultracentrifugation of the Spt16-N histones H3–H4 complex reveals 0.04 Spt16-N a 1:1 stoichiometry. Shown are three peaks with apparent molecular masses corresponding to (x1) free histones H3–H4 dimers, (x2) one Spt16-N molecule plus one H3–H4 dimer, and (x3) two molecules of Spt16-N plus one H3–H4 0.02 tetramer (or two H3–H4 dimers). (B) Gel-filtration profile (Upper) and SDS/ PAGE gel (Lower) for the interaction between Spt16-N and the tailless, glob- ular domains of histones H3–H4. Abs ( λ = 280 nm) 0 901211 131415171116 108 291 2 Elution volume (ml) H3 and H4 tails. Thus, we replaced the functional groups of 55 Spt16-N conserved residues in the central cavity (Fig. 4D) to alanine. 40 These mutants do not affect the dissociation constant for H3 or 35 H4 peptides in vitro (Fig. 4C). This suggests that the region 25 corresponding to the catalytic site of aminopeptidases may not mediate interactions with H3/H4 tails. 15 H3 H4 A second highly conserved patch maps to a groove within the kDa N-terminal lobe (Fig. 1B). Interestingly, this surface is in direct contact with the N terminus of a neighboring molecule in our Fig. 2. Spt16-N interacts with histones H3–H4. (A) GST pull-down assays of crystals. The N terminus of a second molecule in the crystal recombinant core histones with immobilized Spt16-N. Assays were conducted binds the Spt16-N pocket in an extended conformation (Fig. 4 E with increasing stringency of salt concentration in the washing buffer. (B) GST and F). A similar interaction occurs in a second crystal form pull-down assays of native Drosophila melanogaster core histone octamers with (form B; Fig. S4 and Table S1), hinting at a peptide-binding role immobilized GST-Spt16-N. Input and pulldown lanes are from different parts of for this pocket. Mutation of the highly conserved Ser-83 and the same SDS/PAGE gel. (C) Cofractionation of Spt16-N with purified H3–H4 Lys-86 residues, which contribute to this intermolecular crystal histones by size-exclusion chromatography. Spt16-N incubated with H3–H4 yields interaction, does not change the affinity for the H3 tail but two peaks (dark blue; running at a calculated molecular mass of 185 and 90 kDa), reduces binding to the H4 peptide Ϸ15-fold (Fig. 4C). Our corresponding to a tetramer and dimer of H3–H4 bound to two or one molecules of Spt16-N, respectively. Consistently, free H3–H4 also yields two species (at 102 site-directed mutagenesis analysis thus maps the binding of the and 47 kDa), whose molecular masses match those of tetramers and dimers histone H4 tail to this conserved pocket within the N-terminal (medium blue). Light blue shows the elution profiles for Spt16-N alone, which lobe of Spt16-N. runs as a monomer (at 50 kDa). Because histones H3–H4 have N-terminal tails, Distinct histone marks can be recognized by specific protein their retention times are slightly larger than expected for their mass. The void modules. Thus, we tested the specificity of H4 tail peptide volume for the Superdex 200 10/300 GL column is at 8.2 ml. interaction by incubating Spt16-N with H4 peptides carrying known posttranslational modifications of yeast H4 (37). Diacety- lation of H4 K8/K16, a mark of actively transcribed chromatin, stoichiometry (Fig. 4B), suggesting the existence of a specific or monomethylation of H4 K20 reduces but does not ablate the surface (or surfaces) on Spt16-N that is responsible for binding binding of H4 peptides to Spt16-N (Fig. 4A). These data show H3 and H4 tails. We therefore asked whether the two conserved that the Spt16-N module can interact with both native and surface patches in Spt16-N (Fig. 1B) mediate the interaction with modified H4 N-terminal tails.

8886 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712293105 Stuwe et al. Downloaded by guest on October 2, 2021 ABH2B + Spt16-N C Spt16-N + H4 peptide 0 0 H4 (1-35) KD (µM) -1 Spt16-N, wild-type 3.0 ± 0.2 D262A/K264A/S266A 3.9 ± 0.1 Spt16-N + -2 histone peptide KD (µM) F364L/R365A 3.8 ± 0.3 µcal/sec H4 3.0 ± 0.2 -3 S83A/K86A 31 ± 4 H3 11 ± 0.6 H3 (1-38) -2 H2B >200 -4 N = 1.07 ± 0.01 K = 251000 ± 8000 Spt16-N, wild-type 11 ± 0.6 H4 + Spt16-N H4 K8Ac/K16Ac 13 ± 1.3 -5 ∆H = -8200 ± 50

Enthalpy, ∆ H (kcal/mol) Enthalpy, D262A/K264A/S266A 10 ± 1.2 H4 K20me1 8 ± 0.4 ∆S = 3.9 -6 F364L/R365A 11 ± 0.8 040020 608010 120 0231 S83A/K86A 12 ± 1 Time (min) Molar ratio (H4/Spt16)

D S83/K86 E F

F364/R365

S83 K86 BIOCHEMISTRY

D262/K264/S266

Fig. 4. Spt16-N directly binds the N-terminal tails of histone H3 and H4 using different pockets. (A) ITC profile for the binding of H4 N-terminal tail peptide (1–35; black) to Spt16-N in comparison to H2B tail (blue), which does not bind Spt16-N. The Inset shows equilibrium dissociation constants (KD) for different peptides, including histone H4 diacetylated at K8/K16 (H4 K8Ac/K16Ac) or monomethylated at K20 (H4 K20me1). (B) Fitting of the experimental data to an equilibrium binding profile for Ϫ1 the unmodified H4 (1–35) peptide. The Inset shows the stoichiometry of binding (N), the KD, enthalpy ⌬H (kcal⅐mol ), and entropy ⌬S.(C) KD for the binding of H4 and H3 peptides to wild-type and engineered mutant Spt16-N. (D) Overview of the site-directed mutants in the two putative ligand binding pockets of Spt16-N (yellow balls denote the mutated positions). The figure also shows a second Spt16-N molecule (gray) in the asymmetric unit of the crystal that interacts with Spt16-N through the N-terminal peptide sequence (in cyan). The framed box indicates the region of Spt16-N shown in E.(E) The second, conserved pocket of Spt16-N locates to the N-terminal lobe of the module (surface view; color coding as in Fig. 1B) and is in contact with N-terminal residues of a second Spt16-N molecule in the asymmetric unit (ball-and-stick). (F) Key residues in Spt16-N (ball-and-stick, yellow backbone) mediating the crystal contact to the Stp16-N N terminus (ball-and-stick, cyan backbone).

Together, our biochemical and mutational experiments show thus likely bears independent H3 tail and H3–H4 core binding that Spt16-N is a histone binding module for H3 and H4, which surfaces. Together, our combined data implicate the S. pombe interacts with the N-terminal tail of H3 and H4, as well as with Spt16 peptidase fold in an unexpected FACT-mediated binding their globular domains. Furthermore, Spt16-N recognizes H3 function for histones H3 and H4. and H4 tails through distinct surfaces and associates with the S. cerevisiae FACT is an essential nuclear complex involved in globular domains of H3–H4 to form a biochemically defined transcriptional regulation and chromatin remodeling. Genetic protein assembly. experiments in S. cerevisiae suggest that the Spt16 N-terminal region is dispensable for several FACT functions (21). Similarly, Discussion we observe that fission yeast strains expressing ectopically Spt16 The evolutionary history of transcription and chromatin factors mutant proteins that lack the peptidase fold (Spt16-⌬N) are is largely unclear. Spt16-N is a good example of a eukaryotic viable (data not shown). In fact, recent findings indicate that the transcription regulator derived from an ancient enzyme fold. budding yeast N-terminal domain (Spt16-NTD) and the middle Other aminopeptidases have lost their catalytic function to domain of Pob3 (Pob3-M) may mediate partially redundant acquire vital chromatin and transcription roles, including Taf2, functions and account for the viability of either single mutant part of the promoter selectivity factor TFIID, and Ebp1 (28, 38, (29). Indeed, both Spt16-NTD and the middle domain of Pob3 39), a factor involved in transcription and translation. genetically interact with histones, and pob3-M spt16-NTD double Here we identify a H3–H4 globular domain binding role for mutants display synthetic defects (24, 29). the N-terminal domain of the FACT subunit Spt16. Further- At the structural level, S. pombe and S. cerevisiae Spt16-N more, the Spt16-N aminopeptidase module also binds directly to exhibit a high degree of structural similarity (rmsd is 1.5 Å histone H3 and H4 tails. Our structure also reveals a conserved between 410 corresponding C␣ atoms), in particular with regard surface pocket involved in H4 tail binding. Ser-83 and Lys-86 in to the two conserved surface pockets (Fig. 1B), one of which we this pocket directly mediate peptide binding through a contact show is involved in histone H4 tail binding. Our functional with a neighboring molecule in the crystal. Mutation of these analysis of the S. pombe Spt16-N module and the genetics in S. residues reduces H4-tail affinity but retains high-affinity binding cerevisiae together argue for a role of the peptidase domain in to H3 tails and interaction with H3–H4 globular cores. Spt16-N FACT-mediated chromatin remodeling. FACT has been pro-

Stuwe et al. PNAS ͉ July 1, 2008 ͉ vol. 105 ͉ no. 26 ͉ 8887 Downloaded by guest on October 2, 2021 posed to facilitate polymerase progression on chromatin tem- was acquired at beamline BM16 (European Synchrotron Radiation Facility, plates by removing H2A–H2B from nucleosomes and reassem- Grenoble, France). A complete data set for crystal form B was recorded at bling the octamer in its wake (14, 20). We now show that Spt16-N beamline I04 (Diamond Light Source, Didcot, UK). Data processing and scaling were done with XDS (44). associates with histones H3–H4 cores and tails, suggesting that the large subunit of FACT may contribute to the binding, Structure Determination and Refinement. Multiple-wavelength anomalous dis- eviction, and/or deposition of all histones. Alternatively, Spt16 persion data were used to locate eight selenium sites with SHELXD (45) that were interaction with H3–H4 could participate in the tethering of input into SOLVE and RESOLVE (46) for site refinement, phasing, density modi- nucleosome fragments after RNA polymerase passage, promote fication, and phase extension. Secondary structure elements were identified with H2A–H2B deposition, and restore the structural integrity of BUCCANEER (47). The structure was completed in alternating cycles of model chromatin. Our assays show that Spt16-N binds H3–H4 histones correction in COOT (48) and restrained TLS refinement in Refmac5 (49). The structure of crystal form B was determined by molecular replacement with primarily through a 1:1 complex with H3–H4 dimers and a 2:1 PHASER (50). Structural visualization was done with POVSCRIPT/POVRAY (51). complex with H3–H4 tetramers (or a 2:2 complex with H3–H4 dimers, assuming that Spt16-N dimerizes). Future structural ITC. Binding affinities of wild-type and mutant Spt16-N with N-terminal tails of studies of FACT bound to histones will reveal differences and H4, residues 1–35 (N-acetylated, with a C-terminal Tyr), and H3, residues 1–38 similarities to chaperones such as Asf1 (40–42) and promise to (with a C-terminal Tyr), were determined at 25°C by using ITC (MicroCal). Proteins shed further light on the inner workings of this comprehensive and peptides were dialyzed against ITC buffer (50 mM Tris, pH 7.9/25 mM NaCl/1 H2A–H2B and H3–H4 histone binding and nucleosome reor- mM EDTA). Injections consisted of 10 ␮l of peptide (650 ␮M) into 40 ␮M protein ganization complex. at 5-min intervals. Data were analyzed by using Origin (version 5.0). Materials and Methods Histone Refolding and Gel Filtration. Histone refolding was performed as described (43), with modifications: Full-length and globular H3 and H4 were Protein Expression and Purification. S. pombe Spt16 N-terminal domain mixed at equimolar ratios to a final concentration of 1 mg/ml and refolded in (Spt16-N; residues 1- 442) was cloned into pETM11, providing an N-terminal 150 mM NaCl, 25 mM Tris (pH 7.5), and 5 mM ␤-ME. Globular H3 and H4 were 6ϫHis tag and tobacco etch virus (TEV) site (leaving an N-terminal overhang further diluted into the same buffer containing 50 mM NaCl. Histones and of the residues Gly–Met, where Met corresponds to the residue 1 of Spt16). Spt16-N were mixed at equimolar ratios and incubated on ice for 30 min. Spt16-N was grown in E. coli BL21 DE3 to OD ϭ 0.6 and induced with 0.2 mM Proteins were separated on a Superdex 200 10/300 GL column at 150 mM NaCl. isopropyl ␤-D-thiogalactoside in TB at 18°C for 16 h. Selenomethionine- labeled protein was expressed in strain B834 (DE3) at 28°C and induced for 18 h Analytical Ultracentrifugation. Sedimentation velocity experiments were done with 0.5 mM isopropyl ␤-D-thiogalactoside in TB with 40 ␮g/ml seleno-L- at 4°C by using two-channel charcoal centerpieces at 47,000 rpm in a Beckman methionine. Cells were resuspended in 50 mM NaPi (pH 7.4), 500 mM NaCl, 10 Optima XL-A centrifuge fitted with a four-hole AN-60 Ti rotor. Samples of mM imidazole, and 5 mM 2-mercaptoethanol (␤-ME), lysed by sonication, and Spt16-N (9 ␮M) and H3–H4 (9 ␮M) were equilibrated against a buffer con- centrifuged at 45,000 ϫ g for 45 min. The supernatant was loaded onto a Co2ϩ taining 150 mM NaCl and 25 mM Tris (pH 7.5) and loaded into a double-sector affinity chromatography column (Sigma), washed with 50 mM NaPi, 1 M NaCl, quartz cell. Sedimentation velocity profiles were collected by monitoring 20 mM imidazole, and 5 mM ␤-ME, and eluted in the same buffer with 500 mM absorbance at 280 nm. Sedimentation coefficient and molecular mass distri- imidazole. Elutions were concentrated to 10 mg/ml by using an Amicon 10,000 butions were analyzed by the C(s) method (52). Buffer density and viscosity MWCO concentrator. The 6ϫHis tag was cleaved with TEV for 16 h at 4°C. Next, corrections were made according to published data (53). Spt16-N was purified on a Superdex 75 HR16/60 column (GE Healthcare) ␮ equilibrated in 50 mM Na Pi (pH 7.4), 500 mM NaCl, and 5 mM ␤-ME. Fractions GST Pull-Downs. A total of 40 l of glutathione Sepharose FF beads (GE were dialyzed against 20 mM Hepes (pH 7.0), 25 mM NaCl, and 3 mM DTT and Healthcare) were incubated with 50 ␮gofE. coli-expressed, gel-filtration- concentrated to Ϸ13 mg/ml. Site-specific mutations were introduced by PCR purified, GST-fused Spt16-N for 30 min on ice in 150 mM NaCl and 25 mM Tris and purified like wild type. Recombinant histones were purified and refolded (pH 7.5). Next, beads were incubated with refolded H2A–H2B and/or H3–H4 at as described (43). 2-fold excess of histone for1hat4°C. Beads were washed five times with 250 mM NaCl, 25 mM Tris (pH 7.5), and 0.1% Nonidet P-40. The sample was boiled in SDS loading buffer and analyzed by SDS/PAGE. Native Drosophila histones Crystallization and Data Collection. Orthorhombic crystals of selenomethi- were purified from 0- to 12-h embryos (54). onine-labeled Stp16-N (form A; Table S1) were grown at room temperature from hanging drops composed of 1 ␮l of protein and 1 ␮l of crystallization buffer (13% [vol/vol] PEG 2000/100 mM Bis-Tris propane, pH 7.0) suspended ACKNOWLEDGMENTS. We thank K. Luger (Colorado State University, Fort over 0.5 ml of the latter. Unlabeled protein crystals (form B; Table S1) devel- Collins, CO) for histone plasmids; G. Stier (EMBL, Heidelberg) for pETM11 oped in 20% [vol/vol] PEG 300 and 100 mM Mes (pH 5.5). Crystals were vector; staff at beamlines PX01, BM16, and I04 (Diamond, Didcot, UK) for assistance during data collection; and F. Wieland, C. Schultz, T. Gibson, and C. transferred in reservoir solution containing 15% [vol/vol] ethylene glycol and Margulies for comments. This work was supported by the European Molecular frozen in liquid N2. Multiple-wavelength anomalous dispersion data were Biology Laboratory, the European Union Epigenome Network of Excellence collected at beamline PX01 (Swiss Light Source, Villigen, Switzerland) by using (A.G.L.), European Union Marie Curie Research Training Network Chromatin the microdiffractometer and a defocused beam. A higher-resolution data set Plasticity (A.G.L.), and the Peter and Traudl Engelhorn Foundation (M.H.).

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