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• W2076634850 abstract "•Improved structural description of the SAGA coactivator•Localization of the deubiquitination module within SAGA•Identification of a TBP-binding clamp•Mapping of the nucleosome binding interface with SAGA The molecular organization of the yeast transcriptional coactivator Spt-Ada-Gcn5 acetyltransferase (SAGA) was analyzed by single-particle electron microscopy. Complete or partial deletion of the Sgf73 subunit disconnects the deubiquitination (DUB) module from SAGA and favors in our conditions the cleavage of the C-terminal ends of the Spt7 subunit and the loss of the Spt8 subunit. The structural comparison of the wild-type SAGA with two deletion mutants positioned the DUB module and enabled the fitting of the available atomic models. The localization of the DUB module close to Gcn5 defines a chromatin-binding interface within SAGA, which could be demonstrated by the binding of nucleosome core particles. The TATA-box binding protein (TBP)-interacting subunit Spt8 was found to be located close to the DUB but in a different domain than Spt3, also known to contact TBP. A flexible protein arm brings both subunits close enough to interact simultaneously with TBP. The molecular organization of the yeast transcriptional coactivator Spt-Ada-Gcn5 acetyltransferase (SAGA) was analyzed by single-particle electron microscopy. Complete or partial deletion of the Sgf73 subunit disconnects the deubiquitination (DUB) module from SAGA and favors in our conditions the cleavage of the C-terminal ends of the Spt7 subunit and the loss of the Spt8 subunit. The structural comparison of the wild-type SAGA with two deletion mutants positioned the DUB module and enabled the fitting of the available atomic models. The localization of the DUB module close to Gcn5 defines a chromatin-binding interface within SAGA, which could be demonstrated by the binding of nucleosome core particles. The TATA-box binding protein (TBP)-interacting subunit Spt8 was found to be located close to the DUB but in a different domain than Spt3, also known to contact TBP. A flexible protein arm brings both subunits close enough to interact simultaneously with TBP. Transcription of protein coding genes by RNA polymerase II is a tightly regulated process that requires the coordinated action of several protein molecules triggering the assembly of the preinitiation complex (PIC) on gene promoters. Sequence-specific transcriptional activators and posttranslational modifications of nucleosomal histones recruit multisubunit coactivator complexes acting as bridging factors between activators and the PIC (Grünberg and Hahn, 2013Grünberg S. Hahn S. Structural insights into transcription initiation by RNA polymerase II.Trends Biochem. Sci. 2013; 38: 603-611Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Coactivators modify promoter chromatin structure and coordinate PIC assembly with epigenetic chromatin modifications. The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex is a paramount example of coactivators (Grant et al., 1997Grant P.A. Duggan L. Côté J. Roberts S.M. Brownell J.E. Candau R. Ohba R. Owen-Hughes T. Allis C.D. Winston F. et al.Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex.Genes Dev. 1997; 11: 1640-1650Crossref PubMed Scopus (881) Google Scholar). In S. cerevisiae, the 1.8 MDa SAGA complex is composed of 19 distinct subunits, most of which have homologs in higher eukaryotes (Nagy and Tora, 2007Nagy Z. Tora L. Distinct GCN5/PCAF-containing complexes function as co-activators and are involved in transcription factor and global histone acetylation.Oncogene. 2007; 26: 5341-5357Crossref PubMed Scopus (302) Google Scholar). The SAGA complex has a modular organization, as evidenced by genetic complementation studies (Eisenmann et al., 1994Eisenmann D.M. Chapon C. Roberts S.M. Dollard C. Winston F. The Saccharomyces cerevisiae SPT8 gene encodes a very acidic protein that is functionally related to SPT3 and TATA-binding protein.Genetics. 1994; 137: 647-657Crossref PubMed Google Scholar) and electron microscopy (EM) models (Wu et al., 2004Wu P.Y. Ruhlmann C. Winston F. Schultz P. Molecular architecture of the S. cerevisiae SAGA complex.Mol. Cell. 2004; 15: 199-208Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Recently, quantitative proteomics established a subunit interaction network and segmented SAGA into four stable modules (Lee et al., 2011Lee K.K. Sardiu M.E. Swanson S.K. Gilmore J.M. Torok M. Grant P.A. Florens L. Workman J.L. Washburn M.P. Combinatorial depletion analysis to assemble the network architecture of the SAGA and ADA chromatin remodeling complexes.Mol. Syst. Biol. 2011; 7: 503Crossref PubMed Scopus (119) Google Scholar). The SPT module contains four proteins (Spt3, Spt7, Spt8, Spt20), originally identified in a genetic screen for suppressors of promoter mutations (Winston and Sudarsanam, 1998Winston F. Sudarsanam P. The SAGA of Spt proteins and transcriptional analysis in yeast: past, present, and future.Cold Spring Harb. Symp. Quant. Biol. 1998; 63: 553-561Crossref PubMed Scopus (47) Google Scholar) clustered with Ada1 and Tra1. Spt3 and Spt8 were shown to directly interact with TATA-box binding protein (TBP) (Mohibullah and Hahn, 2008Mohibullah N. Hahn S. Site-specific cross-linking of TBP in vivo and in vitro reveals a direct functional interaction with the SAGA subunit Spt3.Genes Dev. 2008; 22: 2994-3006Crossref PubMed Scopus (77) Google Scholar) and to modulate positively or negatively its interaction with particular promoters (Warfield et al., 2004Warfield L. Ranish J.A. Hahn S. Positive and negative functions of the SAGA complex mediated through interaction of Spt8 with TBP and the N-terminal domain of TFIIA.Genes Dev. 2004; 18: 1022-1034Crossref PubMed Scopus (53) Google Scholar). The C terminus of Spt7 can be cleaved by the Pep4 protease to form the SAGA-like (SLIK or SALSA) complex, which also lacks Spt8 that interacts with the cleaved part of Spt7 (Pray-Grant et al., 2002Pray-Grant M.G. Schieltz D. McMahon S.J. Wood J.M. Kennedy E.L. Cook R.G. Workman J.L. Yates 3rd, J.R. Grant P.A. The novel SLIK histone acetyltransferase complex functions in the yeast retrograde response pathway.Mol. Cell. Biol. 2002; 22: 8774-8786Crossref PubMed Scopus (185) Google Scholar, Spedale et al., 2010Spedale G. Mischerikow N. Heck A.J. Timmers H.T. Pijnappel W.W. Identification of Pep4p as the protease responsible for formation of the SAGA-related SLIK protein complex.J. Biol. Chem. 2010; 285: 22793-22799Crossref PubMed Scopus (24) Google Scholar). SLIK was shown to be recruited to HIS3 promoter and activate transcription, thereby counteracting the inhibiting role of SAGA on this promoter (Belotserkovskaya et al., 2000Belotserkovskaya R. Sterner D.E. Deng M. Sayre M.H. Lieberman P.M. Berger S.L. Inhibition of TATA-binding protein function by SAGA subunits Spt3 and Spt8 at Gcn4-activated promoters.Mol. Cell. Biol. 2000; 20: 634-647Crossref PubMed Scopus (110) Google Scholar). The Tra1 subunit is shared with the NuA4 complex and was shown to interact with a large set of activator such as Gcn4 and Gal4 (Brown et al., 2001Brown C.E. Howe L. Sousa K. Alley S.C. Carrozza M.J. Tan S. Workman J.L. Recruitment of HAT complexes by direct activator interactions with the ATM-related Tra1 subunit.Science. 2001; 292: 2333-2337Crossref PubMed Scopus (292) Google Scholar, Knutson and Hahn, 2011Knutson B.A. Hahn S. Domains of Tra1 important for activator recruitment and transcription coactivator functions of SAGA and NuA4 complexes.Mol. Cell. Biol. 2011; 31: 818-831Crossref PubMed Scopus (60) Google Scholar). A TBP-associated factor (TAF) module composed of Taf5, Taf6, Taf9, Taf10, and Taf12 is shared with the transcription factor IID (TFIID) complex and is believed to form a structural core on which the other modules are assembled. A core-TFIID complex composed of a similar Taf subset (Taf5, Taf6, Taf9, Taf4, Taf12) was identified (Bieniossek et al., 2013Bieniossek C. Papai G. Schaffitzel C. Garzoni F. Chaillet M. Scheer E. Papadopoulos P. Tora L. Schultz P. Berger I. The architecture of human general transcription factor TFIID core complex.Nature. 2013; 493: 699-702Crossref PubMed Scopus (111) Google Scholar). Its structure and assembly pathway revealed a dimeric core TFIID assembled from a (Taf5-Taf6-Taf9)2 subcomplex shared with SAGA. The two other Tafs present in SAGA (Taf10 and Taf12) contain a histone-fold domain (HFD) and have SAGA-specific HFD-containing interaction partners, Spt7 and Ada1, respectively (Gangloff et al., 2001Gangloff Y.G. Romier C. Thuault S. Werten S. Davidson I. The histone fold is a key structural motif of transcription factor TFIID.Trends Biochem. Sci. 2001; 26: 250-257Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). SAGA harbors a histone acetyl transferase (HAT) activity carried by the Gcn5 subunit and modulated by the Ada2 and Ada3 subunits (Grant et al., 1997Grant P.A. Duggan L. Côté J. Roberts S.M. Brownell J.E. Candau R. Ohba R. Owen-Hughes T. Allis C.D. Winston F. et al.Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex.Genes Dev. 1997; 11: 1640-1650Crossref PubMed Scopus (881) Google Scholar). Together with Sgf29, these subunits form the HAT module, which is a major regulator of histone H3 acetylation in yeast cells. Gcn5 contains a bromodomain, which binds acetylated lysines in histone tails (Hassan et al., 2002Hassan A.H. Prochasson P. Neely K.E. Galasinski S.C. Chandy M. Carrozza M.J. Workman J.L. Function and selectivity of bromodomains in anchoring chromatin-modifying complexes to promoter nucleosomes.Cell. 2002; 111: 369-379Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar), and Sgf29 holds a double Tudor domain capable of binding H3K4me2/3, two hallmarks of actively transcribed chromatin (Vermeulen et al., 2010Vermeulen M. Eberl H.C. Matarese F. Marks H. Denissov S. Butter F. Lee K.K. Olsen J.V. Hyman A.A. Stunnenberg H.G. Mann M. Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers.Cell. 2010; 142: 967-980Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar). A deubiquitination (DUB) module composed of the catalytic Ubp8 subunit, tightly regulated by the Sgf73, Sgf11, and Sus1 subunits, catalyzes the cleavage of monoubiquitin from lysine 123 of histone H2B (Daniel et al., 2004Daniel J.A. Torok M.S. Sun Z.W. Schieltz D. Allis C.D. Yates 3rd, J.R. Grant P.A. Deubiquitination of histone H2B by a yeast acetyltransferase complex regulates transcription.J. Biol. Chem. 2004; 279: 1867-1871Crossref PubMed Scopus (228) Google Scholar, Henry et al., 2003Henry K.W. Wyce A. Lo W.S. Duggan L.J. Emre N.C. Kao C.F. Pillus L. Shilatifard A. Osley M.A. Berger S.L. Transcriptional activation via sequential histone H2B ubiquitylation and deubiquitylation, mediated by SAGA-associated Ubp8.Genes Dev. 2003; 17: 2648-2663Crossref PubMed Scopus (547) Google Scholar). Ubp8 is inactive until interacting with the other DUB module subunits, indicating that all four components are required for optimal enzymatic activity (Köhler et al., 2008Köhler A. Schneider M. Cabal G.G. Nehrbass U. Hurt E. Yeast Ataxin-7 links histone deubiquitination with gene gating and mRNA export.Nat. Cell Biol. 2008; 10: 707-715Crossref PubMed Scopus (160) Google Scholar). The Sgf73 and Sgf11 subunits harbor the conserved SCA7 and Zinc finger domains, respectively, which have been shown to interact with nucleosomes (Bonnet et al., 2010Bonnet J. Wang Y.H. Spedale G. Atkinson R.A. Romier C. Hamiche A. Pijnappel W.W. Timmers H.T. Tora L. Devys D. Kieffer B. The structural plasticity of SCA7 domains defines their differential nucleosome-binding properties.EMBO Rep. 2010; 11: 612-618Crossref PubMed Scopus (26) Google Scholar, Koehler et al., 2014Koehler C. Bonnet J. Stierle M. Romier C. Devys D. Kieffer B. DNA binding by Sgf11 protein affects histone H2B deubiquitination by Spt-Ada-Gcn5-acetyltransferase (SAGA).J. Biol. Chem. 2014; 289: 8989-8999Crossref PubMed Scopus (19) Google Scholar). The incorporation of a long polyglutamine expansion into ATXN7, the human homolog of Sgf73, leads to type 7 spinocerebellar ataxia (David et al., 1997David G. Abbas N. Stevanin G. Dürr A. Yvert G. Cancel G. Weber C. Imbert G. Saudou F. Antoniou E. et al.Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion.Nat. Genet. 1997; 17: 65-70Crossref PubMed Scopus (695) Google Scholar, Helmlinger et al., 2004Helmlinger D. Hardy S. Sasorith S. Klein F. Robert F. Weber C. Miguet L. Potier N. Van-Dorsselaer A. Wurtz J.M. et al.Ataxin-7 is a subunit of GCN5 histone acetyltransferase-containing complexes.Hum. Mol. Genet. 2004; 13: 1257-1265Crossref PubMed Scopus (193) Google Scholar). Structural information is required to understand the mechanisms of SAGA-dependent gene activation and the crosstalk between the multiple functions integrated in the SAGA complex. EM has been used to determine a 3D model of the SAGA complex and to map 9 of the 19 SAGA subunits (Wu et al., 2004Wu P.Y. Ruhlmann C. Winston F. Schultz P. Molecular architecture of the S. cerevisiae SAGA complex.Mol. Cell. 2004; 15: 199-208Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The atomic structure of the DUB module shows a compact organization with a highly intertwined subunits interaction network (Köhler et al., 2010Köhler A. Zimmerman E. Schneider M. Hurt E. Zheng N. Structural basis for assembly and activation of the heterotetrameric SAGA histone H2B deubiquitinase module.Cell. 2010; 141: 606-617Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, Samara et al., 2010Samara N.L. Datta A.B. Berndsen C.E. Zhang X. Yao T. Cohen R.E. Wolberger C. Structural insights into the assembly and function of the SAGA deubiquitinating module.Science. 2010; 328: 1025-1029Crossref PubMed Scopus (166) Google Scholar). The DUB module forms two functional lobes, organized around the two Ubp8 domains. The catalytic lobe is formed by the C-terminal domain of Ubp8 and the C-terminal zinc-finger domain of Sgf11. The assembly lobe is organized around a long Sgf11 helix. The N-terminal 104 residues of Sgf73 are located between the two Ubp8 domains where they potentiate Ubp8 activity and anchor the DUB module to the SAGA complex. To locate the DUB module within the SAGA complex, we have determined the 3D structure of SAGA complexes in which the N-terminal part of Sgf73 or the full Sgf73 subunit has been deleted to remove the DUB module. The module is found in the vicinity of Gcn5 and Spt7, thus defining a nucleosome interaction interface within the SAGA complex. In our hands, the deletion of the DUB module leads to an increased processing of the C-terminal part of Spt7 and consequently to the loss of Spt8. These subunits were also mapped and found in the same region as the DUB module, which was unexpected since Spt3, the other TBP-interacting protein, is found in a different domain. Saccharomyces cerevisiae SAGA was purified from a wild-type (WT) strain TAP tagged at the C terminus of Spt20, and its protein composition was consistent with previous results (Figure 1A). The Spt7 subunit migrated as two bands with apparent molecular weights of 250 kDa for the full length and 230 kDa for the truncated form of Spt7, which incorporates into the SLIK complex (Pray-Grant et al., 2002Pray-Grant M.G. Schieltz D. McMahon S.J. Wood J.M. Kennedy E.L. Cook R.G. Workman J.L. Yates 3rd, J.R. Grant P.A. The novel SLIK histone acetyltransferase complex functions in the yeast retrograde response pathway.Mol. Cell. Biol. 2002; 22: 8774-8786Crossref PubMed Scopus (185) Google Scholar, Spedale et al., 2010Spedale G. Mischerikow N. Heck A.J. Timmers H.T. Pijnappel W.W. Identification of Pep4p as the protease responsible for formation of the SAGA-related SLIK protein complex.J. Biol. Chem. 2010; 285: 22793-22799Crossref PubMed Scopus (24) Google Scholar). In our conditions, these two bands are of similar intensity, suggesting that SLIK and SAGA are equally represented. Samples were analyzed by multidimensional protein identification technology (MudPIT) (Washburn et al., 2001Washburn M.P. Wolters D. Yates 3rd, J.R. Large-scale analysis of the yeast proteome by multidimensional protein identification technology.Nat. Biotechnol. 2001; 19: 242-247Crossref PubMed Scopus (4081) Google Scholar), and the relative abundance of SAGA subunits was estimated by normalized spectral abundance factors (NSAFs) (Zybailov et al., 2006Zybailov B. Mosley A.L. Sardiu M.E. Coleman M.K. Florens L. Washburn M.P. Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae.J. Proteome Res. 2006; 5: 2339-2347Crossref PubMed Scopus (819) Google Scholar) (Figure 1B). All 19 SAGA subunits were identified among the most abundant proteins, and the NSAF values were in similar ranges, suggesting that all subunits are present in the same copy number except for Sus1, which was detected at higher abundance. The structural homogeneity of the sample was checked by EM of negatively stained molecules (Figure 1C). A sgf73Δ1–104 strain was generated from the WT strain by deleting residues 1–104 of Sgf73 necessary to assemble the DUB module. As expected, the four DUB module subunits were absent from the sgf73Δ1–104 SAGA, as shown by gel electrophoresis (Figure 1A) and mass spectrometry (Figure 1B), except for the Sgf73 subunit for which peptides corresponding to the C terminus were still detected. Interestingly, peptides from the C-terminal part (after position 1,142) of Spt7 were strongly depleted and the upper Spt7 band is missing in SDS-PAGE (Figure 1A). The cleaved part of Spt7 holds the Spt8 binding site (Spedale et al., 2010Spedale G. Mischerikow N. Heck A.J. Timmers H.T. Pijnappel W.W. Identification of Pep4p as the protease responsible for formation of the SAGA-related SLIK protein complex.J. Biol. Chem. 2010; 285: 22793-22799Crossref PubMed Scopus (24) Google Scholar), and consistently, mass spectrometry showed that Spt8 is also missing, indicating that the sgf73Δ1–104 SAGA is fully converted into the SLIK form. Such a comprehensive Spt7 cleavage upon removal of the DUB module was not observed previously (Bian et al., 2011Bian C. Xu C. Ruan J. Lee K.K. Burke T.L. Tempel W. Barsyte D. Li J. Wu M. Zhou B.O. et al.Sgf29 binds histone H3K4me2/3 and is required for SAGA complex recruitment and histone H3 acetylation.EMBO J. 2011; 30: 2829-2842Crossref PubMed Scopus (174) Google Scholar, Köhler et al., 2010Köhler A. Zimmerman E. Schneider M. Hurt E. Zheng N. Structural basis for assembly and activation of the heterotetrameric SAGA histone H2B deubiquitinase module.Cell. 2010; 141: 606-617Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) and may reflect slight differences in genetic background, growth conditions, or purification setting. The proteomic analysis of a second mutant with a full SGF73 deletion (sgf73Δ strain) confirmed that the four subunits of the DUB module were missing and that Spt7 was preferentially cleaved resulting in low Spt8 abundance (Figures 1A and 1B). Negatively stained WT SAGA molecules were processed for EM, and the analysis of 19.226 images revealed characteristic SAGA views showing five domains annotated from I to V and organized into two major lobes containing domains I and II (lobe A) and domains III, IV, and V (lobe B) (Figure 2A). The 3D model calculated from this data set revealed three protein connections between domains I and II in lobe A, as well as large solvent-accessible channels (Figure 2B). Lobe B adopts a molecular clamp-shaped architecture in which domains III and V protrude from a large crescent-shaped domain IV. The major connection between the two lobes occurs through domains II and IV, while a faint connection is observed between domains I and III. In order to analyze possible conformational changes, a 3D classification scheme based on regularized likelihood optimization was used to form four classes (Figure 2C) (Scheres, 2010Scheres S.H. Classification of structural heterogeneity by maximum-likelihood methods.Methods Enzymol. 2010; 482: 295-320Crossref PubMed Scopus (70) Google Scholar). While the structure of lobe A did not fluctuate, lobe B showed large conformational changes. A major variation is evidenced by an opening and closing of the clamp. In the most closed configuration, the two domains are in contact and form a protein ring, while in the more opened state, the two domains are separated by 7 nm. Relative movements between lobes A and B were also evidenced such as rotations along the long molecular axis and around the connection between domains II and IV. To correct for these movements, each lobe was aligned independently to improve the resolution of structural details. Such an approach has been used previously (Sauerwald et al., 2013Sauerwald A. Sandin S. Cristofari G. Scheres S.H. Lingner J. Rhodes D. Structure of active dimeric human telomerase.Nat. Struct. Mol. Biol. 2013; 20: 454-460Crossref PubMed Scopus (99) Google Scholar) and is applicable because the two lobes show little overlap in the SAGA views. While little resolution gain was obtained for lobe A, the structural description of lobe B improved significantly (Figure 2D; see also Figures S1 and S2 and Table S1 available online). In particular, domain III is more extended and harbors a protein density only drafted in the uncorrected model. Domain V grows in size and shows more structural details such as a constriction in the connection to domain IV. Finally, the connection between domains I and III is strengthened when lobe B is analyzed separately. SAGA complexes purified from the sgf73Δ1–104 or sgf73Δ strains were analyzed to map the DUB module. A 3D model of sgf73Δ SAGA was determined from 30.409 negatively stained molecules by analyzing each lobe independently. Lobe A is similar in the mutant and in the WT SAGA models (data not shown), thus excluding the possibility that it hosts the 137 kDa DUB module. In contrast, large differences are detected in lobe B (Figure 3B; see also Figure S3 and Movie S1). The largest difference reflects the absence of domain V the sgf73Δ SAGA complexes (red density marked by an arrowhead in Figure 3B). While domain V is not visible in sgf73Δ SAGA, the analysis of the sgf73Δ1–104 mutant clearly revealed the flexible domain V with similar dimensions than in WT SAGA (Figure 3A). This observation indicates that domain V does not contain the DUB module. A similarly sized volume is found inserted at the bottom of the sgf73Δ SAGA cleft, while it is absent in the WT SAGA (blue density marked by an arrowhead in Figure 3B). This observation shows that the position of domain V is different in the mutant SAGA complexes. It probably folds back into the cleft when the C-terminal part of Sgf73 is removed and produces the additional density, although a more profound reorganization of lobe B cannot be ruled out (Figure 3C). A second large density missing in the mutant SAGA complex is located at the tip of domain III, and the size of this density is compatible with that of the DUB module (red density marked by an asterisk in Figure 3B). The crystal structure of the quaternary complex containing the full-length Ubp8, Sgf11, and Sus1 and the N-terminal fragment of Sgf73 (Köhler et al., 2010Köhler A. Zimmerman E. Schneider M. Hurt E. Zheng N. Structural basis for assembly and activation of the heterotetrameric SAGA histone H2B deubiquitinase module.Cell. 2010; 141: 606-617Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) was fitted into this missing volume in a unique position since the location of the Ubp8 domain composed of residues 281–303 and 326–353 forming three β strands and two loops was discriminative (Figure 3D). The density difference map between the WT and the sgf73Δ SAGA complexes revealed a third significant difference located at the interface between domain III and domain I (red arrow in Figure 3B). This density could correspond to the 189 C-terminal residues of Spt7 (21.6 kDa) and to Spt8 (66 kDa) lost during proteolysis. Consistently, previous labeling experiments mapped the C terminus of Spt7 in lobe III (Wu et al., 2004Wu P.Y. Ruhlmann C. Winston F. Schultz P. Molecular architecture of the S. cerevisiae SAGA complex.Mol. Cell. 2004; 15: 199-208Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). In order to confirm the position of Spt8, subunit-specific antibodies were incubated with purified WT SAGA. The analysis of 1,227 images shows the antibody molecule bound to domain III in a position consistent with the density detected in the difference map (Figures 4A and 4B ). The SAGA coactivator harbors several nucleosome binding motifs all located in lobe B (Figure 4D). In order to verify whether nucleosome core particles (NCPs) can interact with SAGA and to map its binding interface, coactivator complexes were immobilized on calmodulin sepharose beads and incubated with a 7.5-fold molar excess of recombinant NCPs formed from full-length bacterially expressed recombinant Xenopus laevis core histones assembled onto a 196 bp long 601 nucleosome positioning sequence (Syed et al., 2010Syed S.H. Goutte-Gattat D. Becker N. Meyer S. Shukla M.S. Hayes J.J. Everaers R. Angelov D. Bednar J. Dimitrov S. Single-base resolution mapping of H1-nucleosome interactions and 3D organization of the nucleosome.Proc. Natl. Acad. Sci. USA. 2010; 107: 9620-9625Crossref PubMed Scopus (157) Google Scholar). The complexes were briefly washed and eluted before EM preparation and observation. The analysis of 3,909 negatively stained complexes revealed that about 24% of the SAGA particles were bound to NCPs, thus indicating the possibility for SAGA to interact with unmodified NCPs. Class averages calculated from SAGA-NCP images showed a bound density adjacent to domain III and consistent with the size of a nucleosome (Figure 4C). Both round and elongated densities are attached to SAGA, suggesting that the NCP orientation can fluctuate. The architecture of the SAGA coactivator was revisited by analyzing a large image data set and by considering its molecular heterogeneity and flexibility (Figure S2 and Movie S1). Lobe A appears extremely stable, and its organization into two domains interconnected by three protein links did not show significant conformational changes. Antibody labeling experiments showed that the crescent-shaped domain I contains the Tra1 subunit (Wu et al., 2004Wu P.Y. Ruhlmann C. Winston F. Schultz P. Molecular architecture of the S. cerevisiae SAGA complex.Mol. Cell. 2004; 15: 199-208Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). This part of SAGA was found structurally similar to the NuA4 complex, which is also organized into two domains (Chittuluru et al., 2011Chittuluru J.R. Chaban Y. Monnet-Saksouk J. Carrozza M.J. Sapountzi V. Selleck W. Huang J. Utley R.T. Cramet M. Allard S. et al.Structure and nucleosome interaction of the yeast NuA4 and Piccolo-NuA4 histone acetyltransferase complexes.Nat. Struct. Mol. Biol. 2011; 18: 1196-1203Crossref PubMed Scopus (64) Google Scholar). This similarity probably reflects the functional homology between the two complexes in activator binding and HAT activity. Not only is the overall shape conserved but also the number and position of connections with Tra1, suggesting that Tra1 shares the same interaction interfaces with the SAGA or NuA4 subunits. A recent genetic and functional dissection of Tra1 is consistent with this observation since the same nonviable Tra1 mutations both affected SAGA and NuA4 complex stability (Knutson and Hahn, 2011Knutson B.A. Hahn S. Domains of Tra1 important for activator recruitment and transcription coactivator functions of SAGA and NuA4 complexes.Mol. Cell. Biol. 2011; 31: 818-831Crossref PubMed Scopus (60) Google Scholar). The modular organization of SAGA determined by quantitative mass spectrometry (Lee et al., 2011Lee K.K. Sardiu M.E. Swanson S.K. Gilmore J.M. Torok M. Grant P.A. Florens L. Workman J.L. Washburn M.P. Combinatorial depletion analysis to assemble the network architecture of the SAGA and ADA chromatin remodeling complexes.Mol. Syst. Biol. 2011; 7: 503Crossref PubMed Scopus (119) Google Scholar) has proposed an interaction between Tra1 and subunits of the SPT module. Spt3 and Spt20 map in the B lobe at a distance from Tra1 that precludes direct interaction (Wu et al., 2004Wu P.Y. Ruhlmann C. Winston F. Schultz P. Molecular architecture of the S. cerevisiae SAGA complex.Mol. Cell. 2004; 15: 199-208Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Our results, however, indicate that Spt8 and possibly the C terminus of Spt7 form a bridge between domains III and I to contact Tra1, thus giving a hint to the observed interaction. The DUB module is located at the tip of domain III, and the fitting of its atomic coordinates shows that the ubiquitin binding and the catalytic sites are solvent exposed in a suitable orientation to interact with chromatin (Figure 3D). The DUB module is positioned close to Gcn5, which may explain the reported crosstalks between the two enzymatic activities. In mammalian cells, the deletion of Gcn5 impairs the association of the DUB module with SAGA (Atanassov et al., 2009Atanassov B.S. Evrard Y.A. Multani A.S. Zhang Z. Tora L. Devys D. Chang S. Dent S.Y. Gcn5 and SAGA regulate shelterin protein turnover and telomere maintenance.Mol. Cell. 2009; 35: 352-364Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), while in yeast, the full deletion of Sgf73 compromises the HAT activity, which can be restored by expressing human ATXN7 (McMahon et al.," @default.
• W2076634850 created "2016-06-24" @default.
• W2076634850 creator A5004177060 @default.
• W2076634850 creator A5007129745 @default.
• W2076634850 creator A5011086141 @default.
• W2076634850 creator A5012113257 @default.
• W2076634850 creator A5052498269 @default.
• W2076634850 date "2014-11-01" @default.
• W2076634850 modified "2023-10-13" @default.
• W2076634850 title "Mapping the Deubiquitination Module within the SAGA Complex" @default.
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