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• W2013844573 abstract "Article8 June 2006free access Roles for APIS and the 20S proteasome in adenovirus E1A-dependent transcription Mozhgan Rasti Mozhgan Rasti Cancer Research UK Institute for Cancer Studies, The Medical School, The University of Birmingham, Edgbaston, Birmingham, UK Search for more papers by this author Roger JA Grand Roger JA Grand Cancer Research UK Institute for Cancer Studies, The Medical School, The University of Birmingham, Edgbaston, Birmingham, UK Search for more papers by this author Ahmed F Yousef Ahmed F Yousef Departments of Oncology and Microbiology & Immunology, University of Western Ontario, London, Ontario, Canada Search for more papers by this author Michael Shuen Michael Shuen Departments of Oncology and Microbiology & Immunology, University of Western Ontario, London, Ontario, Canada Search for more papers by this author Joe S Mymryk Joe S Mymryk Departments of Oncology and Microbiology & Immunology, University of Western Ontario, London, Ontario, Canada Search for more papers by this author Phillip H Gallimore Phillip H Gallimore Cancer Research UK Institute for Cancer Studies, The Medical School, The University of Birmingham, Edgbaston, Birmingham, UK Search for more papers by this author Andrew S Turnell Corresponding Author Andrew S Turnell Cancer Research UK Institute for Cancer Studies, The Medical School, The University of Birmingham, Edgbaston, Birmingham, UK Search for more papers by this author Mozhgan Rasti Mozhgan Rasti Cancer Research UK Institute for Cancer Studies, The Medical School, The University of Birmingham, Edgbaston, Birmingham, UK Search for more papers by this author Roger JA Grand Roger JA Grand Cancer Research UK Institute for Cancer Studies, The Medical School, The University of Birmingham, Edgbaston, Birmingham, UK Search for more papers by this author Ahmed F Yousef Ahmed F Yousef Departments of Oncology and Microbiology & Immunology, University of Western Ontario, London, Ontario, Canada Search for more papers by this author Michael Shuen Michael Shuen Departments of Oncology and Microbiology & Immunology, University of Western Ontario, London, Ontario, Canada Search for more papers by this author Joe S Mymryk Joe S Mymryk Departments of Oncology and Microbiology & Immunology, University of Western Ontario, London, Ontario, Canada Search for more papers by this author Phillip H Gallimore Phillip H Gallimore Cancer Research UK Institute for Cancer Studies, The Medical School, The University of Birmingham, Edgbaston, Birmingham, UK Search for more papers by this author Andrew S Turnell Corresponding Author Andrew S Turnell Cancer Research UK Institute for Cancer Studies, The Medical School, The University of Birmingham, Edgbaston, Birmingham, UK Search for more papers by this author Author Information Mozhgan Rasti1, Roger JA Grand1, Ahmed F Yousef2, Michael Shuen2, Joe S Mymryk2, Phillip H Gallimore1 and Andrew S Turnell 1 1Cancer Research UK Institute for Cancer Studies, The Medical School, The University of Birmingham, Edgbaston, Birmingham, UK 2Departments of Oncology and Microbiology & Immunology, University of Western Ontario, London, Ontario, Canada *Corresponding author. Cancer Research UK Institute for Cancer Studies, The Medical School, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. Tel.: +44 121 414 4483; Fax: +44 121 414 4486; E-mail: [email protected] The EMBO Journal (2006)25:2710-2722https://doi.org/10.1038/sj.emboj.7601169 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We have determined distinct roles for different proteasome complexes in adenovirus (Ad) E1A-dependent transcription. We show that the 19S ATPase, S8, as a component of 19S ATPase proteins independent of 20S (APIS), binds specifically to the E1A transactivation domain, conserved region 3 (CR3). Recruitment of APIS to CR3 enhances the ability of E1A to stimulate transcription from viral early gene promoters during Ad infection of human cells. The ability of CR3 to stimulate transcription in yeast is similarly dependent on the functional integrity of yeast APIS components, Sug1 and Sug2. The 20S proteasome is also recruited to CR3 independently of APIS and the 26S proteasome. Chromatin immunoprecipitation reveals that E1A, S8 and the 20S proteasome are recruited to both Ad early region gene promoters and early region gene sequences during Ad infection, suggesting their requirement in both transcriptional initiation and elongation. We also demonstrate that E1A CR3 transactivation and degradation sequences functionally overlap and that proteasome inhibitors repress E1A transcription. Taken together, these data demonstrate distinct roles for APIS and the 20S proteasome in E1A-dependent transactivation. Introduction Adenovirus (Ad) E1A protein expression is essential for both viral replication and cellular transformation (Gallimore and Turnell, 2001). Ad E1A proteins are expressed predominantly, from two splice variant transcripts, 12S and 13S, which, in Ad2/5, give rise to proteins of 243 and 289 amino acids respectively. Ad-regulated expression of viral early genes, and some cellular genes, is dependent upon conserved region 3 (CR3), a region unique to 13S E1A (Jones, 1995). Studies investigating CR3 have not only been important in elucidating E1A function, but have also served as a model system for understanding the basis of transactivation per se. Comparison of E1A sequences from different Ad subtypes suggests that CR3 extends from residue 144 to 191 in Ad2/5 (Avvakumov et al, 2002). CR3 contains a C4 zinc-finger, a single Zn2+ ion coordinated by amino acids C154, C157, C171 and C174; mutation of any of the critical C residues results in loss of transactivation function (Culp et al, 1988; Webster et al, 1991). Residues H160 and S172 within the finger domain, residues V147, P150, Y175 and R177, which lie proximal to the finger domain, and a contiguous stretch of amino acids, encompassing residues V183–S188 within the C-terminal region of CR3, are also critical for transactivation (Webster and Ricciardi, 1991). The transactivating properties of CR3 reside in its ability to recruit the necessary components of the cellular transcription machinery specifically to transcription factor-bound promoters. Indeed, the zinc-finger binds TBP, as part of a functional holo-TFIID complex (Lee et al, 1991; Boyer and Berk, 1993; Geisberg et al, 1994). The C-terminal region of CR3 (i.e. V183–S188) also recruits, independently of TBP, multiple TAF components of TFIID (Chiang and Roeder, 1995; Geisberg et al, 1995; Mazzarelli et al, 1997). The C-terminal region of CR3 is also required for promoter targeting of competent E1A-containing transactivation complexes; CR3 binds to promoter-bound transcription factors through their DNA-binding domains (DBD) (Hurst and Jones, 1987; Lee et al, 1987; Lillie and Green, 1989; Liu and Green, 1990, 1994). Phosphorylation of residues S185 and S188 within the C-terminal domain modulates E1A-dependent transactivation (Whalen et al, 1997). The zinc-finger domain of CR3 also binds hSur2 (MED23), a component of Mediator (Boyer et al, 1999). Thus, CR3 is unable to function efficiently as a transactivator of Ad early genes in sur2−/− embryonic stem cells following viral infection or DNA transfection (Stevens et al, 2002). Indeed, the ability of mouse Ad1 to stimulate early gene transcription, and replicate, in sur2−/− mouse embryo fibroblasts is reduced significantly, relative to its capacity to perform these functions in sur2+/+ mouse embryo fibroblasts (Fang et al, 2004). The 26S proteasome is the major non-lysosomal proteolytic machinery in eukaryotes, serving to degrade polyubiquitylated proteins in an ATP-dependent manner (Ciechanover, 1998). The 26S proteasome is assembled from two large macromolecular complexes, the 20S proteolytic particle and the 19S regulatory complex (RC). The 20S proteasome degrades non-ubiquitylated proteins in an energy-independent manner and possesses trypsin-, chymotrypsin- and postglutamyl hydrolase-like activities. The 19S RC can be functionally subdivided into base and lid components. The base complex consists of six homologous AAA ATPases (S4, S6, S6′, S7, S8 and S10b) and three non-ATPase (S1, S2 and S5a) components, whereas the lid of the RC is made of an additional eight non-ATPase subunits. The 19S ATPases regulate the assembly of the 26S proteasome, unfold substrate proteins targeted for degradation and gate protein translocation into the central chamber of the 20S proteasome (Ciechanover, 1998). The realisation that the Saccharomyces cerevisiae protein responsible for rescue of a class of Gal4 activation domain mutants, Sug1, was a component of the 26S proteasome was crucial in establishing a potential role for the proteasome in transcription (Rubin et al, 1996). Significantly, Sug1 had previously been reported to recruit transcription factors such as Gal4 to TBP (Swaffield et al, 1995). The belief that Sug1 functions specifically in a number of diverse transcription programmes was strengthened by the observation that the mammalian orthologue of Sug1, S8, interacted with the thyroid receptor, the retinoic acid receptor (RAR) α, the oestrogen receptor (ER) and other nuclear receptors in a ligand- and AF-2 activation domain-dependent manner (Lee et al, 1995; vom Baur et al, 1996). 26S proteasome-mediated degradation is essential for both RARγ- and ER-dependent transactivation (Lonard et al, 2000; Gianni et al, 2002). Given the emerging role of the proteasome in transcriptional regulation, and the specific ability of 13S E1A to function as a transactivator, we undertook a comprehensive study to determine whether there was a role for the proteasome in CR3-dependent transcriptional activation. Data presented here offer new insights into E1A-dependent transactivation and clearly establish roles for the 19S ATPase, S8, as a component of 19S ATPase proteins independent of 20S (APIS), and the 20S proteasome in Ad E1A-CR3-regulated transcriptional initiation and elongation in vivo. We also show for the first time that E1A transactivation capacity is dependent upon its inherent stability. CR3 transactivation and degradation sequences functionally overlap, suggesting that the 20S proteasome also regulates the termination of E1A transcription programmes. Taken together, these data define key and novel roles for proteasomal complexes in transcription programmes regulated by E1A, and offer insights into proteasome function, per se, in transcriptional regulation. Results The 19S ATPase S8 binds to the CR3 transactivation domain of E1A We have previously shown that S8 binds to the N-terminal region of E1A (Turnell et al, 2000). In order to determine whether S8 can also bind to E1A through CR3, we utilised an N-terminal double point E1A mutant, L1920A, which in the context of 12S E1A does not bind S8 (Rasti et al, 2005). Using GST-w.t.-12S E1A and GST-L1920A-12S E1A fusion proteins we initially confirmed, using pull-downs from A549 cell lysates, that the N-terminal E1A binding proteins S8, TBP and Ran all bind w.t. 12S E1A but do not bind L1920A-12S E1A (Figure 1A, compare lanes 2 and 3). Interestingly, however, S8 and TBP retained significant binding capacity for L1920A-13S, whereas Ran did not (Figure 1A, compare lanes 4 and 5), suggesting that S8 and TBP bind CR3 but Ran does not. The ability of pRB to bind in equal measure to each of the GST-E1A fusion proteins validates the structural integrity of the GST proteins used (Figure 1A, upper panel). Figure 1.S8 binds to CR3 of Ad5 13S E1A. (A) GST pull-downs using w.t. 12S and 13S E1A, and 12S L1920A and 13S L1920A E1A mutants, were performed to assess the in vitro binding capacity of these E1A proteins for pRB, S8, TBP and Ran from A549 cellular lysates. Following pull-down, binding was assessed by Western blotting. (B, C) S8 and TBP retain the ability to bind L1920A 13S E1A in vivo. S8 and TBP complexes were immunoprecipitated from 13S E1A-, and 13S E1A L1920A-, expressing A549 cells, and subjected to Western blotting for E1A. *Nonspecific band recognised by the anti-S8 antibody. WCE, whole-cell extract. Download figure Download PowerPoint In order to establish whether S8 binds to CR3 in vivo, we generated a number of A549-derived, clonal cell lines that constitutively express either w.t. 13S E1A or L1920A-13S E1A. Significantly, immunoprecipitation studies revealed that relative to w.t. 13S E1A, L1920A-13S E1A retained appreciable binding capacity for S8 (Figure 1B) and TBP (Figure 1C), indicating that these proteins bind CR3 specifically, in vivo. Using cell lysis conditions that dissociate the 19S proteasome from the 20S proteasome (Grand et al, 1999), we next determined, by GST pull-down, the binding site within CR3 for S8. These experiments revealed that the S8 binding site extends from amino acid 169 to 188 (Figure 2A, upper panel). S8 retained appreciable binding capacity for the zinc-finger mutant C157S (Figure 2A, upper panel), and had w.t. CR3 binding capacity for the H160Y and Y175F zinc-finger mutants (Figure 2A, middle panel). S8 also bound to CR3 species from all six human Ad subgroups (Figure 2A, lower panel), suggesting that this is a conserved function in E1A. TBP had a similar propensity to bind these same CR3 mutant proteins (Supplementary Figure S1A). Figure 2.Binding of S8 and 20S proteasomal components to Ad5 CR3 E1A mutants, and to CR3s from different Ad subgroups. Following GST pull-down, binding capacity was assessed by Western blotting for S8 (A) and 20S proteasomes (B). The 20S proteasome antibody recognises α1–α3 and α5–α7 subunits. In lanes 1 and 2 of the upper panel of (A), GST and GST-Ad5 CR3 were incubated in lysis buffer alone. (C) Coomassie-stained gel showing the purity of the E1A proteins used. *Nonspecific band recognised by the anti-S8 antibody. WCE, whole-cell extract. The corresponding regions of CR3 used during this study were as follows: Ad5, residues 139–204; Ad3, residues 132–209; Ad4, residues 128–206; Ad9, residues 120–198; Ad12, residues 124–210; Ad40, residues 117–193. Download figure Download PowerPoint As S8 and TBP physically interact (Swaffield et al, 1995), we next examined by selective siRNA treatment whether S8 and TBP bind independently to CR3. Significantly, TBP retained full binding capacity for CR3 following the knock-down of S8, and S8 retained full binding capacity for CR3 following the knock-down of TBP (Supplementary Figure S2A and B). As S8 is one of six, homologous ATPases that comprise the base of the 19S RC, we also examined the capacity of CR3 and TBP to bind S4, S6, S6′, S7, S8 and S10b in vitro. Interestingly, TBP bound to all the 19S ATPases (Supplementary Figure S2C), although it bound to S8 with the highest capacity. CR3, on the other hand, bound S8 strongly, but had little or no capacity to bind other 19S ATPases (Supplementary Figure S2C). CR3, thus binds to S8 independently of TBP, and selectively binds S8 in preference to other 19S ATPases. The 20S proteasome binds to the CR3 transactivation domain independently of S8 Given the ability of CR3 to bind S8, we wished to determine whether 20S proteasomes similarly bound to CR3. Using the protocol outlined above, we assayed the relative abilities of the CR3-E1A mutants to bind 20S proteasomes from A549 cell lysates. Under these conditions, the 20S proteasome bound to CR3 (Figure 2B, upper panel). 20S binding to CR3 differed appreciably from that of S8. The binding site within CR3 for 20S extends from amino acid 161 to 177 (Figure 2B, upper panel). In contrast to S8, the 20S proteasome had little or no affinity for the zinc-finger mutants, H160Y and Y175F (Figure 2A and B, compare middle panels). The 20S proteasome did however bind to CR3 species from all Ad subgroups, suggesting that this is a conserved function of E1A (Figure 2A and B, compare lower panels). Taken together, these data suggest that S8 function and 20S proteasome function, in the context of E1A transcription, are separable. MED23 had a similar, but not identical, propensity as the 20S proteasome to bind these same CR3 mutants (Supplementary Figure S1B). CR3 binds preferentially to ‘free’ 20S proteasomes in vivo Given that the 20S core particle can exist in vivo as a distinct entity, we wished to determine which 20S species were targeted by CR3. To do this, we fractionated 26S proteasomes from 20S proteasomes derived from A549 and 13S-L1920A A549 cell lysates by fast protein liquid chromatography (FPLC) (Figure 3). 26S proteasomes and 20S proteasomes from A549 and 13S-L1920A A549 cells eluted with identical profiles (cf. Figure 3A and B), suggesting that E1A binding does not affect 26S proteasome assembly. Immunoprecipitation studies confirmed this observation (Supplementary Figure S3A). Significantly, immunoprecipitation of 20S proteasomes from fractionated 13S-L1920A A549 cells revealed that E1A bound preferentially to ‘free’ 20S proteasomes, and that only a minor proportion of 20S bound E1A as a component of the 26S proteasome (Figure 3C). Figure 3.Binding of 20S proteasomes to 13S L1920A in vivo. A549 (A) and 13S-L1920A A549 (B) cellular lysates were fractionated upon a Superose 6 HR 10/30 column by FPLC (Materials and methods). E1A, and 26S, and 20S, proteasome-containing fractions were identified by Western blotting. Fractions containing 26S proteasomes or 20S proteasomes were pooled, and immunoprecipitation was performed either with an antibody that immunoprecipitates 20S proteasomal components or a normal IgG for control immunoprecipitations. Immunoprecipitates were separated upon a urea gel, and E1A identified by Western blotting (C). The blots indicate that L1920A 13S E1A binds significantly better to bulk 20S proteasomes (× 1 exposure) than 20S proteasomes associated with 26S proteasomes (× 20 exposure). Download figure Download PowerPoint CR3 binds preferentially to APIS-associated S8 in vivo The 19S RC exists predominantly in vivo associated with one or both ends of the 20S proteasome as functional 26S complexes. The base component of the 19S RC, of which S8 is a part, has also recently been shown to exist as a distinct functional species, APIS, that is not associated with 20S proteasomes (Gonzalez et al, 2002; Figure 4A). Using fractionated eluates from 13S-L1920A A549 cells (Figure 4A), we endeavoured to resolve whether CR3 targets S8 associated with 26S proteasomes or S8 associated with APIS. Intriguingly, immunoprecipitation studies revealed that CR3 bound preferentially to S8 associated with APIS (Figure 4B, upper panel); only a minor proportion of S8 bound to CR3 was through the 26S proteasome (Figure 4B, upper panel, compare lane 2 with lanes 6 and 8). Significantly, a ‘minor fraction’ of 20S proteasomes co-eluting with APIS were found not to be associated with E1A; CR3 targeted ‘free’ 20S proteasomes (Figure 4B, lower panel). To confirm that APIS components associate with E1A in vivo, we immunoprecipitated 19S base components S8, S2, S6, S6′ and S10b from 13S-L1920A A549 cells and subsequently Western blotted these samples for E1A. Immunoprecipitation revealed that 19S base components are found associated with E1A in 13S-L1920A A549 cells (Figure 4C, upper panel). Reciprocal immunoprecipitation similarly revealed the association of 19S base components S2 and S10b with E1A (Figure 4C, lower panel). Consistent with these findings, E1A expression did not affect association of S8 with other 19S base components (Supplementary Figure S3B), indicating that E1A targets the whole APIS complex. The 19S base non-ATPase component S2 co-fractionated with APIS (Figure 4A) and also bound E1A (Figure 4C), establishing S2 as an integral component of APIS. Taken in their entirety, these studies indicate that CR3 targets 20S proteasomes and APIS, independently in vivo, to perform distinct functions. Figure 4.Binding of S8 as a component of APIS to 13S L1920A in vivo. (A) 13S-L1920A A549 cellular lysates were fractionated by FPLC and E1A-, 26S-, 20S- and APIS-containing fractions identified by Western blotting. Long exposures (long) were performed to identify all fractions containing 26S, 20S and APIS. Fractions comprising 26S proteasomes, or 20S proteasomes or APIS were pooled and immunoprecipitation was performed either with an antibody that immunoprecipitates S8 (B, upper panel), an antibody that immunoprecipitates 20S proteasomal components (B, lower panel) or a normal IgG for control immunoprecipitations (B). Immunoprecipitates were separated upon a urea gel, and E1A identified by Western blotting (B). The blots indicate that L1920A 13S E1A binds significantly better to S8 through APIS than through 26S proteasomes (B, upper panel), and that L1920A 13S E1A binds significantly better to bulk 20S proteasomes rather than 20S proteasomes associated with 26S (B, lower panel). L1920A 13S E1A also associates with other 19S base (APIS) components in vivo (C). S8, S2, S6, S6′ and S10b were immunoprecipitated from 13S-L1920A A549 cellular lysates and immunoprecipitated E1A* identified by Western blotting. Download figure Download PowerPoint S8 functions to enhance CR3-dependent transactivation in vitro To determine whether CR3 utilises S8 for its transactivation function, we initially investigated the effects of S8 overexpression upon the ability of a Gal4DBD-CR3 construct to transactivate a Gal4-responsive luciferase reporter gene in HCT116 cells. Interestingly, low-level expression of exogenous FLAG-tagged human S8 potentiated CR3 transactivation (Figure 5A, compare lanes 1 and 3 with lanes 4 and 5), whereas high-level expression of FLAG-tagged human S8 repressed CR3 transactivation (Figure 5A, compare lanes 1 and 3 with lanes 6–8). As low levels of exogenous S8 enhanced CR3 transactivation, we next determined by RNA interference (RNAi) the requirement for endogenous S8 in CR3-dependent transactivation. Treatment of HCT116 cells with non-silencing siRNAs allowed efficient transactivation by exogenously expressed Gal4DBD-CR3 (Figure 5B, compare lanes 1 and 2). Interestingly, however, knock-down of S8 expression by RNAi dramatically reduced the ability of DBD-CR3 to transactivate the Gal4-responsive reporter gene (Figure 5B, compare lanes 2 and 6). Knock-down of TBP expression by RNAi similarly reduced the ability of DBD-CR3 to transactivate the Gal4-responsive reporter gene (Figure 5B, compare lanes 2 and 4). Figure 5.Requirement for S8 and TBP in 13S E1A transactivation function. (A) Effect of S8 expression upon E1A transactivation function. HCT116 cells were transfected with a constant amount of pcDNA3-Gal4DBD-CR3, and a Gal4-responsive luciferase reporter, with increasing amounts of pcDNA3-N-FLAG-S8. At 24 h post-transfection, cell lysates were prepared and luciferase activities measured. Total S8 and N-FLAG S8 were quantified by Western blotting. (B) Effect of TBP and S8 knock-down upon E1A transactivation. Following knock-down, HCT116 cells were transfected with a constant amount of pcDNA3-Gal4DBD and a Gal4-responsive luciferase reporter or pcDNA3-Gal4DBD-CR3 and a Gal4-responsive luciferase reporter. At 24 h post-transfection, cell lysates were prepared and luciferase activities measured. S8 and TBP levels were determined by Western blotting. (C) Following knock-down, HCT116 cells were transfected with an Ad5 E4 promoter-tethered CAT reporter and pcDNA3 Ad5 13S E1A. At 24 h post-transfection, cell lysates were prepared and CAT activities measured. In all instances, transfected DNA levels were equalised using pcDNA3 alone. Results are the mean±s.d. from three independent experiments. E1A, S8 and TBP levels were determined by Western blotting. The ability of pLE2 to transactivate the Ad5 E4 promoter was also inhibited significantly following knock-down of TBP or S8 (data not shown). Download figure Download PowerPoint Given these findings, we next investigated whether knock-down of S8 (or TBP) expression by RNAi affected 13S E1A-dependent transactivation of the Ad5 E4 promoter. Significantly, knock-down of either S8 or TBP expression by RNAi attenuated 13S E1A-dependent transactivation of the E4 promoter (Figure 5C). Importantly, exogenous expression of FLAG-tagged human S8, or the ablation of endogenous S8 expression by RNAi, did not affect the transactivation capacity of Gal4DBD-CBP (Supplementary Figure S4A and B), suggesting that not all transactivators utilise S8 for transcription. Taken together, these experiments demonstrate that S8 function is integral to CR3 transactivation function in mammalian cells. Functional 19S ATPases are required for CR3 transactivation in yeast Recent studies have utilised 19S conditional mutants of S. cerevisiae to demonstrate a functional requirement for Sug1 and Sug2, as components of APIS, in transcription initiation and transcription elongation (Gonzalez et al, 2002; Ezhkova and Tansey, 2004; Lee et al, 2005). Using these same yeast mutants, we investigated whether the ability of CR3 to stimulate transcription in S. cerevisiae (Shuen et al, 2002) was dependent on the function of these 19S base proteins. Following transformation of w.t. yeast and sug1-25 and sug2-1 strains with Gal4DBD-CR3 and a Gal4-responsive reporter possessing the GAL1 regulatory region upstream of the bacterial LacZ reporter gene, and their subsequent growth selection, we assayed CR3 transactivation capacity in these cells. In w.t. cells, CR3 efficiently stimulated transcription, whereas CR3 was unable to stimulate transcription in sug1-25 and sug2-1 mutant cells (Figure 6A), despite the Gal4-CR3 protein being expressed to similar levels (Figure 6B). These data unequivocally establish a requirement for Sug1 and Sug2 in CR3-dependent transcription in yeast, and are consistent with a role for orthologous proteins in CR3-dependent transcription in human cells. Figure 6.A requirement for Sug1 and Sug2 in CR3 transactivation function in yeast. W.t., sug1-25 and sug2-1 yeast strains were transformed with a Gal4-reporter construct and Gal4DBD or Gal4DBD-CR3. Cell lysates were subsequently prepared and β-galactosidase assays (A) performed to determine the transactivation capacity of CR3 in mutant sug1-25, mutant sug2-1 and the congenic w.t. strain. Western blotting analyses were also performed (B) with an anti-GAL4DBD antibody to determine the levels of E1A protein expression. Results are the mean±s.d. from three independent experiments. Download figure Download PowerPoint S8 function is required for efficient CR3 transactivation during Ad infection As S8 interacts directly with CR3 in vitro and in vivo, and regulates CR3 transactivation function, we wished to determine whether S8 modulated the ability of CR3 to transactivate viral early promoters during Ad infection. To do this, we initially treated A549 cells with either non-silencing siRNAs or with siRNAs directed against S8. At 24 h post-transfection, we infected cells with w.t. Ad5 at a multiplicity of infection (m.o.i.) of one plaque forming unit (PFU)/cell. At the appropriate time post-infection, we quantified Ad early gene expression. Significantly, the mRNA levels of 13S E1A, the 2.2 kb E1B transcript, E3A and E4 orf6/7 were all reduced significantly in Ad-infected cells where S8 expression was reduced by RNAi, relative to their levels in Ad-infected control cells, as measured by quantitative real-time polymerase chain reaction (PCR) (Figure 7A). Consistent with these findings, Ad early protein expression was also reduced appreciably following S8 knock-down, relative to cells expressing normal levels of S8 (Figure 7B, compare lanes 1–6 with lanes 7–12). These data provide substantive evidence to suggest that S8 functions in vivo during Ad infection to regulate CR3 transactivation function. Figure 7.The effect of S8 knock-down upon Ad early region gene, and gene product, expression during Ad infection. Following knock-down, A549 cells were infected with w.t. Ad5 at 1 PFU/cell. At the indicated time post-infection, total RNA was prepared and the absolute levels of 13SE1A, the 2.2 kb E1B transcript, E3A and E4 orf6/7 mRNAs were determined (A) by quantitative real-time PCR. In each instance, Ad early mRNA levels in non-silencing controls were ascribed a value of 100% expression. mRNA levels in knock-down cells were calculated on this basis. Values are the mean±s.d. from three independent experiments. At the appropriate time post-infection, cell lysates were prepared and the relative levels of S8, E1A, E1B-55K, E1B-19K, E2-72K and E4ORF6 proteins were determined (B) by Western blotting. Download figure Download PowerPoint Recruitment of S8 and 20S proteasomes to Ad early promoters during Ad infection We next investigated by chromatin immunoprecipitation (ChIP) whether E1A, S8 or the 20S proteasome could associate with Ad early promoters during viral infection. A549 cells were thus either mock-infected or infected with w.t. Ad5 at an m.o.i. of 1 PFU/cell. At 24 h post-infection, protein–DNA complexes were recovered by ChIP and the specific recruitment of E1A, S8 and the 20S proteasome to early region promoter elements was determined by PCR (see Supplementary Figure S5 for schematic representation of promoter regions amplified). Interestingly, these analyses showed that in addition to E1A, S8, α4 and α6 subunits of the proteasome were all found associated with Ad early promoters during viral infection (Figure 8, panels 1–5, respectively; compare lanes 1–5 with lanes 6–10). The apparent difference between α4 an" @default.
• W2013844573 created "2016-06-24" @default.
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• W2013844573 date "2006-06-08" @default.
• W2013844573 modified "2023-10-01" @default.
• W2013844573 title "Roles for APIS and the 20S proteasome in adenovirus E1A-dependent transcription" @default.
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