Structural insights into the assembly of the PNAS PLUS deacetylase-associated Sin3L/Rpd3L complex

Michael D. Clarka, Ryan Marcuma, Richard Gravelineb, Clarence W. Chana, Tao Xiea, Zhonglei Chena, Yujia Dinga, Yongbo Zhanga, Alfonso Mondragóna, Gregory Davidb, and Ishwar Radhakrishnana,1

aDepartment of Molecular Biosciences, Northwestern University, Evanston, IL 60208-3500; and bDepartment of Pharmacology, New York University School of Medicine, New York City, NY 10016

Edited by Kevin Struhl, Harvard Medical School, Boston, MA, and approved May 28, 2015 (received for review February 26, 2015)

Acetylation is correlated with decondensation and receptor corepressor (NCoR), factor (TF) IIIB] transcriptional activation, but its regulation by domain on the other hand. However, both Sin3A and Sin3B— (HDAC)-bearing corepressor complexes is poorly understood. the paralogous subunits targeted by HDAC1 and HDAC2 in the Here, we describe the mechanism of assembly of the mammalian Sin3/Rpd3 complexes—have no readily recognizable SANT do- Sin3L/Rpd3L complex facilitated by Sds3, a conserved subunit main. Furthermore, a core subunit in the Sin3L/Rpd3L complex deemed critical for proper assembly. Sds3 engages a globular, called Sds3 was previously shown to be critical for the interaction helical region of the HDAC interaction domain (HID) of the scaf- between Sin3 and HDAC1/2 (paralogous and synonymous sds3 folding Sin3A through a bipartite motif comprising a helix complexes are separated by a slash), because deletions and an adjacent extended segment. Sds3 dimerizes through not and mutations adversely affected complex integrity and HDAC only one of the predicted coiled-coil motifs but also, the segment recruitment, respectively (6, 7). These observations suggested a preceding it, forming an ∼150-Å-long antiparallel dimer. Contrary unique mode of HDAC recruitment into the Sin3/Rpd3 complexes. The Sin3/Rpd3 complexes are ancient, with the core subunits to previous findings in yeast, Sin3A rather than Sds3 functions in conserved in organisms in the fungal, plant, and animal king- recruiting HDAC1 into the complex by engaging the latter through doms (1, 8). Whereas the larger 1.2- to 2-MDa mammalian a highly conserved segment adjacent to the helical HID subdomain. Sin3L/Rpd3L complex comprising seven unique subunits and at In the resulting model for the ternary complex, the two copies of the least as many paralogs is recruited to the promoter regions of HDACs are situated distally and dynamically because of a natively by sequence-specific DNA-binding transcription factors, the smaller unstructured linker connecting the dimerization domain and the 0.5- to 0.6-MDa Sin3S/Rpd3S complex comprising five unique Sin3A interaction domain of Sds3; these features contrast with the subunits and at least three paralogs is targeted to the intragenic static organization described previously for the NuRD (nucleosome regions of actively transcribed genes through interactions with remodeling and deacetylase) complex. The Sds3 linker features sev- specific chromatin signals (9–23). Both complexes share Sin3A/B, eral conserved basic residues that could potentially maintain the com- HDAC1/2, and RBBP4/7 subunits and rely largely on HDAC ac- plex on chromatin by nonspecific interactions with DNA after initial tivity to affect transcriptional repression; we note that there is now recruitment by sequence-specific DNA-binding repressors. increasing evidence that Sin3A and Sin3B partition into the Sin3L/ Rpd3L and Sin3S/Rpd3S complexes, respectively (20, 23). transcription repression | histone deacetylase | corepressor complex | protein–protein interaction | structural biology Significance

istone deacetylation constitutes the primary mechanism of Gene transcription in eukaryotes is regulated by enzymes that Herasing marks on , leading to a chro- posttranslationally add or remove acetyl groups from histones and matin environment that is repressive to gene transcription. His- render the underlying DNA more or less accessible to the tran- tone deacetylases (HDACs) exhibit limited substrate specificity scription machinery. How histone deacetylases (HDACs), the en- and rely on transcription factors with sequence-specific DNA- zymes responsible for deacetylation that are commonly found in binding and/or chromatin-binding activities for their targeting multiprotein complexes, are assembled and targeted to their sites 2+ specificity. Among 11 known Zn -dependent HDACs in mam- of action to affect transcription repression is largely unknown. We mals, only HDAC1, HDAC2, and HDAC3 are constitutively nu- show biochemically and structurally how two key subunits of a clear, regulating the transcription of a broad array of genes that conserved HDAC complex recruit multiple copies of HDACs into BIOCHEMISTRY impact fundamentally on cellular physiology and organism de- the complex in a manner that allows the enzymes to explore a velopment (1–3). These HDACs are commonly found in multi- large conformational space when the complex is targeted to protein corepressor complexes, with the closely related HDAC1 specific genomic loci. This complex seems to be tailored for effi- and HDAC2 partitioning broadly into the Sin3L/Rpd3L, Sin3S/ cient deacetylation of nucleosomes that are situated far apart. Rpd3S, NuRD (nucleosome remodeling and deacetylase), and CoREST (corepressor of REST transcription factor) complexes, Author contributions: M.D.C., R.M., R.G., G.D., and I.R. designed research; M.D.C., R.M., whereas HDAC3 is found exclusively in SMRT/NCoR (silencing R.G., C.W.C., T.X., Z.C., Y.D., Y.Z., and G.D. performed research; M.D.C., R.M., R.G., C.W.C., of retinoid and thyroid hormone receptor/nuclear receptor T.X., Z.C., Y.D., A.M., G.D., and I.R. analyzed data; and M.D.C., R.M., R.G., C.W.C., G.D., and I.R. wrote the paper. corepressor) complexes. Little is known regarding the structure and organization of these complexes, although molecular insights into The authors declare no conflict of interest. HDAC recruitment into these complexes are beginning to emerge. This article is a PNAS Direct Submission. High-resolution structures of HDAC1 and HDAC3 in com- Data deposition: The atomic coordinates, crystallographic structure factors, and NMR chemical shift and restraint tables have been deposited in the RCSB (Research Collabo- plex with the MTA1 (metastatic tumor antigen 1) and SMRT ratory for Structural Bioinformatics) , www.pdb.org (PDB ID codes subunits in the NuRD and SMRT/NCoR complexes, respectively 2N2H and 4ZQA) and the Biological Magnetic Resonance Bank (BMRB ID code 25599). (4, 5), revealed a shared structural theme involving the catalytic 1To whom correspondence should be addressed. Email: [email protected]. domain of the deacetylases on the one hand and a SANT This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. [switching-defective protein 3 (Swi3), adaptor 2 (Ada2), nuclear 1073/pnas.1504021112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1504021112 PNAS | Published online June 29, 2015 | E3669–E3678 Downloaded by guest on September 26, 2021 form soluble aggregates, even when fused to maltose binding protein (MBP), MBP-HID constructs spanning residues 601–830, 550–742, and 601–742 of Sin3A coeluted with Sds3 SID after coexpression and copurification in size-exclusion chromatography (SEC) assays (Fig. S1 A–C). In contrast, the MBP-HID construct spanning residues 661–742 failed to coelute in lock step with the Sds3 SID (Fig. S1D). Collectively, these results imply that the minimal Sds3-binding site resides within the segment spanning residues 601–742. Solution NMR spectroscopy was used to further define the Sin3A–Sds3 interaction. The 1H-15N–correlated spectrum of Sin3A HID601-742 is characterized by reasonable chemical shift dispersion of amide proton resonances, which is a hallmark of a folded domain (Fig. S2A). In the presence of one equivalent of Sds3 SID201-234, a subset of cross-peaks exhibits significant changes in chemical shift characteristic of a specific interaction. Fig. 1. Domain organization of Sin3 and Sds3 proteins and solution struc- The corresponding Sds3 SID201-234 spectrum features poor am- ture of the Sin3A HID–Sds3 SID complex. (A) Domain map of Sin3 and Sds3 α ide proton chemical shift dispersion, characteristic of an in- proteins with each domain drawn approximately to scale. (B, Left)C traces trinsically disordered segment, but the dispersion improves of the NMR ensemble of 20 conformers following a best-fit superposition of A backbone atoms in ordered regions and (B, Right) a representative structure significantly on binding to the HID (Fig. S2 ), implying the of the ensemble. The HID segments are blue, and those of the SID are in acquisition of a folded conformation. Additional analysis of the magenta. NMR spectra revealed the presence of flexible residues at the N and C termini of both domains. Hence, constructs for both do- mains were further optimized to improve the quality of the NMR 608-729 205-228 Early studies mapped an ∼300-residue segment within Sin3A spectra. Sin3A HID and Sds3 SID (henceforth des- for efficient interactions with HDAC1/2 (24) (Fig. 1A). This ignated simply as Sin3A HID and Sds3 SID) were deemed segment, designated the HDAC interaction domain (HID), was suitable for structural analysis, because these constructs inter- found to be necessary and sufficient for HDAC1/2 recruitment, acted with comparable affinity in the submicromolar range as the is unique to Sin3 homologs, and shares no obvious sequence or original constructs used for NMR according to isothermal ti- structural similarity to well-characterized protein domains or tration calorimetry (ITC) data (Table 1 and Fig. S2B). motifs. The HID is also the presumed site of interaction with several subunits of the Sin3L/Rpd3L complex, including Sds3, Structure of the Sin3A HID–Sds3 SID Complex. The solution struc- ture of the Sin3A HID–Sds3 SID complex was determined using RBBP4/7, SAP130, and ARID4A/B (7, 14, 25). The Sin3 poly- 1 1 peptides, thus, function as molecular scaffolds for the assembly a combination of H- H NOE-based distance and backbone of the complex and also, molecular adapters bridging subunits chemical shift-based torsion angle restraints (Table 2). An en- that engage in interactions with diverse DNA-bound factors and semble of 20 converged structures in good agreement with input chromatin signals on the one hand and HDAC activities on the restraints and with excellent backbone and covalent geometry other hand. Consistent with a fundamental role in transcription was selected for additional analysis. The Sin3A HID comprises regulation and their essential role in Sin3L/Rpd3L complex six helices, with the two longest helices-α1 and -α5 forming an stability, sin3, /2, and sds3 KOs are embryonically lethal, intramolecular coiled-coil stalk (Fig. 1); a cluster of short helices, whereas acute somatic deletions of these genes cause profound including α2, α3, and α4, along with the intervening loops pack defects in cell cycle progression and development (26–28). against α1 and α5 to form the globular head. Helix-α6 imme- The Sds3 polypeptide has at least two paralogs in higher eu- diately follows α5, is largely solvent-exposed, and makes few karyotes, including the suppressor 1 contacts with the rest of the domain. DALI (distance alignment (BRMS1), which suppresses metastasis of several different forms matrix method) found no closely related structural homologs for of cancer at multiple steps of this process, and p40, a BRMS1- like (BRMS1L) protein (29, 30). All three proteins share two K – predicted coiled-coil motifs and a conserved ∼30-residue Sin3A/B Table 1. Equilibrium d values for Sin3A HID Sds3 SID complexes

interaction domain (SID) that is necessary and sufficient for direct Reactants Kd (μM) interactions with Sin3A/B (31) (Fig. 1A). Other than an intact SID, the coiled-coil domains of Sds3 seem to be required for efficient Sds3 SID201-234 + Sin3A HID601-742 0.93 ± 0.03 recruitmentofHDACactivityinreporter assays (7). Little is known Sds3 SID + Sin3A HID 0.34 ± 0.16 regarding the molecular mechanisms by which the Sds3/BRMS1 Sds3 SID + Sin3A HID L620A 2.25 ± 0.45 proteins affect HDAC recruitment and Sin3L/Rpd3L assembly. Sds3 SID + Sin3A HID V623A 0.41 ± 0.03 Here, we describe the solution structure of an Sin3A HID Sds3 SID + Sin3A HID I673A 53 ± 24 subdomain in complex with the Sds3 SID and a crystal structure Sds3 SID + Sin3A HID K677A 1.31 ± 0.38 of the minimal dimerization domain of Sds3 to clarify the Sds3 SID + Sin3A HID I691A 1.20 ± 0.10 function of the respective domains in HDAC recruitment and Sds3 SID + Sin3A HID I695A 2.20 ± 0.87 Sin3L/Rpd3L complex assembly. In the process, we uncover Sds3 SID + Sin3A HID K698A 0.91 ± 0.24 some unexpected similarities and differences with the mecha- Sds3 SID + Sin3A HID R699A 41.5 ± 5.91 nism of HDAC1/2 recruitment in the NuRD corepressor com- Sds3 SID + Sin3A HID M702A 0.53 ± 0.10 plex; we also show that the mode of Sds3 dimerization is shared Sds3 SID + Sin3A HID A710F 0.51 ± 0.21 by the BRMS1/BRMS1L proteins. Sin3A HID + Sds3 SID D213Q 0.66 ± 0.11 201-234 Results Sin3A HID + Sds3 SID E214R 2.27 ± 1.60 Sin3A HID + Sds3 SID D219R201-234 ndb Mapping of the Minimal Interacting Domains of Sin3A and Sds3. To + 201-234 biochemically map the interaction interface between mammalian Sin3A HID Sds3 SID L223R ndb Sin3A and Sds3, several constructs of the HID spanning residues Unless specified otherwise, all experiments were performed with Sds3 550 and 830 were made based on sequence conservation patterns SID205-228, and mutants were assayed in the context of Sin3A HID608-729; and secondary structure prediction and coexpressed with the Sds3 values represent averages and SDs from at least three independent measure- SID (residues 201–234). Whereas the HID constructs tended to ments. ndb, No detectable binding.

E3670 | www.pnas.org/cgi/doi/10.1073/pnas.1504021112 Clark et al. Downloaded by guest on September 26, 2021 Table 2. NMR structure determination statistics for the Sin3A involving the backbone include the carbonyl groups of N208 and PNAS PLUS HID–Sds3 SID complex L210 of the SID that are targeted by the side-chain e-amino Metric Value group of K703 of the HID (Fig. S3). In line with expectation, residues that contribute extensively to Restraint statistics the protein–protein interface and engage in specific noncovalent NOE-based distance restraints 2,812 interactions are either invariant or highly conserved in both Sin3 Unambiguous NOE-based restraints 1,999 and Sds3 homologs (including BRMS1 and BRMS1L) from yeast B C Intraresidue 1,094 to (Fig. 2 and ). Instances where they seem not be Sequential (j i − j j = 1) 385 conserved (e.g., L207 and Y209 of Sds3) can be attributed to < j − j ≤ compensatory changes in character of the complementary sur- Medium range (1 i j 4) 272 – Long range (j i − j j > 4)* 167 face (e.g., L620 and V623 in Sin3). The Sin3 Sds3 interaction is, Intermolecular restraints 81 thus, largely conserved, although some of the features have evolved through covariation. Ambiguous NOE-based restraints 813 Hydrogen-bonding distance restraints 114 – ϕ ψ Functional Analysis of Sin3A HID Sds3 SID Complex. To test the Torsion angle restraints 200 (100 ,100 ) predictions of the NMR structure, selected individual residues Structure quality of NMR ensemble contributing to the protein–protein interface in Sin3A HID were Restraint satisfaction mutated and evaluated for binding to Sds3 SID in ITC assays. rms Differences for distances (Å) 0.014 ± 0.001 The strongest reductions in binding affinity were observed for rms Differences for torsion angles (°) 0.202 ± 0.063 R699A and I673A (>100-fold), consistent with their critical Deviations from ideal covalent geometry contributions to the interaction (Fig. 2D and Table 1). The ef- Bond lengths (Å) 0.003 ± 0.000 fects of mutating hydrophobic residues at the periphery of the Bond angles (°) 0.463 ± 0.013 protein–protein interface were more modest, with reductions in Impropers (°) 1.256 ± 0.067 affinity in the three- to sevenfold range, which is in line with Ramachandran plot statistics (%) expectation (Table 1 and Fig. S4). Complementary single-site, Residues in most favored regions 84.0 nonconservative substitutions of the Sds3 SIDs, such as D219R Residues in additional allowed regions 14.3 and L223R, completely abrogated binding to the Sin3A HID in Residues in generously allowed regions 0.9 ITC assays (Fig. 2E), consistent with their roles in stabilizing the Residues in disallowed regions 0.8 Sin3A–Sds3 complex by electrostatic and hydrophobic in- Average atomic rmsds from average structure (Å) teractions, respectively. In comparison, Sds3 mutant D213Q had All atoms 3.46 no significant effect on binding affinity, whereas Sds3 mutant † All atoms except in disordered regions 1.23 E214R only had a small effect, consistent with the noninvolve- α ′ ment of these side chains in complex-stabilizing roles (Fig. 2 C Backbone atoms (N, C ,C) E All residues 3.16 and and Table 1). Additional confirmation for the NMR All residues in secondary structure elements 0.66 structure was obtained by Sin3A coimmunoprecipitation (coIP) † experiments conducted with Flag-tagged Sds3 WT and mutant All residues except disordered regions 0.43 proteins in HEK293T cells. Whereas the WT Sds3 could asso- *A subset of long-range restraints includes intermolecular restraints. ciate with endogenous Sin3A, the L211R, D219R, and L220R Sds3 †Disordered regions include the three nonnative residues at the N termini of mutants exhibited greatly diminished binding because of the non- both proteins: residues 205–206 and 227–228 of Sds3 and residues 608–615 conservative nature of the substitutions and the critical roles played and 724–729 of Sin3A. by these residues at the protein–protein interface (Fig. 2E).

Sds3 Harbors a Dimerization Function. Because Sds3 harbors two Sin3A HID, although the intramolecular coiled-coil feature is putative coiled-coil motifs at the sequence level, we asked shared with several proteins in the Protein Data Bank, including whether the protein could self-associate to form higher-order flagellar export chaperones, histone demethylases, histidine- oligomers. A construct spanning the coiled-coil and SID domain containing phosphotransfer proteins, viral nucleocapsid proteins, (CCSID) eluted with a molecular mass (∼45 kDa) consistent and photosystem II proteins among others. with a dimer in SEC-coupled multiangle light scattering (MALS) The Sds3 SID forms a bipartite structural motif comprising an experiments (Fig. 3A and Table 3); similarly, a 1:1 mixture of N-terminal segment in an extended conformation spanning res- MBP-tagged Sin3A HID and Sds3 CCSID also eluted with a idues A205–T212 followed immediately by a 13-residue helix, αA molecular mass of a 2:2 heterodimer (∼195 kDa). To test (Fig. 1B). Sds3 residues in these segments engage a shallow whether coiled-coil motif 1 (CC1) or CC2 was important for di- groove punctuated by pockets on the surface of the Sin3A HID, merization activity, proline mutations were introduced at positions with the N-terminal segment interacting principally with the stalk D75P and E156P, respectively, in the middle of two predicted BIOCHEMISTRY region, whereas the helical segment engages exclusively the head coiled-coil motifs either singly or combined and evaluated for (Fig. 2A). The nature of these contacts is primarily hydrophobic, dimerization activity. Whereas the Sds3 CCSID D75P protein with the side chains of L207, Y209, L211, and L223 of the SID coeluted with MBP-Sin3A HID as an ∼192-kDa dimer, like the interacting with cavernous pockets on the surface of the HID WT protein (Fig. 3B and Table 3), both the E156P and the formed, in large part, by the side chains of I673, Y674, I681, and D75P,E156P double mutant coeluted with MBP-Sin3A HID, W707, whereas the SID side chains of L210, I216, L220, T222, with masses of ∼98 and ∼109 kDa, respectively, characteristic of and L226 target shallower surfaces of the HID. The sole solvent- a monomeric Sin3A–Sds3 complex and implying that CC2 but excluded charged residue of the SID at the protein–protein not CC1 was important for dimerization activity. interface, D219, engages in an intermolecular salt bridge and To further refine the boundaries of the Sds3 dimerization hydrogen-bonding interactions through its side chain with those domain, constructs of CC1, CC2, and a segment spanning the of R699 and Y675 of the HID, respectively (Fig. 2A and Fig. S3); CC2 and the linker region connecting CC1 and CC2 (LnkCC2) these interactions are augmented by an electrostatic interaction were generated and tested for dimerization activity. Surprisingly, between the side chains of E218 of the SID and R699 of the both CC1 and CC2 constructs eluted with molecular masses HID. The close proximity of the polypeptide backbones of SID consistent with those of monomers (∼9 kDa each) (Fig. 3B), and HID at the stalk–head junction affords an intermolecular whereas the LnkCC2 construct eluted with the size expected for hydrogen-bonding interaction between the amide and carbonyl a dimer (∼20 kDa), implying that the linker segment other than groups of L211 and I673, respectively. Additional interactions CC2 was essential for dimerization.

Clark et al. PNAS | Published online June 29, 2015 | E3671 Downloaded by guest on September 26, 2021 Fig. 2. The Sin3A HID–Sds3 SID interface, patterns of sequence conservation within the interacting segments, and functional analysis of the interaction in vitro and in cells. (A) Views of the Sin3A HID residues (blue) that make up the interface in (Upper) stick and (Lower) molecular surface representations. The Sin3-interacting side chains and the peptide backbone of Sds3 SID are also shown (magenta). Complementary views of the interface are shown in Fig. S3. MUSCLE-guided multiple sequence align- ments of (B) Sin3 and (C) Sds3 homologs from vari- ous organisms. Residues are shaded according to their level of conservation. Colored asterisks imply intermolecular noncovalent interactions involving backbone and/or side chain atoms: green, hydrogen bonding; orange, electrostatic; red, hydrophobic. (D) ITC analysis of various Sin3A HID mutants. Rep- resentative titrations for various mutant and WT proteins are shown. Symbols denote raw data, whereas the continuous lines correspond to fits. The

fitted equilibrium dissociation constants (Kd values) for the various proteins are cataloged in Table 1. (E) ITC analysis of selected Sds3 SID mutants. Rep- resentative titrations for mutant proteins are shown. (F) CoIP assays conducted in HEK293T cells to assess the ability of various FLAG-tagged Sds3 con- structs to pull down endogenous Sin3A. (Upper)Cell lysates (input) and (Lower) the immunoprecipitated proteins (IPs) were resolved by SDS/PAGE and visual- ized by Western blot using anti-Flag and anti-Sin3A antibodies. Dm, Drosophila melanogaster;Dr,Danio rerio;Hs,Homo sapiens;Mm,Mus musculus;Rn, Rattus norvegicus;Sc,Saccharomyces cerevisiae;Sp, Schizosaccharomyces pombe;Xl,Xenopus laevis.

Structural Analysis of Sds3 Dimerization Domain. To gain additional engage in electrostatic interactions with those of E153, K143, insights into the structure of the dimerization domain, the and K132; R109 forms salt bridges with E131 and E138. The LnkCC2 construct was crystallized. Excellent diffraction-quality R109–E131 salt bridge seems to be especially crucial, because crystals were obtained, and the structure was solved by single both R109 and E131 are invariant in not only Sds3 orthologs but isomorphous replacement with anomalous scattering using data also, the paralogs BRMS1 and p40/BRMS1L and likely dictate collected for native crystals and a selenomethionine derivative. the specificity of the interaction and the antiparallel orientation Data up to 1.65 Å were used for structure solution and refine- of the (Fig. S5 B and C). ment (Table 4). The final Rwork and Rfree values were 16.0% and 20.9%, respectively, with close to ideal covalent geometry and all Functional Analysis of Sds3 Dimerization Domain. To test the pre- residues in the most favored regions of the Ramachandran plot. dictions of the crystal structure, we mutated L102, V124, F139, In the refined model, each monomer in the dimer forms a and I161; the latter is a conserved residue that could potentially continuous helix spanning residues G90–L170, with the helices be involved in engaging CC1 but is exposed to solvent in our organized in a head-to-tail antiparallel arrangement (Fig. 3C). structure. Each of the residues was, in turn, mutated to gluta- Analysis of knobs-and-holes packing involving side chains at the mate in the context of a construct spanning the two coiled-coil dimer interface using the SOCKET program reveals a coiled coil motifs to have a significant adverse impact on dimerization and that extends from residue Y106 to residue E138 in the central subjected to SEC-MALS. As expected, both V124E and F139E segment. However, the dimer interface extends farther in both exhibited broad elution profiles, with molecular masses that were directions: from T91 near the N terminus of the construct to inconsistent with stable dimer formation, unlike the WT con- K157 (Fig. 3C and Fig. S5A). The segment from K158 to G172 is, struct (Fig. 3D and Table 3). In contrast, both L102E and I161E thus, fully exposed to solvent, and bending in excess of 10° in the eluted with similar masses, consistent with the formation of a local helical axis is noted for each residue from K157 to M160. dimer, like the WT construct. The lack of any perturbation in the Whether these features are preserved in the construct harboring dimerization function for L102E is because L102 is located near an intact CC1 segment is unknown, because attempts to crystallize K142 (Fig. 3C and Fig. S5A), potentially allowing for a favorable the segment spanning both CC1 and CC2 were unsuccessful. The electrostatic interaction with the newly substituted glutamate dimer interface, which extends over a surface area of ∼1,380 Å2, residue that might have rescued the function of an otherwise is dominated by hydrophobic interactions and a few electrostatic deleterious mutation. Although the I161E mutation is not in- interactions, including two salt bridges. Key residues engaging herently destabilizing to the dimer, it nevertheless eluted some- in hydrophobic interactions include the side chains of Y95, what earlier than the WT protein (Fig. 3D), perhaps because of an L102, L117, V124, Y128, A135, F139, L146, and L150, all of alteration in the shape of the molecule caused by the mutation. which are invariant or highly conserved in Sds3 orthologs (Fig. The structure of the Sds3 dimer is most likely conserved in its 3C and Fig. S5B). The side chains of R98, D103, and E114 paralogs given the high degree of sequence conservation in this

E3672 | www.pnas.org/cgi/doi/10.1073/pnas.1504021112 Clark et al. Downloaded by guest on September 26, 2021 PNAS PLUS BIOCHEMISTRY

Fig. 3. Structural and functional characterization of the dimerization domain of Sds3. (A) SEC-MALS analysis of an His6-tagged Sds3 construct spanning the CCSIDs (residues 61–234) in the absence (red) and the presence (blue) of MBP-tagged Sin3A HID (601–742). Chromatograms shown are the measured dif- ferential refractive indices. The molecular masses of the HID–CCSID complex and apo-CCSID measured by light scattering are indicated by the curves shown in darker colors within each peak. *Minor peaks correspond to aggregated Sin3A HID. **Minor peaks correspond to monomer apo-Sin3A HID. #Minor peaks

correspond to an impurity. (B) SEC-MALS analysis of (Left) mutant and WT His6-tagged Sds3 constructs (in the background of CCSID: 61–234) in complex with MBP-Sin3A HID (601–742) and (Right) various Sds3 segments harboring putative oligomerization motifs (LnkCC2: 90–174; CC2: 121–174; CC1: 61–104). Chromatograms shown are the measured differential refractive indices. The curves on top of or overlapping with each peak are the measured molecular mass ranges. (C, Top) Crystal structure of the minimal dimerization domain of Sds3 (residues 90–174) with the backbone in a ribbon representation. (C, Middle) Key residues at the dimer interface are shown in stick representation along with the molecular surface of the interacting protomer. (C, Bottom) Close-up views of the intersubunit contacts made by the side chains of these residues. These views are rotated by 90° along the horizontal axis from those shown in Middle.

(D) SEC-MALS analysis of His6-tagged WT and mutant Sds3 constructs spanning the two coiled-coil motifs (CC: 61–174) and harboring glutamic acid mutations at the indicated positions. (E) CoIP assays conducted in HEK293T cells to assess the ability of various FLAG-tagged Sds3 and BRMS1 constructs to pull down various HA-tagged BRMS1 constructs. (Upper) Cell lysates (input) and (Lower) the immunoprecipitated proteins (IPs) were resolved by SDS/PAGE and visu- alized by Western blot using either anti-HA or anti-Flag antibodies.

Clark et al. PNAS | Published online June 29, 2015 | E3673 Downloaded by guest on September 26, 2021 Table 3. Predicted and experimentally determined molecular masses of various Sin3A–Sds3 complexes and Sds3 constructs Molecular species Apparent MM (kDa) Expected MM (kDa)*

61-234 601-742 His6-Sds3 + MBP-Sin3A HID 195 ± 9.7 83.6 61-234 601-742 His6-Sds3 CCSID D75P + MBP-Sin3A HID 192 ± 12 83.6 61-234 601-742 His6-Sds3 CCSID E156P + MBP-Sin3A HID 98 ± 4.6 83.6 61-234 601-742 His6-Sds3 CCSID D75P, E156P + MBP-Sin3A HID 109 ± 4.8 83.6 61-234 His6-Sds3 CCSID 45.3 ± 2.6 23.5 61-104 His6-Sds3 CC1 8.7 ± 0.2 7.82 90-174 His6-Sds3 LnkCC2 20.0 ± 0.6 12.8 121-174 His6-Sds3 CC2 8.9 ± 0.2 9.01 61-174 His6-Sds3 33.4 ± 0.3 16.7 61-174 His6-Sds3 L102E 33.8 ± 5.1 16.7 61-174 His6-Sds3 V124E 21.8 ± 1.5 16.7 61-174 His6-Sds3 F139E 24.5 ± 2.2 16.7 61-174 His6-Sds3 I161E 40.3 ± 0.5 16.7

MM, molecular mass. *All expected molecular masses are given for the monomeric form of the protein or protein complex.

region, especially at the dimer interface (Fig. S5C). However, To map the segment(s) of Sin3A necessary for efficient in- unlike Sds3, both BRMS1 and BRMS1L harbor two proline teraction with HDAC1, we first analyzed the sequences of the residues near the N terminus of this region, at least one of which ∼300-residue HID of Sin3 orthologs and paralogs from a broad is conserved. We asked whether BRMS1 could heterodimerize range of species. These analyses revealed a highly conserved with Sds3 in coIP assays. After their overexpression in HEK293T segment at the N terminus of the HID, including a sequence cells, HA-tagged BRMS1 could be efficiently immunopreci- motif (CxRLGxSYRAL; residues 552–562) that bore some re- pitated by Flag-tagged Sds3 (Fig. S5D), indicative of a direct semblance to the ELM2-specific motif (EIRVGxxYQAxI) pre- interaction between the two proteins. To test whether the viously shown to engage HDAC1 (Fig. S6). Sin3A deletion interaction was through the dimerization domains in the respec- constructs spanning residues 550–600 and 743–850, corre- tive proteins, we repeated these binding assays after introducing sponding to the segments immediately N- and C-terminal, re- a glutamate substitution at R115 (the equivalent of R109 in Sds3 spectively, to the region involved in binding Sds3, were engineered that engages in key intersubunit electrostatic interactions; com- along with a construct lacking residues 552–564. All three con- pare with above) in BRMS1. Whereas the WT BRMS1 protein structs were cotransfected, in turn, with Flag-tagged HDAC1 and interacted efficiently with WT Sds3, the BRMS1 R115E mutant tested for binding in coIP assays. As expected, the Δ550–600 exhibited greatly diminished binding in these immunoprecipita- construct lacking the highly conserved segment of HID failed to tion experiments (Fig. 3E). Similarly, the BRMS1 R115E mutant coIP HDAC1, whereas the Δ552–564 showed diminished ability to bound poorly to the WT BRMS1 protein, whereas the differ- bind HDAC1 (Fig. 4C); the Δ743–850 construct, however, showed entially tagged WT BRMS1 proteins interacted efficiently with similar HDAC1-binding activity as the WT protein. Collectively, each other. Collectively, these results suggest that the BRMS1 these results suggest that the HDAC1 interface on Sin3A resides proteins can form homodimers and heterodimers with Sds3 using within residues 550–742, with ∼50 residues at the N terminus of the same dimerization interface. this segment making significant contributions to the interaction. To test whether this segment of Sin3A could directly recruit Roles of Sds3 and Sin3 in HDAC Recruitment. Previous studies sug- histone deacetylase activity, we engineered GAL4-Sin3A fusions gested a critical role for Sds3 in recruiting HDAC activity into of residues 550–742, 601–742, and 662–742 and tested their the Sin3L/Rpd3L complex. For example, in sds3Δ yeast strains, function in transcriptional repression assays. Whereas the Sin3A the Sin3 and Rpd3 proteins were found to elute in separate 550–742 construct repressed transcription more than 17-fold, the chromatographic fractions, whereas in mammals, Sds3 knock- 601–742 and 662–742 constructs did so by only 2- or 4-fold, downs were shown to modestly diminish the activity of associated implying that residues 550–600 were critical for efficient HDAC HDACs (6, 7). Therefore, we asked whether Sin3A could interact recruitment (Fig. 4D). Notably, the 601–742 construct, which with HDAC1 efficiently in the absence of Sds3. To test this idea, we harbors an intact Sds3 interaction site, was unable to efficiently first cotransfected HEK293T cells with WT Myc-tagged Sin3A and support repression. an Sin3A R699A mutant defective for Sds3 binding with Flag-tagged Sds3. Whereas the WT Sin3A was efficiently immunoprecipitated Sds3 Can Bind Nucleic Acids. Sequence analysis of Sds3 orthologs by Sds3, the R699A mutant was not, which was expected (Fig. 4A). from a broad range of species indicated the presence of two We then cotransfected the same Sin3A constructs separately with highly conserved basic regions (CBRs) separated by a short Flag-tagged HDAC1. Unexpectedly, both the WT and the R699A proline-rich linker in the segment immediately after the di- mutant were efficiently immunoprecipitated by HDAC1 (Fig. 4A). merization domain (Fig. S7A). We first tested the solvent ac- These results, thus, imply that the interaction between Sin3A and cessibility of this segment by conducting limited trypsinolysis for HDAC1 is stable and independent of Sds3. an Sds3 construct (residues 43–234) encompassing this region. To further confirm that the interaction between HDAC1 Trypsin digestion resulted in close to no protection of this seg- and Sds3 was indirect, we cotransfected full-length constructs of ment (Fig. S7C); indeed, the C termini of the two transiently Flag-tagged Sds3 and untagged HDAC1 along with or without protected fragments both mapped to the first basic residue R183 Myc-tagged Sin3A. Although HDAC1 could be efficiently in CBR1. We then asked whether a GST-Sds3 fusion protein immunoprecipitated by Sds3 in the presence of Sin3A, HDAC1 (residues 172–209) harboring this strongly basic segment could failed to be immunoprecipitated when Sin3A was absent (Fig. bind to dsDNA. The GST-Sds3 fusion protein at micromolar con- 4B), implying that Sds3 cannot directly interact efficiently with centrations could efficiently shift a 168-bp DNA duplex in EMSAs HDAC1. These results also show that both Sds3 and HDAC1 (Fig. S7D). This result is in contrast to the negative control, GST, can simultaneously engage Sin3A. which failed to bind DNA under the same conditions.

E3674 | www.pnas.org/cgi/doi/10.1073/pnas.1504021112 Clark et al. Downloaded by guest on September 26, 2021 Table 4. X-ray crystallographic statistics for Sds3 LnkCC290-174 PNAS PLUS Native Se-Met1 Se-Met2 Se-Met3 Se-Met4 Se-Met5

Data collection Detector type/source Rayonix Rayonix Rayonix Rayonix Rayonix Rayonix CCD/APS CCD/APS CCD/APS CCD/APS CCD/APS CCD/APS Wavelength (Å) 0.97856 0.97872 0.97872 0.97872 0.97872 0.97872 Resolution range* (Å) 35.50–1.65 35.55–1.80 32.76–1.80 35.62–1.80 35.57–1.85 32.52–1.90 (1.68–1.65) (1.84–1.80) (1.84–1.80) (1.84–1.80) (1.89–1.85) (1.94–1.90)

Space group C2221 C2221 C2221 C2221 C2221 C2221 a (Å) 44.46 44.40 44.30 44.11 44.48 44.14 b (Å) 49.39 48.75 48.67 47.53 48.46 48.11 c (Å) 106.50 106.60 106.70 106.90 106.70 106.50 Measured reflections* 52,223 159,630 156,576 149,021 133,563 130,366 (2,558) (9,523) (9,376) (8,585) (7,963) (8,245) Unique reflections* 13,534 (667) 11,093 (654) 10,999 (647) 10,765 (620) 10,192 (603) 9,222 (579) Completeness* (%) 94.2 (96.5) 99.8 (100.0) 99.6 (99.4) 99.8 (100.0) 99.8 (100.0) 99.5 (99.7) Anomalous completeness* 99.8 (100.0) 99.7 (99.7) 99.8 (100.0) 99.8 (100.0) 99.6 (99.8) Mean [I/σ(I)]* 11.3 (2.8) 17.4 (4.4) 16.8 (4.1) 12.9 (2.6) 16.9 (5.3) 17.8 (6.6) Multiplicity* 3.9 (3.8) 14.4 (14.6) 14.2 (14.5) 13.8 (13.8) 13.1 (13.2) 14.1 (14.2) Anomalous multiplicity* 7.7 (7.7) 7.6 (7.6) 7.4 (7.3) 7.0 (6.9) 7.6 (7.5)

Rmeasure* 0.056 (0.319) 0.099 (0.609) 0.099 (0.607) 0.115 (0.996) 0.100 (0.468) 0.099 (0.378) MFID 0.153 0.162 0.206 0.135 0.169 Phasing Se sites 33333 Phasing power Dispersive (centric/accentric) 2.715/2.262 5.385/3.389 0.475/0.422 1.541/1.360 2.174/1.873 Anomalous (accentric) 2.530 2.741 1.814 2.456 2.547 FOM (centric/accentric) 0.575/0.217 0.575/0.217 0.575/0.217 0.575/0.217 0.575/0.217 Refinement No. of reflections working/test 12,803/685 (950/46) R (working set; %) 16.0

Rfree (%) 20.9 Structure quality Protein atoms 866 rmsds In bond lengths (Å) 0.013 rmsds In bond angles (°) 0.781 Ramachandran plot—most 100 favored regions (%)

APS, Advanced Photon Source; FOM, figure of merit; MFID, mean fractional isomorphous difference. *All numbers in parentheses are for the highest resolution shell.

Discussion However, in line with our findings, coIP experiments reported The results from our structural and functional analyses permit us previously with the mammalian homologs suggested that Sin3 to propose a molecular model for the assembly of the Sin3L/ could recruit HDAC1 just as efficiently with or without Sds3 (7). Rpd3L complex. In this model, Sds3 interacts with a folded It is conceivable that the functions of yeast Sds3 and its metazoan globular domain of Sin3A/B, whereas Sin3A/B uses a distinct counterparts have diverged during evolution. Furthermore, the neighboring segment to directly engage HDAC1 (Fig. 5) in a direct involvement of Sin3 in HDAC recruitment implies that manner structurally analogous to that observed for the MTA1– this interaction is conserved in both Sin3S/Rpd3S and Sin3L/Rpd3L complexes, because the two complexes share Sin3, HDAC1/2, HDAC1 interaction in the NuRD complex (4). Because the pat- BIOCHEMISTRY tern of sequence conservation in Sin3 extends to solvent-exposed and RBBP4/7, whereas the Sin3S/Rpd3S complex lacks Sds3; also, none of the other known subunits in the latter complex residues in the α1-and α6-helices of the Sin3A–Sds3 complex share a sequence motif that could engage Sin3 HID in an (Fig. S6), we propose that some of these residues could likely analogous manner. directly bolster the interaction with HDAC1/2; this idea is fur- Why then is Sds3 critical for the function of the Sin3L/Rpd3L ther supported by the fact that the fragments encompassing – – complex, which was suggested by the high level of sequence residues 550 601 or 550 621 of Sin3A are insufficient to repress conservation in diverse organisms? It is plausible that it provides transcription in reporter assays (Fig. S8). One key difference a unique dimerization function that places the two HDACs – between the MTA1 HDAC1 interaction and this mode of re- recruited to the complex not only far apart (>150 Å) (Fig. 5) cruitment is that the Sin3–HDAC1 interaction seems to be from each other but also, tethered to the dimerization domain by independent of inositol tetraphosphate (IP4). Indeed, sequence a flexible linker (∼35 residues between the dimerization and SID analysis suggests that an SANT domain, typically involved in domains, which potentially could increase the distance even promoting the latter interaction, is absent in Sin3; perhaps, the farther). This feature contrasts with the close proximity of the IP4 requirement arose later in evolution or is mediated by an- HDACs—recruited by a structurally unrelated dimerization other subunit of the Sin3L/Rpd3L complex. function—at a fixed distance relative to each other (∼90 Å be- The absence of a direct role for Sds3 in HDAC recruitment to tween the two catalytic sites) in the NuRD complex (4). Perhaps, the Sin3L/Rpd3L complex was completely unexpected, because deacetylases in different complexes have evolved distinct modes previous studies conducted in yeast had indicated otherwise (6). of engaging their substrates with the NuRD complex, preferring

Clark et al. PNAS | Published online June 29, 2015 | E3675 Downloaded by guest on September 26, 2021 Fig. 4. Biochemical analysis of interactions in- volving Sds3, HDAC1, and Sin3 and a model for the assembly of the Sin3L/Rpd3L complex. (A) CoIP as- says of Myc-tagged (MT) WT and mutant Sin3A proteins by (Left) Flag-tagged Sds3 and (Right) Flag- tagged HDAC1. Cell lysates (input) and the immu- noprecipitated proteins (IPs) were resolved by SDS/ PAGE and visualized by Western blot using anti-Myc and anti-Flag antibodies. (B) CoIP assays of un- tagged HDAC1 and/or MT-Sin3A by Flag-tagged Sds3. Proteins were resolved and visualized as in A but also, used an HDAC1-specific antibody. (C) CoIP assays of MT-Sin3A WT and various internal de- letions to identify the segments involved in direct HDAC1 interactions. Proteins were resolved and vi- sualized as in A.(D) Transcriptional repression assays conducted in HEK293T cells with GAL4 DBD fusions of different Sin3A constructs using a luciferase reporter and a thymidine kinase promoter harboring four tan- dem GAL4 DNA-binding sites. The error bars represent SDs from three independent measurements.

adjacent acetylated nucleosomes that are situated in closer BRMS1 could recruit ARID4A/B to the complex in a manner proximity in contrast to the Sin3L/Rpd3L complex, which might dependent on intact CC1 and CC2, raising the possibility that prefer or tolerate nucleosomes that are farther apart (Fig. 5). CC1 and CC2, which are in close proximity in the Sds3-based Three of four known HDAC1/2-containing complexes, in- model for the dimer, form a recruiting platform for these types of cluding the Sin3L/Rpd3L, NuRD, and CoREST complexes, rely interactions (37). However, it has been shown that Sds3 is unable on DNA-bound repressors for their genome-targeting activity, to suppress metastasis in the absence of BRMS1 (38). In this whereas the Sin3S/Rpd3S complex has dedicated chromatin- regard, we note that CBR1 found in Sds3 is not conserved in binding proteins. The Sin3L/Rpd3L complex was the first com- BRMS1 (Fig. S7B), implying functional divergence for these plex shown to deacetylate histones in a highly localized manner segments in the respective proteins. (32). After its recruitment to a defined , the complex has also been shown to be anchored to chromatin, even after the Materials and Methods departure of the associated repressor, although the source of this Production of Sin3 and Sds3 Polypeptides. The gene sequences encoding activity is not known (33). Based on the nonspecific DNA- mammalian Sin3A HID608-729 and Sds3 SID205-228 were inserted into the binding activity of the CBRs, we propose that Sds3 could pMCSG21 and pMCSG7 (39) vectors, respectively, sequenced to verify integrity, contribute to this chromatin-anchoring activity, enhancing the and coexpressed in Escherichia coli BL21(DE3) cells at 20 °C. The proteins were + residence time of the complex on chromatin substrates to facil- copurified by Ni2 affinity chromatography under denaturing conditions. After

itate complete deacetylation in preparation for long-term re- elution and removal of the denaturant, the His6 tags were cleaved using pression of associated loci. In this regard, we note that Sds3 has tobacco etch virus protease, and the proteins were purified to homogeneity by been implicated in the genesis of pericentric heterochromatin, reversed-phase HPLC. Uniformly, 15N- and/or 13C-labeled proteins were pro- and sds3 mutations (including both null and ΔSID mutations) duced using the same procedure, except that the cells were grown in M9 15 13 lead to cellular lethality caused by the resulting defects in minimal medium containing N-ammonium sulfate and/or C-D-glucose, re- segregation (26). spectively. Protein identities were confirmed by electrospray ionization MS. Sds3 is related to the BRMS1 and BRMS1L proteins, which Mammalian Sin3A HID601-742, Sin3A HID550-830, Sds3 CCSID61-234,Sds3 seem to have arisen sometime more recently during metazoan CCSID43-234, Sds3 SID201-234, and Sds3172-209 were subcloned, expressed, and evolution. Although the BRMS1 protein plays important roles in purified in a similar manner as above, except that Sin3A HID601-742 was metastasis suppression of a variety of cancers (34), the precise subcloned in pMCSG23 in addition to pMCSG21 (39). Coexpressed MBP- and

molecular role of this protein in the context of the Sin3L/Rpd3L His6-tagged proteins were copurified by amylose affinity chromatography complex has been elusive. Structures of BRMS1 CC1 motif have under native conditions. Apo-Sds3 CCSID61-234, Sds3 CCSID43-234, the three provided contrasting views of higher-order association, with one proline CCSID mutants, CC161-104, CC2121-174, LnkCC290-174,CC61-174, and the segment of the protein crystallizing as a hexamer, a slightly dif- various glutamate mutants in the CC61-174 background were expressed singly + ferent segment crystallizing as a dimer, and each association and purified by Ni2 affinity chromatography under denaturing conditions. using distinct surfaces (35, 36). We propose that a model for Sin3A HID and Sds3 coiled-coil and SID mutants were generated using the BRMS1 dimerization based on the structure of the Sds3 di- QuikChange site-directed mutagenesis protocol (Agilent); all mutations were merization domain is likely to be physiologically relevant. In- confirmed by DNA sequencing. The mutant proteins were expressed and deed, previous genetic and biochemical studies have shown that purified using the protocols for the corresponding WT proteins.

E3676 | www.pnas.org/cgi/doi/10.1073/pnas.1504021112 Clark et al. Downloaded by guest on September 26, 2021 PNAS PLUS

Fig. 5. A model for the assembly of an Sin3L/Rpd3L subcomplex comprising the Sin3A, Sds3, and HDAC1/2 subunits. The thick blue lines indicate segments of Sin3A that presumably engage HDAC1 largely in an extended conformation. Natively unstructured seg- ments corresponding to the Sds3 CBRs that presumably engage DNA are shown as dashed lines in magenta. Given the flexibility inherent in this segment, the two HDACs in the dimeric complex can potentially engage the same nucleosome.

ITC. ITC experiments were performed on a MicroCal iTC200 Calorimeter 100 mM imidazole (pH 7.2–7.8). The crystals were cryoprotected using the crystal (Malvern). Titrations were performed at 20 °C in 50 mM sodium phosphate growth condition with 25% (vol/vol) glycerol in place of ethanol, the crystalli- buffer (pH 7.7). Proteins were dialyzed overnight against the buffer used for zation precipitant, before freezing in liquid nitrogen. Diffraction data were titrations. Protein concentrations were determined spectrophotometrically collected using a Rayonix CCD Detector (Rayonix) at Life Sciences-Collaborative (40). WT and mutant Sin3 HID proteins (in cell) and the Sds3 SID polypeptides Access Team beamlines (APS, Argonne National Laboratory) at 100 K, indexed, (in syringe) were at initial concentrations of 20–40 μM and 0.2–0.4 mM, re- integrated with XDS (52) or MOSFLM (53), and scaled with AIMLESS (54). Ex- spectively. A single-site binding model in Origin 7.0 was used for analysis. perimental phases were determined with AutoSHARP (55) by single isomorphous replacement with anomalous scattering from five Sds3 LnkCC290-174 SeMet Sin3A HID–Sds3 SID Complex Generation and NMR Sample Preparation. NMR derivative crystals, which were grown and frozen as the native crystals. After samples of Sin3A HID and Sds3 SID were prepared by dissolving the respective solvent flattening by AutoSHARP (55), a nearly complete initial model was proteins in 20 mM sodium phosphate buffer (pH 6.7) containing 4 mM DTT- generated using Buccaneer (56) in CCP4 (57). Iterative rounds of model building

d10, 10% (vol/vol) D2O, 100 mM guanidine hydrochloride, and 0.2% NaN3.A and refinement were performed using Coot (58) and Refmac5 (59), respectively. 1.2-fold excess unlabeled Sds3 SID was added to 15N-/13C-labeled Sin3A HID; Finally, TLS (translation, libration, and small movements) refinement was done similarly, unlabeled Sin3A HID was added to 15N-/13C-labeled Sds3 SID followed using four groups for the structure. The final atomic coordinates for Sds3 by ultrafiltration to remove excess Sds3 SID. Equimolarity was verified by re- LnkCC290-174 were validated using the MolProbity (60) web server. R factors in cording 2D 1H-15N heteronuclear single quantum coherence (HSQC) spectra. For Table 4 were computed as suggested in ref. 61. The structure was analyzed using

D2O experiments, samples were lyophilized and dissolved in 99.996% D2O. SOCKET (62), HELANAL-Plus (63), and MONSTER (50) webservers.

NMR Spectroscopy and Structure Determination. NMR data were acquired on a CoIP Assays. Point mutations were introduced using Q5 Site-Directed Mu- Varian Inova or Agilent DirectDrive 600-MHz Spectrometer equipped with a tagenesis (New England Biolabs) or QuikChange (Agilent) protocol in ex- cold probe at 30 °C. The concentrations used for the structure determination pression plasmids encoding FLAG-tagged mouse Sds3, HA-tagged BRMS1, were in the range of 0.45–1 mM. NMR data processing and analysis were and Myc-tagged Sin3A; all mutations were verified by DNA sequencing. For performed using Felix 98.0 (Felix NMR) and Sparky (41), respectively. Back- coIPs, HEK293T cells were transfected with the indicated plasmids; 48 h after bone and side-chain 1H, 15N, and 13C resonance assignments for Sds3 SID in transfection, cells were washed two times in ice cold PBS and lysed in lysis buffer complex with Sin3A HID were obtained by analyzing 2D 1H-15NHSQC,2D (20 mM Hepes, pH 7.9, 150 mM KCl, 5% (vol/vol) glycerol, 1 mM DTT, 0.1 mM zinc 1 13 15 13 H- C constant time-HSQC, 3D N-edited NOESY, 3D C-edited NOESY, and acetate, 2 mM MgCl2, 2 mM EDTA, 0.2% Nonidet P-40) with protease inhibitors. 2D 15N,13C double half-filtered NOESY (42). Whole-cell extracts were incubated at 4 °C with anti-FLAG M2 affinity gel (Sigma Backbone and side-chain 1H, 15N, and 13C resonance assignments for the Aldrich) for 4 h. After five washes in high-salt buffer (lysis buffer with 300 mM Sin3A HID in complex with Sds3 SID were obtained by analyzing 2D 1H-15N KCl), the samples were boiled in SDS/PAGE loading buffer, and the proteins were HSQC, 1H-13C HSQC, 15N,13C double half-filtered NOESY, CB(CGCD)HD, and resolved by SDS/PAGE and transferred to a nitrocellulose membrane. The blot CB(CGCDCE)HE and 3D HNCA, HN(CO)CA, CBCA(CO)NH, HNCACB, C(CO)NH- was probed with primary anti-Myc (M5546, 1:500 dilution; Sigma-Aldrich), anti- TOCSY, HNCO, HCCH-COSY, and HCCH-TOCSY spectra (43, 44). FLAG (F3165, 1:500 dilution; Sigma-Aldrich), anti-HA (H3663, 1:500 dilution; Backbone ϕ-andψ-dihedral angle restraints for structure calculations were Sigma-Aldrich), or anti-HDAC1 antibodies (sc-7872, 1:400 dilution; Santa Cruz α α β derived from a combined analysis of the 1H , 13C , 13C , 13C′, and backbone 15N Biotechnology) followed by secondary rabbit anti-mouse HRP-conjugated anti- chemical shifts using TALOS+ (45); only residues with reliability scores of 10 in body (OB617005, 1:1,000 dilution; Thermo Fisher Scientific) or HRP-conjugated helical segments were restrained. NOE-based distance restraints were derived goat anti-rabbit (OB405405, 1:1,000 dilution; Thermo Fisher Scientific; only used from two sets of three spectra recorded for each protein in the complex, in- for anti-HDAC1 antibodies). The blots were developed using West Pico Chemi- 15 13 cluding 3D N-edited NOESY (τm = 75 ms) recorded in H2O, 3D C-edited luminescent Substrate (34080; Thermo Fisher Scientific). 13 13 aliphatic NOESY (τm = 60 ms), and 3D C-filtered, C-edited NOESY (τm = 140 ms) recorded in D2O. A small subset of intermolecular NOEs involving a minor Luciferase Assays. Subconfluent HEK293T cells were transfected with GAL4- conformer was detected in the latter experiment; however, the NOEs involving Sin3A fusion constructs along with the firefly luciferase reporter plasmid driven the major conformer were self-consistent and well-resolved from those of the by four GAL4 binding sites upstream of the thymidine kinase promoter and the minor conformer, and they were used for structure determination. Renilla luciferase or β-gal reporter plasmid driven by a CMV promoter for BIOCHEMISTRY Structures were determined using ARIA 1.2 in conjunction with CNS 1.1 starting normalization purposes. Cells were lysed 48 h posttransfection, and whole-cell from an initial structure with extended backbone conformations (46–48). All NOEs extracts were assayed for firefly luciferase activity using the Dual-Luciferase were calibrated automatically and assigned iteratively by ARIA; the assignments Reporter Assay (Promega) and an automated luminometer. Transfection effi- were checked manually for errors. Eighty conformers were calculated; 40 con- ciencies were normalized using the Renilla luciferase or β-gal. formers with the lowest restraint energies were refined in a shell of water, and 20 conformers with the lowest restraint energies and violations and ideal co- 43-234 Limited Proteolysis. Purified His6-tagged Sds3 CCSID was used for limited valent geometry were selected. The final conformers were analyzed using CNS trypsinolysis. Reactions were performed at a 150:1 (wt/wt) protein:enzyme (46), PROCHECK (49), MONSTER (50), and scripts written in house. ratio at room temperature in 200 mM (NH4)2CO3 (pH 8) buffer, stopped using liquid nitrogen, lyophilized, and analyzed by SDS/PAGE and electro- SEC-MALS. SEC-MALS experiments were performed and analyzed exactly as spray ionization MS. described previously (51). Proteins or protein mixtures of 37–100 μMin20mM Tris buffer (pH 7.4) containing 200 mM NaCl and 2 mM DTT or 50 mM Tris buffer EMSAs. Purified GST or GST-Sds3172-209 was incubated with a 168-bp DNA (pH 8.0), 200 mM NaCl, and 2 mM tris(2-carboxyethyl)phosphine were used. sequence in 0.2× TBE (Tris/Borate/EDTA) buffer and resolved by native PAGE in the same buffer, and DNA bands were visualized using ethidium bromide. Crystallography and Structure Determination. The best diffracting crystals of 90-174 Sds3 LnkCC2 were grown by vapor diffusion against 15% (vol/vol) ACKNOWLEDGMENTS. We thank Drs. Robert Eisenman, Donald Ayer, David ethanol, 200 mM MgCl2, and 100 mM imidazole (pH 7.4), although high- Jones, and Kelly Mayo for generously sharing expression constructs and other quality crystals were obtained in 15–22.5% (vol/vol) ethanol, 200 mM MgCl2,and resources. We also thank Annie Bruns, Janny Concha Urday-Zaa, Rebecca

Clark et al. PNAS | Published online June 29, 2015 | E3677 Downloaded by guest on September 26, 2021 Imhoff, Qianyi Luo, Graham Winston, and Maciek Zmyslowski for their Molecular Biophysics Training Grant T32GM008382. C.W.C. was supported by an contributions. We thank the Lurie Cancer Center at Northwestern for Achievement Rewards for College Scientists Foundation Fellowship. Funding for supporting structural biology research. M.D.C. and Y.D. were supported by this work was provided by NIH Grant R01GM64715 (to I.R.) and American Heart Northwestern Summer Undergraduate Research grants. R.M. was supported by Association Grant 14GRNT20170003 (to I.R.).

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