![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 17, Issue 10, 4228-4236, October 2006
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,
,
*Biochemie-Zentrum der Universität Heidelberg, INF328, D-69120, Heidelberg, Germany; University Children's Hospital Heidelberg, INF150, D-69120, Heidelberg, Germany; Centro de Investigación Príncipe Felipe, E-46013, Valencia, Spain; and ||Cell Signaling Unit, Departament de Ciencies Experimentals i de la Salut, Universitat Pompeu Fabra, E-08003, Barcelona, Spain
Submitted February 3, 2006;
Revised June 20, 2006;
Accepted July 11, 2006
Monitoring Editor: Susan Wente
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
cells. Thus, the Sus1–Sgf11–Ubp8 module could work at the junction between SAGA-dependent transcription and nuclear mRNA export. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Rodríguez et al., 2004; Aguilera, 2005). Gaining access to chromatin is a prerequisite for transcription and requires a conversion from inactive to active chromatin states. Histone-modifying enzymes impose a pattern of posttranslational modifications that can either activate or repress transcription, depending on the type and location of the modification on the histone octamer (Jenuwein and Allis, 2001; Cosgrove et al., 2004; Mellor, 2005). The 1.8-MDa Spt/Ada/Gcn5 acetyltransferase (SAGA) complex is one of the best studied examples of a histone modifier, with acetylating and deubiquitinylating activities, that regulates RNA polymerase II (Grant et al., 1997; Henry et al., 2003).
The SAGA complex contains a small protein Sus1, which was shown to be present also in the Sac3–Thp1–Cdc31 mRNA export complex (Fischer et al., 2004; Rodríguez-Navarro et al., 2004). The Sac3–Thp1–Cdc31–Sus1 complex binds to nucleoporins at the nuclear basket, and in concert with the export receptor Mex67-Mtr2, it mediates nuclear export of messenger ribonucleoprotein particles (Fischer et al., 2004). Consequently, Sus1 has functional roles in both mRNA export and transcriptional regulation. This suggests that Sus1 could be a physical bridging factor between a transcriptional coactivator and nuclear pore-associated mRNA export factors (Rodríguez et al., 2004). Recently, Sus1 was shown to be involved in the repositioning and subsequent confinement of dynamic motility of the GAL1 locus to the nuclear periphery upon transcriptional activation (Cabal et al., 2006).
SAGA is organized into distinct domains that have been extensively studied by biochemical, genetic, and structural methods. Electron-microscopic (EM) analyses of human TATA-binding protein (TBP)-associated factor-containing complex and yeast SAGA complexes together with immuno-EM labeling of subunits have revealed specialized structural and functional modules. These modules include components for histone acetylation and deubiquitinylation, activator interactions, TBP regulation, and components that constitute the SAGA structural backbone (Brand et al., 1999; Wu et al., 2004).
SAGA-dependent modification of histones plays an important role in the regulation of gene expression. The SAGA acetyltransferase Gcn5 has been implicated in acetylation of histones H3 and H2B, which is necessary for full transcriptional activation (Candau et al., 1997; Grant et al., 1999; Berger, 2002; Carrozza et al., 2003). Ubp8, the SAGA-deubiquitylating enzyme, removes ubiquitin moieties from histone H2B (Holstege et al., 1998; Sanders et al., 2002; Henry et al., 2003; Daniel et al., 2004). Different studies have demonstrated that Lys-123 at the carboxy terminus of H2B is ubiquitinylated by the E2/E3 enzymes Rad6/Bre1 (Robzyk et al., 2000; Hwang et al., 2003; Wood et al., 2003). H2B monoubiquitinylation induces a trans-tail methylation of histone H3 at Lys-4 and Lys-79 mediated by Set1 and Dot1, respectively (Briggs et al., 2002; Sun and Allis, 2002). H2B ubiquitinylation rises early during gene activation and declines shortly afterward. Both the addition and subsequent removal of ubiquitin seem to be required for optimal gene activation (Henry et al., 2003).
Recently, Sgf11, a SAGA subunit (Lee et al., 2004; Powell et al., 2004), has been shown to be important for Ubp8 association with SAGA. Several lines of evidence suggest that Sgf11 and Ubp8 form a distinct functional module inside of SAGA required for deubiquitinylation of H2B (Ingvarsdottir et al., 2005; Lee et al., 2005). Moreover, a recent study has shown that Ubp8 differentially regulates Lys-4 methylation of histone H3 in vivo (Shukla et al., 2006).
In this study, we show that Sus1 binds to the SAGA complex through Ubp8 and Sgf11 and thereby extend the concept of a deubiquitinylating module into a heterotrimeric subcomplex. Moreover, Sus1 is required for Ubp8 binding to the GAL1 promoter under conditions of transcription activation. In vivo, Sus1 is required for H2B deubiquitinylation and global H3 methylation. Furthermore, deletion of SGF11 from cells aggravates the growth and mRNA export defects that are detected in sus1 cells. Together, the data suggest that Sus1, Ubp8, and Sgf11 are organized in a functional module of the SAGA complex that could couple transcription with mRNA export.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Chromosomal integration of green fluorescent protein (GFP) (HIS3/KANMX6 marker), TAP (TRP1 marker), MYC (HIS3 marker), and hemagglutinin (HA) (HIS3 marker) as C-terminal tags was performed as described previously (Longtine et al., 1998; Gavin et al., 2002). TAP-tagged strains with deletions were obtained by polymerase chain reaction (PCR)-targeted disruption of the gene of interest or by mating individual TAP-tagged strains and deletion strains. Gene deletion strains that were not obtained from EUROSCARF were made by transformation with a PCR disruption cassette derived from pFA6a-KanMX6 (Longtine et al., 1998) and from pRS425-LEU2 (Brachmann et al., 1998) plasmids. Strains were grown under standard conditions. For growth analysis, yeast cells were diluted to 0.5 OD600, and serial dilutions (1:10) were spotted onto YPD and incubated at various temperatures.
|
). TAP-purified complexes were analyzed by SDS-PAGE by using Novex 4–12% gradient gels (Invitrogen, Carlsbad, CA) and visualized by staining with PageBlue protein-staining Coomassie (MBI Fermentas, Hanover, MD). For isolation of a Sus1–Sgf11–Ubp8 subcomplex, IgG-bound Sus1-TAP was incubated with a buffer containing 1 M MgCl2, 50 mM Tris-HCl, pH 6.0, 0.5 mM dithiothreitol for 1 h at 4°C. Resin was then washed with 50 bed volumes of the same buffer followed by 5 volumes of TAP buffer. All other steps were performed essentially as published previously (Gavin et al., 2002). The protein bands were excised, digested with trypsin, and analyzed by matrix-assisted laser desorption ionization/time of flight (TOF)/TOF mass spectrometry (Voyager 4700; Applied Biosystems, Foster City, CA). Total amounts of H3 and H4 were analyzed using anti-TAP (Open Biosystems, Huntsville, AL), anti-H3, and anti-H4 (Abcam, Cambridge, United Kingdom) antibodies following the manufacturer's protocol.
Global levels of ubH2B were assessed by using YZ6276, YZ6276sus1, YZ6276ubp8, and YZ6276sgf11 strains expressing FLAG-tagged H2B (Robzyk et al., 2000). Whole-cell extracts were prepared in 20% trichloroacetic acid to precipitate histones, and the resuspended precipitates were either used directly for Western blot analysis or used for immunoprecipitation with anti-FLAG M2 agarose (Sigma-Aldrich, St. Louis, MO). The immunoprecipitates were washed twice in immunoprecipitation (IP) buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.5% NP-40, 0.5% bovine serum albumin, protease inhibitors) and three times in IP buffer without bovine serum albumin. Elution was performed with 300 ng of FLAG peptide (generous gift from Dr. Saus, Centro de Investigación Príncipe Felipe), and input and eluate samples were subjected to SDS-PAGE and Western blotting with anti-H2B (Abcam) and anti-FLAG antibodies (Sigma-Aldrich), respectively. Global levels of H3 methylation were tested with antibodies against K4 monomethylated H3 and K79 trimethylated H3 (Abcam) following the manufacturer's protocol.
Chromatin Immunoprecipitation (ChIP)
PCR assays were performed as described previously (De Nadal et al., 2004). In all ChIP experiments, yeast cultures were grown to early log phase (0.6 OD600) before cells were transferred to YPG for 5 h. The oligonucleotides used to amplify the GAL1 chromosomal region were located from –537 to –216 base pairs with respect to ATG. TEL corresponds to a 177-base pair region located 500 base pairs before the right end of chromosome VI. Quantification was performed using Quantity One software (Bio-Rad, Hercules, CA).
Miscellaneous
Sus1-GFP localization was performed with exponentially growing cells using a Leica DM6000B fluorescence microscope (Leica, Wetzlar, Germany) with a 63x PL APO objective. Localization of poly(A)+ RNA by in situ hybridization was performed using a Cy3 end-labeled oligo(dT)50 as described in Santos-Rosa et al. (1998).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Notably, when Ada2-TAP (Figure 1A) or Taf6-TAP (our unpublished data) were affinity purified from a yeast strain lacking chromosomal SUS1 (sus1), most of the SAGA subunits still coprecipitated, but a band at 
53 kDa, which we identified as Ubp8, was specifically absent (our unpublished data). To demonstrate that binding of Ubp8 to SAGA depends on Sus1, we purified Ada2-TAP from wild-type and sus1 cells that express myc-tagged Ubp8. Mass spectrometry of the corresponding band in the wild-type strain and Western blot analysis revealed that Ubp8 does not copurify with SAGA when Sus1 is absent (Figure 1A). These data suggested that Sus1 could be required for the association of Ubp8 with SAGA.
|
strain, but it was readily detectable when isolated from an isogenic UBP8+ wild-type strain (Figure 1A). Together, these analyses revealed that both Sus1 and Ubp8 are required for their binding to the SAGA complex.
The observation that Ubp8 is necessary for binding of Sus1 to SAGA prompted us to test whether Sus1 is still associated with its second complex, the Sac3–Thp1–Cdc31 complex, when Ubp8 is absent. It was suggested that Sus1 via Cdc31 binds to this mRNA export complex that is located at the nuclear site of the NPC (Fischer et al., 2004). Sus1 together with Cdc31 binds to the Ct-interacting domain motif, which is present in the Sac3 carboxy-terminal domain. Sus1-TAP affinity purified from the ubp8 strain was still associated with the subunits of the mRNA export complex (Sac3, Thp1, and Cdc31), but remarkably, members of the SAGA complex were absent (Figure 1B). However, a protein of 11 kDa, which was identified as the SAGA component Sgf11 by mass spectrometry, was still coenriched with Sus1-TAP isolated from the ubp8 strain. Ubp8 association with SAGA requires a putative zinc (Zn) finger domain in the N-terminal region. To determine whether this domain was also required for the Sus1 interaction with SAGA, we TAP tagged Sus1 in two Ubp8 mutants with substitutions in its Zn finger domain (Ingvarsdottir et al., 2005). As shown for the complete deletion of Ubp8, these Zn finger mutants also cause the loss of Sus1 from SAGA (Figure 1B). Together, these results suggest that wild-type Ubp8 is required for interaction of Sus1 with the SAGA complex.
To test whether Sgf11 is also required to recruit Sus1 to the SAGA complex, we affinity purified Sus1-TAP from an sgf11 strain. Similar to the ubp8 strain, Sus1-TAP no longer coprecipitated the SAGA complex when isolated from the sgf11 strain, but interaction with the Sac3 complex was not impaired (Figure 1B). Notably, Ubp8 does not copurify with Sus1 in the absence of Sgf11, whereas Sgf11 is still present in Sus1-TAP when Ubp8 is deleted or mutated (see above). These data demonstrate that the interaction of Sus1 with SAGA requires Ubp8 and Sgf11 and suggest that Sgf11 could be the direct binding partner of Sus1.
A Salt-Stable Sus1–Sgf11–Ubp8 Subcomplex Can Be Dissociated from SAGA
To further investigate whether Sus1, Sgf11, and Ubp8 could constitute a stable subcomplex of SAGA, we affinity purified Sus1-TAP from yeast lysates, but before eluting the immobilized Sus1 from IgG-Sepharose with tobacco etch virus (TEV) protease, the beads were treated with increasing concentrations of MgCl2. Interestingly, most of the SAGA subunits (e.g., Tra1 and Spt7) were released from beads bound with Sus1 at 1 M MgCl2, whereas Ubp8, Sgf11, Cdc31, Thp1, and Sac3 were specifically retained along with Sus1 (Figure 1C). Thus, high MgCl2 concentrations could dissociate most SAGA subunits from Sus1, but Ubp8 and Sgf11 stayed bound. Moreover, Sus1 remained associated with its second complex (Sac3, Thp1, and Cdc31) under conditions of high MgCl2. Notably, when immobilized Sus1-TAP was incubated with 1.2 M MgCl2, Ubp8 but not Sgf11 was dissociated (our unpublished data), supporting the idea that Sgf11 could be more tightly bound to Sus1 than Ubp8. Together, the biochemical data suggest that Sus1, Sgf11, and Ubp8 form a structural entity within the SAGA holoenzyme.
Sus1 and Ubp8 Are Codependent for Their Recruitment to the GAL1 Promoter
Previously, we showed that Sus1 is recruited to the GAL1 promoter after transcriptional activation with galactose (Rodríguez-Navarro et al., 2004). That Sus1 does not coprecipitate with SAGA in the absence of Ubp8 suggested that Ubp8 might be required for Sus1 recruitment to chromatin. To test this possibility, we carried out ChIP assays of Sus1 to the GAL1 promoter after induction with galactose in wild-type and ubp8 cells. After galactose induction, Sus1 immunopurified from wild-type cells specifically purified a DNA fragment corresponding to the GAL1 promoter, indicating that Sus1 is bound to the chromatin. Interestingly, in ubp8 cells no specific purification of the DNA fragment containing the GAL1 promoter region compared with the control fragment (TEL) was observed. This analysis revealed that Sus1 recruitment to the GAL1 promoter depends on Ubp8 (Figure 2A).
|
To further understand how Sus1 binds to chromatin, we analyzed whether SAGA integrity is necessary for promoter recruitment of Sus1. Therefore, we carried out ChIP assays of Sus1 in wild-type and spt20 cells. Sus1 was not present at the GAL1 promoter in an spt20 mutant (Figure 2C), which is known to have a severely disrupted SAGA structure (Grant et al., 1997; Sterner et al., 1999). Thus, we conclude that Sus1 cannot be targeted to the GAL1 promoter independently but requires a functionally intact SAGA complex. These data are complementary to a recent report demonstrating that Ubp8 recruitment to chromatin equally depends on SPT20 (Shukla et al., 2006).
Considering the potential role of Sus1 in histone modification, we next asked whether it could also be involved in SAGA targeting to chromatin. To test this possibility, we performed ChIP assays of two different SAGA subunits in wild-type and sus1 cells. Although a slight reduction in the binding of Spt20 and Ada2 to the GAL1 promoter was observed for the sus1 mutant (Figure 2, D and E), we conclude that Sus1 is dispensable for SAGA recruitment to chromatin. These results corroborate the recent findings from Shukla et al. (2006) in which it was shown that Ubp8 does not affect Spt20, Taf10, or Taf12 recruitment to the GAL1 upstream-activating sequence.
Intranuclear Localization of Sus1 Depends Upon Ubp8 and Sgf11
Sus1 is bound to two different complexes: the SAGA complex, which shows a predominantly intranuclear localization, and the Sac3 complex, which is associated with the nuclear pore. Accordingly, Sus1 is located in the nucleoplasm with a modest concentration at the nuclear periphery in wild-type cells (Rodríguez-Navarro et al., 2004). Interestingly, the localization of Sus1-GFP at the nuclear periphery significantly increased in ubp8- or sgf11-deleted cells (Figure 3). In addition, we analyzed the preferential Sus1 localization in the SAGA mutant ada2. As expected, deletion of Ada2 did not cause a change in the intranuclear localization of Sus1 (Figure 3). These data suggest that Sus1 is redistributed to the nuclear envelope, possibly the NPC (i.e., the Sac3 complex), when binding to the SAGA complex and that recruitment to chromatin are inhibited.
|
; Lee et al., 2005) suggested to us that Sus1 could also be important for the deubiquitinylation of histones. Hence, SUS1 was disrupted in a strain that expressed FLAG-tagged H2B to facilitate detection of ubiquitinylated and unmodified H2B by Western blot analysis (Robzyk et al., 2000). As anticipated, although similar levels of unmodified H2B were detected in whole-cell extracts, the amount of ubiquitinylated H2B increased significantly in a sus1 strain compared with wild type (Figure 4A). In addition, we found that ubH2B levels in sus1 are similar to those present in ubp8 and sgf11, reinforcing the functional link between the three members of the SAGA subcomplex. Therefore, we conclude that Sus1 is required for H2B deubiquitinylation.
|
; Sun and Allis, 2002), we analyzed the role of Sus1 in H3 methylation. H3 K4 monomethylation and H3 K79 trimethylation levels in total yeast extract were compared in wild-type and sus1 cells. These analyses revealed that the amount of H3 methylation at K4 and K79 residues is increased in cells lacking SUS1 (Figure 4B). Thus, Sus1 acts in the same pathway as Ubp8 with respect to H2B deubiquitinylation and H3 methylation.
Coenrichment of Histones During SAGA Purification Depends on Sus1 and Ubp8
Notably, Sus1-TAP copurifies Coomassie-stainable amounts of histones H2A, H2B, H3, and H4 (Rodríguez-Navarro et al., 2004). To investigate the role of Sus1–Ubp8–Sgf11 for the interaction of SAGA with histones, we compared the amount of coenriched histones H3 and H4 during affinity purification of Sus1-TAP from wild-type and ubp8 or sgf11 cells. Whereas similar levels of H3 and H4 were present in the lysates of each strain, coprecipitation of these histones with Sus1-TAP after the TEV and EGTA elutions was largely abolished when either UBP8 or SGF11 was disrupted (Figure 5A). To find out whether purification of a bona fide SAGA subunit also leads to coenrichment of histones, we tested affinity-purified Ada2-TAP. As expected, Ada2 significantly coenriched H3 and H4 (Figure 5B). Interestingly, the copurification of histones H3 and H4 was drastically reduced when Ada2-TAP was isolated from sus1 or ubp8 cells (Figure 5B). In contrast, Ada2-TAP still coprecipitated the other SAGA subunits in the sus1 or ubp8 mutants (e.g., Tra1 or Ada3; also see Figure 1, A and B). Together, the data show that Sus1, Sgf11, and Ubp8 are required for the copurification of histones with the SAGA complex. Thus, it is possible that this subcomplex establishes an affinity between SAGA and its histone substrates on a biochemical level.
|
cells grow very slowly and exhibit an mRNA export defect (Figure 6A; also see Rodríguez-Navarro et al., 2004). In contrast, sgf11 and ubp8 cells are neither impaired in growth nor in nuclear mRNA export at 37°C (Figure 6A). Next, we constructed a sus1/sgf11 double mutant. We speculated that this strain could exhibit an enhanced mRNA export defect, because Sus1 and Sgf11 coprecipitate even in the absence of ubp8 (see above). Indeed, deletion of SGF11 in a sus1 strain caused a synergistically enhanced mRNA export at 30°C and exacerbated the growth defect of sus1 at 39°C (Figure 6B). Interestingly, the ubp8sus1 double mutant did not show any synthetic effect regarding mRNA export and growth (Figure 6B). These data suggest that Sgf11 has a function more closely related to Sus1, including a role in nuclear mRNA export.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Sus1, Ubp8, and Sgf11 are physically and functionally linked as a module in the SAGA complex. Previous studies have described that Ubp8 depends on a putative zinc finger domain in its N terminus to bind to SAGA (Ingvarsdottir et al., 2005). However, stable association also requires the small 11-kDa subunit Sgf11 (Ingvarsdottir et al., 2005; Lee et al., 2005). Our work extends these findings and demonstrates that Sus1 contributes yet another surface for SAGA association, because its absence disrupts the interaction of Ubp8 and Sgf11 with the SAGA complex. The interdependence of Sus1, Ubp8, and Sgf11 for SAGA association could be explained in at least two ways. One possibility is that the SAGA interaction is cooperative such that all three subunits undergo a sequential or concerted conformational change before SAGA incorporation. Alternatively, Sus1, Ubp8, and Sgf11 could form a common SAGA binding surface that only displays sufficient SAGA affinity if all three members are present. Interestingly, we found that Sus1 is able to bind to Sgf11 in the absence of Ubp8, and no association of Sus1 to Ubp8 was observed when Sgf11 was deleted. In addition, Ubp8 could be dissociated from Sus1–Sgf11 by an increase in salt concentration. This implies that Sus1 directly interacts with Sgf11, and this dimer may form a template for Ubp8. Nevertheless, we cannot exclude that the Sus1–Ubp8–Sgf11 deubiquitinylating module comprises additional factors, which are less tightly bound. Whether the heterotrimeric subcomplex is preassembled or undergoes a step-by-step incorporation during SAGA biogenesis remains to be shown.
Loss of Sus1 causes an increase in the levels of H2B ubiquitinylation and H3 methylation. We therefore conclude that Sus1 works in the same pathway as Ubp8. Ubp8 activity is context (i.e., SAGA) dependent, because it is not able to deubiquitinylate H2B in vivo or in vitro on its own (Lee et al., 2005). The H2B ubiquitinylation level increases at a given promoter during gene activation and declines shortly afterward (Henry et al., 2003). How this sophisticated interplay of ubiquitin addition and removal is regulated in time and space is not well understood, even though the enzymatic components have been defined. Is Ubp8 constitutively active, or can it be stimulated by other factors? SAGA association could be required either for targeting the enzymatic activity or to relieve a certain form of Ubp8 inhibition. The increase in H2B ubiquitinylation upon Sus1 deletion could be a simple consequence of Ubp8 dissociation. This would imply that Sus1 exerts its function only by anchoring Ubp8 and Sgf11 to SAGA. However, it is also possible that Sus1 acts as a direct regulator of Ubp8 activity. Concomitantly with an increase in global H2B ubiquitinylation, we found that H3 methylation is also affected in a sus1 deletion mutant. The observed increase in K4 monomethylation and K79 trimethylation could possibly be correlated with alterations in gene expression (9% of the yeast genes) that we previously measured in sus1 cells by DNA macroarray analysis (Rodríguez-Navarro et al., 2004).
The three-dimensional architecture of SAGA has an elongated, curved shape and comprises five domains. The Taf subunits together with Spt7 and Ada1 constitute a core structure. This core structure is surrounded by domains that function in TBP regulation, activator binding, and histone acetylation. Distinct regulatory functions are spatially separated from each other and illustrate the principle of SAGA modularity (Wu et al., 2004; for review, see Timmers and Tora, 2005). The precise position of Sus1, Ubp8, or Sgf11 within SAGA is not known. However, we assume that this subcomplex is probably not a core structural component because deletion of sus1, ubp8, or sgf11 did not perturb the overall integrity of SAGA. This distinguishes the Sus1 subcomplex from SAGA subunits, such as Spt20, Spt7, or Ada1. Deletion of any of these proteins exhibits a strong disruptive effect on SAGA structure (Roberts and Winston, 1996; Grant et al., 1997; Horiuchi et al., 1997; Roberts and Winston, 1997; Sterner et al., 1999; Wu and Winston, 2002). Interestingly, the dissociation of Ubp8 upon Sus1 deletion also did not affect SAGA composition when Ada2 or Taf6 were used as baits for purifications, suggesting a peripheral location of Sus1 within a separate module.
Our ChIP analyses suggest an in vivo relevance for Sus1 function, because it is necessary for Ubp8 recruitment to chromatin. Conversely, Ubp8 is important for Sus1 binding to chromatin. Our study together with recent reports demonstrates that Sus1, Ubp8, and Sgf11 are dispensable for SAGA recruitment to the GAL1 promoter, consistent with the biochemical dispensability of the module for SAGA integrity (Ingvarsdottir et al., 2005; Lee et al., 2005; Shukla et al., 2006). Along this line, we found an altered nuclear localization of Sus1 when it was uncoupled from SAGA by ubp8 or sgf11 deletions. This shift in Sus1 localization suggests interdependence between the association of Sus1 with SAGA and its location within the Sac3 complex at the nuclear pore.
Coenrichment of histones in a SAGA purification critically depends on the presence of all members of the subcomplex. This was unexpected, because several other mechanisms have been described by which SAGA can be targeted and bound to DNA. The 400-kDa protein Tra1, for example, directs SAGA to a promoter via its interaction with transcription activators. In addition, several histone-fold-containing subunits, such as Taf6, Taf12, and Ada1, seem to be involved (Brown et al., 2001; Hall and Struhl, 2002; Klein et al., 2003; Bhaumik et al., 2004). Our biochemical data suggest that the deubiquitinylating module may have an intrinsic affinity for histones (possibly nucleosomes). This affinity could be important for orienting the Ubp8 catalytic site with respect to the monoubiquitinated lysine at position 123 of H2B and might be involved in stabilizing the SAGA-nucleosome interaction once it has been formed. In contrast, the Sus1 subcomplex does not seem to be strictly required for chromatin targeting in vivo when analyzed by ChIP. The discrepancy between our TAP purifications and the ChIP assay may be due to major differences between these techniques. However, it seems also possible that our biochemical observations reflect an additional SAGA function that is independent of its role at the promoter of genes.
Sus1, together with other protein complexes, could couple chromatin modification, transcription initiation, and nuclear mRNA export machineries at the level of the NPC. However, it is not yet clear how Sus1 could connect histone modification and mRNA export. To strengthen the idea that the Sus1 module is indeed linked to both transcription and mRNA export, we tested whether deletion of Sus1 would have a synthetically enhanced mRNA export defect in combination with Sgf11, its putative direct partner. We did observe an enhanced defect when deletions of sgf11 and sus1 were combined. This opens the possibility that Sgf11, a bona fide SAGA component, could have a role in both processes. Interestingly, we have shown that ubp8 deletion does not cause an exacerbation of the sus1 single deletion phenotype. This result suggests that Sgf11 has a function more closely related to Sus1.
Our study has defined a critical mRNA export factor, Sus1, as an important part of a SAGA-deubiquitinylating module. Thus, the interaction between SAGA and the NPC-associated mRNA export machinery could involve the deubiquitinylating module. This insight may help to develop a more accurate view of how transcription and mRNA export are mechanistically coupled.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-02-0098) on July 19, 2006.
These authors contributed equally to this work.
Address correspondence to: Susana Rodríguez-Navarro (srodriguez{at}cipf.es)
Abbreviations used: NPC, nuclear pore complex; SAGA, Spt/Ada/Gcn5 acetyltransferase.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Berger, S. L. (2002). Histone modifications in transcriptional regulation. Curr. Opin. Genet. Dev 12, 142–148.[CrossRef][Medline]
Bhaumik, S. R., Raha, T., Aiello, D. P., Green, M. R. (2004). In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer. Genes Dev 18, 333–343.
Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J., Hieter, P., Boeke, J. D. (1998). Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–132.[CrossRef][Medline]
Brand, M., Leurent, C., Mallouh, V., Tora, L., Schultz, P. (1999). Three-dimensional structures of the TAFII-containing complexes TFIID and TFTC. Science 286, 2151–2153.
Briggs, S. D., Xiao, T., Sun, Z. W., Caldwell, J. A., Shabanowitz, J., Hunt, D. F., Allis, C. D., Strahl, B. D. (2002). Gene silencing: trans-histone regulatory pathway in chromatin. Nature 418, 498.[CrossRef][Medline]
Brown, C. E., Howe, L., Sousa, K., Alley, S. C., Carrozza, M. J., Tan, S., Workman, J. L. (2001). Recruitment of HAT complexes by direct activator interactions with the ATM-related Tra1 subunit. Science 292, 2333–2337.
Cabal, G. G., et al. (2006). SAGA interacting factors confine sub-diffusion of transcribed genes to the nuclear envelope. Nature 441, 770–773.[CrossRef][Medline]
Candau, R., Zhou, J. X., Allis, C. D., Berger, S. L. (1997). Histone acetyltransferase activity and interaction with ADA2 are critical for GCN5 function in vivo. EMBO J 16, 555–565.[CrossRef][Medline]
Carrozza, M. J., Utley, R. T., Workman, J. L., Cote, J. (2003). The diverse functions of histone acetyltransferase complexes. Trends Genet 19, 321–329.[CrossRef][Medline]
Cosgrove, M. S., Boeke, J. D., Wolberger, C. (2004). Regulated nucleosome mobility and the histone code. Nat. Struct. Mol. Biol 11, 1037–1043.[CrossRef][Medline]
Daniel, J. A., Torok, M. S., Sun, Z. W., Schieltz, D., Allis, C. D., Yates, J. R. 3rd, Grant, P. A. (2004). Deubiquitination of histone H2B by a yeast acetyltransferase complex regulates transcription. J. Biol. Chem 279, 1867–1871.
De Nadal, E., Zapater, M., Alepuz, P. M., Sumoy, L., Mas, G., Posas, F. (2004). The MAPK Hog1 recruits Rpd3 histone deacetylase to activate osmoresponsive genes. Nature 427, 370–374.[CrossRef][Medline]
Dover, J., Schneider, J., Tawiah-Boateng, M. A., Wood, A., Dean, K., Johnston, M., Shilatifard, A. (2002). Methylation of histone H3 by COMPASS requires ubiquitination of histone H2B by Rad6. J. Biol. Chem 277, 28368–28371.
Fischer, T., Rodríguez-Navarro, S., Pereira, G., Racz, A., Schiebel, E., Hurt, E. (2004). Yeast centrin Cdc31 is linked to the nuclear mRNA export machinery. Nat. Cell Biol 6, 840–848.[CrossRef][Medline]
Gavin, A. C., et al. (2002). Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, 141–147.[CrossRef][Medline]
Grant, P. A., et al. (1997). Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex. Genes Dev 11, 1640–1650.
Grant, P. A., Eberharter, A., John, S., Cook, R. G., Turner, B. M., Workman, J. L. (1999). Expanded lysine acetylation specificity of Gcn5 in native complexes. J. Biol. Chem 274, 5895–5900.
Hall, D. B. and Struhl, K. (2002). The VP16 activation domain interacts with multiple transcriptional components as determined by protein-protein cross-linking in vivo. J. Biol. Chem 277, 46043–46050.
Henry, 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. (2003). Transcriptional activation via sequential histone H2B ubiquitylation and deubiquitylation, mediated by SAGA-associated Ubp8. Genes Dev 17, 2648 2663.
Holstege, F. C., Jennings, E. G., Wyrick, J. J., Lee, T. I., Hengartner, C. J., Green, M. R., Golub, T. R., Lander, E. S., Young, R. A. (1998). Dissecting the regulatory circuitry of a eukaryotic genome. Cell 95, 717–728.[CrossRef][Medline]
Horiuchi, J., Silverman, N., Pina, B., Marcus, G. A., Guarente, L. (1997). ADA1, a novel component of the ADA/GCN5 complex, has broader effects than GCN5, ADA2, or ADA3. Mol. Cell. Biol 17, 3220–3228.[Abstract]
Hwang, W. W., Venkatasubrahmanyam, S., Ianculescu, A. G., Tong, A., Boone, C., Madhani, H. D. (2003). A conserved RING finger protein required for histone H2B monoubiquitination and cell size control. Mol. Cell 11, 261–266.[CrossRef][Medline]
Ingvarsdottir, K., Krogan, N. J., Emre, N. C., Wyce, A., Thompson, N. J., Emili, A., Hughes, T. R., Greenblatt, J. F., Berger, S. L. (2005). H2B ubiquitin protease Ubp8 and Sgf11 constitute a discrete functional module within the Saccharomyces cerevisiae SAGA complex. Mol. Cell. Biol 25, 1162–1172.
Jenuwein, T. and Allis, C. D. (2001). Translating the histone code. Science 293, 1074–1080.
Klein, J., Nolden, M., Sanders, S. L., Kirchner, J., Weil, P. A., Melcher, K. (2003). Use of a genetically introduced cross-linker to identify interaction sites of acidic activators within native transcription factor IID and SAGA. J. Biol. Chem 278, 6779–6786.
Lee, K. K., Florens, L., Swanson, S. K., Washburn, M. P., Workman, J. L. (2005). The deubiquitylation activity of Ubp8 is dependent upon Sgf11 and its association with the SAGA complex. Mol. Cell. Biol 25, 1173–1182.
Lee, K. K., Prochasson, P., Florens, L., Swanson, S. K., Washburn, M. P., Workman, J. L. (2004). Proteomic analysis of chromatin-modifying complexes in Saccharomyces cerevisiae identifies novel subunits. Biochem. Soc. Trans 32, 899–903.[CrossRef][Medline]
Longtine, M. S., McKenzie, A. 3rd, Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P., Pringle, J. R. (1998). Additional modules for versatile and economical PCR based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961.[CrossRef][Medline]
Mellor, J. (2005). The dynamics of chromatin remodeling at promoters. Mol. Cell 19, 147–157.[CrossRef][Medline]
Powell, D. W., Weaver, C. M., Jennings, J. L., McAfee, K. J., He, Y., Weil, P. A., Link, A. J. (2004). Cluster analysis of mass spectrometry data reveals a novel component of SAGA. Mol. Cell. Biol 24, 7249–7259.
Reed, R. (2003). Coupling transcription, splicing and mRNA export. Curr. Opin. Cell Biol 15, 326–331.[CrossRef][Medline]
Roberts, S. M. and Winston, F. (1996). SPT20/ADA5 encodes a novel protein functionally related to the TATA-binding protein and important for transcription in Saccharomyces cerevisiae. Mol. Cell. Biol 16, 3206–3213.[Abstract]
Roberts, S. M. and Winston, F. (1997). Essential functional interactions of SAGA, a Saccharomyces cerevisiae complex of Spt, Ada, and Gcn5 proteins, with the Snf/Swi and Srb/mediator complexes. Genetics 147, 451–465.[Abstract]
Robzyk, K., Recht, J., Osley, M. A. (2000). Rad6-dependent ubiquitination of histone H2B in yeast. Science 287, 501–504.
Rodríguez-Navarro, S., Fischer, T., Luo, M. J., Antunez, O., Brettschneider, S., Lechner, J., Perez-Ortin, J. E., Reed, R., Hurt, E. (2004). Sus1, a functional component of the SAGA histone acetylase complex and the nuclear pore-associated mRNA export machinery. Cell 116, 75–86.[CrossRef][Medline]
Rodríguez, M. S., Dargemont, C., Stutz, F. (2004). Nuclear export of RNA. Biol. Cell 96, 639–655.[CrossRef][Medline]
Sanders, S. L., Jennings, J., Canutescu, A., Link, A. J., Weil, P. A. (2002). Proteomics of the eukaryotic transcription machinery: identification of proteins associated with components of yeast TFIID by multidimensional mass spectrometry. Mol. Cell. Biol 22, 4723–4738.
Santos-Rosa, H., Moreno, H., Simos, G., Segref, A., Fahrenkrog, B., Pante, N., Hurt, E. (1998). Nuclear mRNA export requires complex formation between Mex67p and Mtr2p at the nuclear pores. Mol. Cell. Biol 18, 6826–6838.
Shukla, A., Stanojevic, N., Duan, Z., Sen, P., Bhaumik, S. R. (2006). Ubp8p, a histone deubiquitinase whose association with SAGA is mediated by Sgf11p, differentially regulates lysine 4 methylation of histone H3 in vivo. Mol. Cell. Biol 26, 3339–3352.
Sterner, D. E., Grant, P. A., Roberts, S. M., Duggan, L. J., Belotserkovskaya, R., Pacella, L. A., Winston, F., Workman, J. L., Berger, S. L. (1999). Functional organization of the yeast SAGA complex: distinct components involved in structural integrity, nucleosome acetylation, and TATA-binding protein interaction. Mol. Cell. Biol 19, 86–98.
Sun, Z. W. and Allis, C. D. (2002). Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast. Nature 418, 104–108.[CrossRef][Medline]
Timmers, H. T. and Tora, L. (2005). SAGA unveiled. Trends Biochem. Sci 30, 7–10.[CrossRef][Medline]
Wood, A., et al. (2003). Bre1, an E3 ubiquitin ligase required for recruitment and substrate selection of Rad6 at a promoter. Mol. Cell 11, 267–274.[CrossRef][Medline]
Wu, P. Y., Ruhlmann, C., Winston, F., Schultz, P. (2004). Molecular architecture of the S. cerevisiae SAGA complex. Mol. Cell 15, 199–208.[CrossRef][Medline]
Wu, P. Y. and Winston, F. (2002). Analysis of Spt7 function in the Saccharomyces cerevisiae SAGA coactivator complex. Mol. Cell. Biol 22, 5367–5379.
This article has been cited by other articles:
|
|
 |
|
  C. Gonzalez-Aguilera, C. Tous, B. Gomez-Gonzalez, P. Huertas, R. Luna, and A. Aguilera The THP1-SAC3-SUS1-CDC31 Complex Works in Transcription Elongation-mRNA Export Preventing RNA-mediated Genome Instability Mol. Biol. Cell, October 1, 2008; 19(10): 4310 - 4318. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. E. Grund, T. Fischer, G. G. Cabal, O. Antunez, J. E. Perez-Ortin, and E. Hurt The inner nuclear membrane protein Src1 associates with subtelomeric genes and alters their regulated gene expression J. Cell Biol., September 8, 2008; 182(5): 897 - 910. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Chen and K. Madura Centrin/Cdc31 Is a Novel Regulator of Protein Degradation Mol. Cell. Biol., March 1, 2008; 28(5): 1829 - 1840. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Chekanova, K. C. Abruzzi, M. Rosbash, and D. A. Belostotsky Sus1, Sac3, and Thp1 mediate post-transcriptional tethering of active genes to the nuclear rim as well as to non-nascent mRNP RNA, January 1, 2008; 14(1): 66 - 77. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Laprade, D. Rose, and F. Winston Characterization of New Spt3 and TATA-Binding Protein Mutants of Saccharomyces cerevisiae: Spt3 TBP Allele-Specific Interactions and Bypass of Spt8 Genetics, December 1, 2007; 177(4): 2007 - 2017. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||
