The Proteasome Regulates HIV-1 Transcription by Both Proteolytic and Nonproteolytic Mechanisms

Molecular Cell

Article The Proteasome Regulates HIV-1 Transcription by Both Proteolytic and Nonproteolytic Mechanisms Irina Lassot,1,3 Daniel Latreille,1,3 Emilie Rousset,1 Marion Sourisseau,1 Laetitia K. Linares,2 Christine Chable-Bessia,1 Olivier Coux,2 Monsef Benkirane,1 and Rosemary E. Kiernan1,* 1 Laboratoire de Virologie Mole´culaire, Institut de Ge´ne´tique Humaine, Centre National de la Recherche Scientifique UPR1142, Montpellier, France 2 Centre de Recherche de Biochimie Macromole´culaire, Centre National de la Recherche Scientifique UPR1086, Montpellier, France 3 These authors contributed equally to this work. *Correspondence: [email protected] DOI 10.1016/j.molcel.2006.12.020

SUMMARY

Although the proteasome facilitates transcription from several yeast promoters, it is unclear if its role is proteolytic or which subunits are involved. We show that the proteasome regulates the HIV-1 promoter in both proteolytic and nonproteolytic modes. In the absence of transcription factor, Tat, proteasome was associated with promoter and coding regions, and its proteolytic activity regulated the level of basal transcription emanating from the promoter. Tat switched the proteasome to a nonproteolytic mode by recruiting a proteasome-associated protein, PAAF1, which favors proteasome dissociation into 19S and 20S particles. Gel filtration chromatography showed that expression of both Tat and PAAF1 enhanced the abundance of a 19S-like complex in nuclear extracts. 19S, but not 20S, subunits were strongly recruited to the promoter in the presence of Tat and PAAF1 and coactivated Tat-dependent transcription. 19S components facilitated transcriptional elongation and may be involved in clearance of paused transcriptional elongation complexes from the promoter. INTRODUCTION Increasing evidence, mostly obtained in yeast, suggests that the ubiquitin/proteasome system (UPS) is directly involved in the regulation of transcription. The 26S proteasome is composed of a 20S core particle (CP) and a 19S regulatory particle (RP) that consists of a ‘‘base’’ and ‘‘lid’’ (reviewed in Pickart and Cohen [2004]). The base consists of six subunits that possess both ATPase and RNA/DNA helicase activity (Rpt1-6) and two non-ATPase subunits (Rpn1 and Rpn2), while the lid consists of eight non-ATPase subunits (Rpn39 and Rpn11 and -12). Another non-ATPase subunit, Rpn10, is required for 19S stability. A combined genome-wide location analysis

and transcriptional profiling approach in yeast revealed that proteasome association correlated with highly transcribed genes (Auld et al., 2006). However, it appears that UPS does not perform the same function at all promoters. Inhibition of proteasome function activated genes involved in protein degradation, stress response, and mitochondrial function, whereas histone genes, mating genes, and those involved in amino acid and protein synthesis were downregulated (Auld et al., 2006; Dembla-Rajpal et al., 2004; Fleming et al., 2002). Similarly, a recent study using genome-wide chromatin immunoprecipitation (ChIP) analyses in yeast revealed that, although proteasome subunits are associated with the majority of genes, several hundred genes crosslinked to either the 20S or 19S subunit, but not to both (Sikder et al., 2006). Thus, proteasome, or proteasome subcomplexes, may perform different functions at different classes of promoters. UPS may also have different functions within the same family of promoters. For example, proteasome-mediated receptor degradation is required for active nuclear receptor-mediated transcription, possibly to ensure receptor turnover on the promoter (Gianni et al., 2002). However, the proteasome represses, rather than activates, GR-mediated transcription (Kinyamu et al., 2005). For those genes activated by proteasome, there is evidence that ubiquitylation and degradation of activators, such as Gcn4, Gal4, and Ino2/4, are required to stimulate expression from target promoters (Lipford et al., 2005). It has been further demonstrated in yeast that ubiquitylation and degradation of Gal4 are required for hyperphosphorylation of RNAPII CTD, and loss of Gal4 ubiquitylation leads to defects in mRNA maturation (Muratani et al., 2005). In addition to proteolytic functions, mounting evidence indicates that the proteasome has nonproteolytic functions important in transcription from certain yeast promoters. A subcomplex of 19S RP consisting of the ATPase subunits, APIS (AAA+ ATPases of the proteasome independent of 20S), is recruited to actively transcribing genes in yeast. It was proposed that the ATP-dependent protein chaperonin activity of APIS remodels RNAPII transcriptional elongation complexes at pause sites throughout the coding region (Ferdous et al., 2001; Gonzalez et al., 2002). Several recent studies have pieced together a role

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Figure 1. The 19S and 20S Subunits of the Proteasome Have Distinct Effects on Transcription from the HIV-1 Promoter (A) Ablation of proteasome components by RNA interference. HeLa-LTR-luc cells were subjected to three consecutive rounds of transfection with siRNAs directed against proteasome components or a control siRNA (Sc) as indicated on the figure. Following the final round of transfection, cells were transduced by overnight treatment with GST-Tat. Cell extracts were harvested 24 hr later and analyzed by direct western blotting using the antibodies indicated. (B) Subunits of the 19S particle are required for Tat-mediated transactivation. Cells transfected with siRNAs as in (A) were assayed for luciferase activity. Fold Tat transactivation was calculated relative to transfection in the absence of Tat. (C) 20S a4 regulates basal transcription from the HIV-1 promoter. HeLa-LTR-luc cells were transfected as described in (A) and assayed for luciferase activity. Shown are luciferase values normalized to the value obtained in control cells, which was attributed a value of 1.

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for UPS in linking histone modifications to transcriptional activation. It has been shown that monoubiquitylated H2B recruits 19S RP that subsequently facilitates H3 methylation (Ezhkova and Tansey, 2004). 19S RP then recruits SAGA histone acetyltransferase complex and appears to stimulate SAGA/coactivator interactions leading to enhanced histone H3 acetylation and transcriptional activation (Lee et al., 2005). While the models proposed for UPS in the process of transcription are tantalizing, many unanswered questions remain. It is not clear how the proteolytic and nonproteolytic activities of UPS, in some cases reported for the same promoter, are regulated. The contribution of the different proteasome subcomplexes, such as 20S CP, 19S RP, or APIS, in transcription is controversial (Gillette et al., 2004; Gonzalez et al., 2002; Lipford and Deshaies, 2003; Morris et al., 2003). In addition, much of the evidence to date has been obtained in yeast, and the relevance of UPS in other transcription systems is not clear. The human immunodeficiency virus type 1 (HIV-1) transactivator protein, Tat, functions through binding to a short leader RNA, TAR. Tat regulates processive transcription from the HIV-1 long terminal repeat (LTR) by recruiting coactivator complexes, such as p300, PCAF, and the positive transcription elongation factor, P-TEFb, to mediate hyperphosphorylation of RNAPII CTD and stimulate transcriptional elongation (reviewed in Taube et al. [1999]). Tat transcriptional activity is regulated by both ubiquitin (Bres et al., 2003) and interactions with proteasomal subunits Rpt1 (Mss1), Rpt3 (Tbp7), and Rpt5 (Tbp1) (Nelbock et al., 1990; Ohana et al., 1993; Shibuya et al., 1992). Thus, Tat-mediated transcription of the HIV-1 LTR is a convenient model to address the role of UPS in transcription from a highly inducible promoter in mammalian cells. We show that the proteasome has both proteolytic and nonproteolytic functions on the HIV-1 LTR that are regulated by Tat. In the absence of Tat, proteasome components are associated with the promoter and coding regions and proteasome activity limits HIV-1 transcription. Tat orchestrates conversion of the proteasome to a nonproteolytic mode by recruiting a proteasome-associated protein, PAAF1 (Park et al., 2005), which promotes dissociation of 26S into 19S and 20S particles and inhibits proteasome activity. The abundance of a 19S-like complex in nuclear extracts was enhanced by expression of Tat and PAAF1. 19S, but not 20S, subunits were strongly recruited to the HIV-1 promoter by Tat in a PAAF1-dependent manner and coactivated Tat-dependent transcription from the LTR. 19S may be required to remodel paused RNAPII transcriptional complexes and thereby stimulate transcriptional elongation.

RESULTS Proteasome Has Both Proteolytic and Nonproteolytic Functions in HIV-1 Transcription To determine the role of the proteasome in HIV-1 transcription, RNA interference directed against several different components of 19S RP and 20S CP was analyzed for its effect on Tat-mediated transactivation of the HIV-1 LTR. Expressions of the different 19S or 20S components were significantly diminished by transfection of targeted siRNAs (Figure 1A). Ablation of ATPases Rpt1, Rpt4, Rpt5, and Rpt6, as well as a 19S lid subunit, Rpn7, significantly diminished Tat transactivation of an LTR-luciferase reporter construct (Figure 1B) without affecting basal transcription in the absence of Tat (data not shown). In contrast, knockdown of 20S a4 expression enhanced basal transcription 2- to 3-fold on average (up to 10-fold in some experiments) without significantly affecting transactivation by Tat (Figure 1C). 26S-dependent proteolytic activity of cell extracts treated with the same siRNAs was reduced approximately equivalently (see Figure S1 in the Supplemental Data available with this article online). Uptake and subcellular localization of GST-Tat was not significantly affected by any of the siRNAs (data not shown). These data suggest a basic difference between the roles of 19S and 20S subunits in HIV-1 transcription that could not be correlated to an effect on protein degradation. To further analyze the role of proteasome in HIV-1 transcription, cells were treated or mock treated with the proteasome inhibitor MG132 in the presence or absence of Tat. Analysis was performed by quantitative RT-PCR of luciferase mRNA since MG132 has been reported to inhibit synthesis of luciferase protein (Deroo and Archer, 2002). Proteasome inhibition enhanced basal transcription from the LTR by 34-fold (Figure 1D, gray bars). In the presence of Tat, an increase in transcription was also observed but the efficiency of transactivation by Tat was diminished (Figure 1D; 9.8-fold transactivation in untreated cells compared to 3.8-fold in MG132-treated cells). MG132 treatment did not significantly affect either the uptake and subcellular localization of GST-Tat or the quantity of GAPDH mRNA levels (data not shown). Thus, MG132 inhibition paralleled that of 20S a4 knockdown in that basal transcription was increased, and both show an intrinsic difference to knockdown of 19S subunits. The effect of overexpressing 19S and 20S subunits on HIV-1 transcription was then tested. All HA-tagged 19S subunits tested significantly coactivated Tat-dependent transcription without affecting basal transcription (Figure 1E and data not shown). Overexpression of Flag-20S

(D) Inhibition of proteasome activity increases HIV-1 transcription. HeLa-LTR-luc cells were treated with GST or GST-Tat as indicated followed by treatment with MG132 or carrier as indicated below the figure. Values represent the amount of luciferase reporter gene mRNA normalized to the amount of GAPDH mRNA in each sample. Values shown above the columns represent fold transactivation by GST-Tat relative to GST alone. (E) Overexpression of 19S subunits enhances Tat-mediated transcription. HeLa-LTR-luc cells were transfected with 500 ng of plasmid expressing HA-Rpt1-6, HA-Rpn9, or Flag-20S b4, as indicated, in the presence or absence of 100 ng of pTat101Flag. Shown is coactivation of Tat-dependent transcription where transactivation by Tat in control cells was attributed a value of 1. Western blot of cell extracts using anti-HA, anti-Flag, and antitubulin antibodies is shown below the graph. All graphs represent mean and standard error obtained from at least three independent experiments.

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Figure 2. Proteasomal ATPases Are Recruited to the HIV-1 Promoter in Response to Tat (A) Schematic diagram showing the locations of primers used to amplify sequences present in chromatin immunoprecipitates by Q-PCR. HIV-1 DNA sequences are depicted as a black line; NF-kB and Sp1 sites within the HIV-1 promoter are indicated by hatched and striped boxes, respectively; transcription start site is shown by the arrowhead; and luciferase reporter gene is shown as a white box. TAR RNA stem loop is indicated at the 50 end of the transcript in gray. Primers used to amplify promoter-proximal and coding region sequences are indicated on the figure. (B) HeLa-LTR-luc cells were treated with GST (gray bars) or GST-Tat101 (black bars) and then analyzed by ChIP by using the antibodies indicated on the figure. Regions proximal to the promoter (top panel) and within the coding sequence (bottom panel) were amplified by Q-PCR.

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b4 had no significant effect on either basal or Tat-mediated transcription (Figure 1E and data not shown). In contrast to wild-type Rpt5, a mutant that contains an N-terminal deletion failed to coactivate Tat (Figure S2A) and interacted significantly less efficiently with both TatFlag and another proteasomal ATPase, Rpt1 (Rpt1-GFP; Figure S2B). These results show that an interaction between Tat and proteasomal ATPases is required for coactivation of HIV-1 transcription. Taken together, these results suggest that the proteasome plays different roles in HIV-1 transcription that are dependent on the presence of Tat. Proteasome-mediated protein degradation downregulates transcriptional output from the LTR in the absence of Tat, whereas 19S appears to be required for Tat-mediated transcription. Recruitment of Proteasome Components to the HIV-1 Promoter Is Altered in Response to Tat Given that the proteasome, or subunits thereof, appear to regulate HIV-1 transcription differently in the presence or absence of Tat, we examined which subunits were associated with the HIV-1 promoter during basal and Tat-dependent transcription. ChIP analysis was performed on HeLa-LTR-luc cells treated with GST or GST-Tat by using anti-GST, anti-19S subunits, and anti-20S a4 antibodies followed by Q-PCR of sequences in promoter-proximal and coding regions using the primers indicated (Figure 2A). In the absence of Tat (Figure 2B, gray bars), 19S subunits were associated with both promoter-proximal and coding regions when compared to mock controls (top and bottom panels). 20S a4 was weakly associated with the promoterproximal region and absent from the coding region (Figure 2B) when compared to mock controls. In the presence of GST-Tat (black bars), a significant increase in the association of 19S components Rpt5, Rpt6, Rpn7, and, to a lesser degree, Rpn10 was observed in the promoter-proximal region (Figure 2B, top panel). Surprisingly, analysis within the coding region showed that association of all of the 19S subunits, with the exception of Rpt5, was diminished in the presence of Tat (Figure 2B, bottom panel and data not shown). In contrast to 19S subunits, association of 20S a4 with either the promoter-proximal or coding region was not significantly increased in the presence of Tat (Figure 2B). Thus, Tat specifically recruits proteasomal 19S components to the promoter-proximal region. An inverse correlation was observed between the degree of transactivation by Tat and proteasome recruitment to the promoter (Figure S3). Comparison of experiments in which Tat-mediated transactivation was high (79-fold) or low (2-fold) following transduction with the same quantity of GST-Tat showed that recruitment of GST-Tat did not significantly differ between high or low transactivation conditions. In contrast, both a 19S subunit (Rpt6) and a 20S subunit (20S a4) were highly recruited, particularly

to the coding region, under low transactivation conditions. High-level recruitment of Rpt6 and 20S a4 was independent of Tat, since both proteins were highly represented on HIV-1 sequences in the absence of Tat (Figure S3, white bars). These data suggest that proteasome is highly present, especially at promoter-distal sites, under conditions that are unfavorable for transcription. Transcription may be more frequently abortive under unfavorable conditions, which results in ubiquitylation of arrested RNAPII (Somesh et al., 2005), and subsequent recruitment of 26S proteasome (Gillette et al., 2004). Tat Induces a Redistribution of Proteasome Subunits Since 19S components coactivated Tat transactivation (Figure 1) and are enriched at the promoter-proximal region in the presence of Tat (Figure 2B), we analyzed the distribution of proteasome complexes in nuclear extracts in the presence and absence of Tat by gel filtration chromatography. In control S3 cells (Figure 2C, panels depicted as ), 19S and 20S subunits were mainly localized in high molecular weight fractions that contain high proteolytic activity. These fractions likely correspond to 26S proteasome and other 20S-containing complexes. Extracts of Tat-expressing S101 cells (Figure 2C, +Tat) contained the putative 26S proteasome but were also highly enriched in a lower molecular weight complex that contained 19S ATPases and non-ATPases Rpn10 and Rpn2 (data not shown) but lacked 20S a4. The lower molecular weight complex was devoid of proteolytic activity, and, although it appears to resemble 19S RP, is biochemically distinguishable from 19S RP (Figures S4A and S4B). Since properties such as globularity and electrostatic charge influence the profile of a complex in these analyses, it is possible that the complex present in fractions 16–18 may represent a modified form of 19S. Therefore, we will refer to this complex as ‘‘19S-like.’’ Furthermore, gel filtration chromatography of nuclear extract from S101-overexpressing 19S subunits (HA-Rpt5 and HA-Rpt6) showed an increase in the abundance of the 19S-like complex relative to control S101 (Figure S4C). In contrast, overexpression of Flag-20S b4 had no effect. Thus, coactivation of Tat-dependent transcription by subunits of the 19S, but not 20S, (Figure 1E) may be due, in part, to their ability to facilitate formation of the 19S-like complex, which is in turn recruited by Tat to the HIV-1 promoter. A Proteasome Disassembly Factor, PAAF1, Is a Tat Cofactor Enrichment of a 19S-like complex in cells expressing Tat (Figure 2C) is reminiscent of the reported activity of a recently described proteasome-interacting protein, proteasomal ATPase-associated factor 1 (PAAF1) (Park et al., 2005). PAAF1 reportedly downregulates 26S proteolytic

(C) Tat induces a redistribution of cellular proteasome components. Nuclear extracts (NE) of S3 and S101 cells, depicted as  and +Tat, respectively, were analyzed by gel filtration chromatography followed by SDS/PAGE and western blot using the antibodies indicated. An aliquot of each fraction was analyzed for peptidase activity, as indicated below the figure. Elution positions of molecular weight markers are shown above the figure.

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Figure 3. A Proteasome-Associated Protein, PAAF1, Is a Coactivator of Tat (A) PAAF1 induces proteasome dissociation in vitro. Purified 26S proteasome (denoted as RP2CP on the figure) was incubated with GST or GSTPAAF1 for 1 hr at 37 C. Products were analyzed by nondenaturing PAGE followed by in-gel peptidase activity assay (left panel). Products were transferred to PVDF and analyzed by western blot using anti-Rpt4 antibody. A faster-migrating complex that represents 19S RP is denoted by an asterisk on the figure (right panel). (B) Tat interacts with PAAF1. Tat-HA was immunoprecipitated with anti-HA antibody, and immunoprecipitates were analyzed by western blot using anti-Flag (top panel). An aliquot of cell extract was analyzed by direct western blot using anti-Flag and anti-HA antibodies (middle and bottom panels). (C) PAAF1 is a coactivator of Tat-mediated transcription. HeLa-LTR-luc cells were transfected with or without plasmid expressing Tat101Flag (100 ng) in the presence of plasmid expressing HA-PAAF1 (500 ng) or empty vector. Extracts were assayed for luciferase activity. Fold activation refers to the increase in Tat transactivation over that seen with Tat101Flag alone, which was normalized to 1. Expression of HA-PAAF1 in cell extracts is shown below. (D) Endogenous PAAF1 is required for optimal Tat-mediated transactivation. HeLa-LTR-luc cells were transfected with control (Sc) or PAAF1-specific (PAAF1) siRNA. Following treatment with GST or GST-Tat, cell extracts were analyzed for luciferase activity, which was normalized to protein content (left panel). An aliquot of cells was analyzed by Q-RT-PCR for the amount of PAAF1 mRNA, which was normalized to GAPDH mRNA in the same samples (right panel).

activity by preventing association between 20S and 19S particles (Park et al., 2005). Indeed, incubation of purified 26S proteasome with GST-PAAF1 led to a significant decrease in 26S peptidase activity in vitro (Figure 3A, left panel). Western blot analysis of the same gel using anti-

Rpt4 antibody showed that the level of 26S proteasome (RP2CP) was reduced while a faster-migrating complex (denoted by asterisk on the figure), which likely represents 19S RP, was correspondingly increased in the presence of GST-PAAF1 (Figure 3A, right panel). Thus, we wondered

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whether PAAF1 might be recruited by Tat to inhibit proteasome activity and generate a 19S-like complex to potentiate transcription. We first investigated whether Tat and PAAF1 interact in vivo. Tat-HA interacted with FlagPAAF1 in overexpression experiments (Figure 3B). We next tested whether PAAF1 could coactivate Tat in HIV-1 transcription. Overexpression of PAAF1 led to a 15-fold increase in Tat-mediated transactivation (Figure 3C), while knockdown of endogenous PAAF1 significantly reduced Tat-dependent transactivation (Figure 3D, left panel). Knockdown of endogenous PAAF1 was verified by Q-RT-PCR of PAAF1 mRNA in cells transfected with control or PAAF1-specific siRNAs (Figure 3D, right panel). PAAF1 had no effect on basal transcription in either overexpression (data not shown) or RNA interference experiments. We next determined whether or not the increase in the 19S-like complex observed in the presence of Tat was dependent on PAAF1. S3 or S101 cells were transfected with empty vector or vector expressing Flag-PAAF1 and analyzed by gel filtration chromatography (Figure 4A). Overexpression of Flag-PAAF1 in S3 cells increased the abundance of the 19S-like complex (fractions 15–17) compared to control S3 cells. In S101 cells, Flag-PAAF1 overexpression did not further increase the amount of 19S (data not shown). It is possible that other factors, which may regulate 19S formation in vivo, may be rate limiting and thus overexpression of Flag-PAAF1 alone in S101 cells, which already contain a significant amount of 19S-like complex, does not lead to the formation of additional complexes. Flag-PAAF1 eluted with high molecular weight fractions (9–13) that contain 26S (peak proteolytic activity at fraction 11) as well as other complexes. This distribution may reflect the association of Flag-PAAF1 with multiple different complexes that have both proteolytic and nonproteolytic functions. Flag-PAAF1 was not detected in fractions corresponding to the19S-like complex in either S3 or S101 cells, suggesting either that PAAF1 does not stably interact with this complex or that the interaction may occur locally, such as at promoter regions. Since a 19S-like complex was enriched by overexpression of Tat or Flag-PAAF1 (Figures 2C and 4A), we analyzed whether the distribution of 20S CP was also affected. Although 20S distribution was not significantly different when analyzed by gel filtration chromatography followed by western blot, this analysis cannot distinguish between different 20S-containing complexes such as 26S proteasome-containing one or two 19S RP or 20S associated with one or two PA28 subunits. To better analyze 20S complexes, fractions spanning the peak of proteolytic activity (fractions 7–14) obtained after gel filtration chromatography were subjected to nondenaturing PAGE, followed by ‘‘in-gel’’ peptidase activity assays (Figure S5). 20S CP has very low specific peptidase activity, but it can be ‘‘activated’’ by treatment with SDS (Glickman et al., 1998), thus facilitating localization of 20S CP. By this analysis, S3 control cells were found to contain mostly 26S proteasome. Very little 20S CP was detected, even after SDS treatment, suggesting that 20S CP was mostly engaged

in 26S proteasome. In the presence of Flag-PAAF1, 20S CP was readily detectable, even in the absence of SDS. Interestingly, 20S CP was abundant in S101 cells, even in the absence of Flag-PAAF1. Furthermore, in S101 cells expressing Flag-PAAF1, virtually no high molecular weight form of 26S proteasome could be detected, and 20S CP was readily apparent. These data show that overexpression of Flag-PAAF1 alone or together with Tat enhanced the dissociation of 20S CP from higher-order complexes in a manner that correlates with the abundance of the 19S-like complex in the same cells. To further analyze the role of PAAF1 in formation of a 19S-like complex, S101 cells were transfected with control or PAAF1-specific siRNA and analyzed by gel filtration chromatography (Figure 4B). PAAF1 mRNA was reduced 2-fold after normalization to GAPDH in both S3 and S101 cells, as measured by Q-PCR (data not shown). The abundance of 19S (fractions 16–18) was significantly diminished in PAAF1 knockdown cells (Figure 4B). Thus, formation of the 19S-like complex optimally depends on the presence of both Tat and PAAF1. Tat-Mediated Recruitment of 19S to the HIV-1 Promoter Is Dependent on PAAF1 The data suggest that Tat interacts with PAAF1 to promote disassembly of the 26S proteasome liberating 19S RP that is subsequently recruited to the HIV-1 promoter by Tat. ChIP analysis was performed to determine whether PAAF1 is associated with the HIV-1 promoter. Thus, HeLa-LTR-luc cells were transfected with empty vector or Flag-PAAF1 in the presence or absence of GST-Tat as indicated and analyzed by ChIP. Flag-PAAF1 was expressed equivalently in cells treated with GST or GSTTat as measured by anti-Flag western blot of cell extracts (data not shown). GST-Tat was enriched 4- to 6-fold at promoter-proximal and coding regions independent of Flag-PAAF1 expression (Figure 5A). Thus, recruitment of GST-Tat was unaffected by overexpression of FlagPAAF1. In contrast, recruitment of Flag-PAAF1 to HIV-1 sequences was highly dependent on Tat. Flag-PAAF1 was enriched 5-fold at a promoter-proximal region and 3-fold in the coding region, in the absence of Tat (Figure 5B, ), while in the presence of Tat, Flag-PAAF1 was enriched 25-fold at a promoter-proximal region and 35fold in the coding region (Figure 5B, +). Thus, PAAF1 was highly recruited to HIV-1 DNA sequences by Tat. To determine whether PAAF1 is required for the specific recruitment of 19S to the HIV-1 promoter in the presence of Tat (see Figure 2B, black bars), control cells or cells knocked down for PAAF1 were analyzed by ChIP for 19S recruitment. Knockdown of endogenous PAAF1 was verified by Q-RT-PCR of PAAF1 mRNA in cells transfected with control siRNA or siRNA specific for PAAF1 (Figure 5C, right panel). Recruitment of Tat was not significantly affected by knockdown of PAAF1 (Figure 5C, left panel; anti-GST). Interestingly, recruitment of 19S components Rpt5 and Rpt6 were also not significantly affected by PAAF1-knockdown. In contrast, 20S b7 was highly

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Figure 4. Formation of a 19S-like Complex Is Dependent on PAAF1 (A) Nuclear extracts from control () or Flag-PAAF1-expressing (+) S3 cells were analyzed by gel filtration chromatography followed by SDS/PAGE and western blot using the indicated antibodies. Fractions containing peak peptidase activity are indicated below. (B) Formation of a 19S-like complex is dependent on PAAF1. S101 cells were transfected with control or PAAF1-specific siRNA as indicated and analyzed by gel filtration chromatography followed by SDS/PAGE and western blot using anti-Rpt4 and anti-20S a4 antibodies. Fractions containing peak peptidase activity are indicated below.

recruited to the promoter in PAAF1 knockdown cells compared to control cells in the presence of Tat (Figure 5C). These data suggest that Tat-mediated recruitment of 19S, independent of 20S, requires PAAF1. In the absence of PAAF1, Tat recruits mostly 26S proteasome that negatively regulates HIV-1 transcription (Figure 1D).

19S RP Is Required for Optimal Transcriptional Elongation Following initiation from the HIV-1 promoter, RNAPII encounters a strong pause site resulting in the synthesis a 59 nt RNA hairpin, TAR. Tat binds to TAR in a complex with the transcription elongation factor, P-TEFb, that

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Figure 5. Tat-Mediated Recruitment of a 19S Subcomplex to the HIV-1 Promoter Is Dependent on PAAF1 (A) Recruitment of Tat to the LTR is unaffected by PAAF1. HeLa-LTR-luc cells, transfected with pFlag-PAAF1 or empty vector, then treated with GST or GST-Tat101 as indicated, were analyzed by ChIP by using anti-GST antibodies. Sequences within the promoter-proximal and coding regions were amplified by Q-PCR. Shown is fold enrichment relative to immunoprecipitations performed in the absence of Tat. (B) Tat recruits PAAF1 to HIV-1 DNA sequences. The same samples analyzed in (A) were immunoprecipitated by using anti-Flag antibodies and amplified by Q-PCR. Shown is fold enrichment relative to immunoprecipitations performed in the absence of Flag-PAAF1. (C) PAAF1 is required for the specific recruitment of a 19S subcomplex to the HIV-1 promoter in the presence of Tat. HeLa-LTR-luc cells were transfected with control (Sc) or PAAF1-specific (PAAF1) siRNA and then treated with GST or GST-Tat as indicated. Samples were analyzed by ChIP using the indicated antibodies followed by Q-PCR of the promoter-proximal region (left panel). The amount of PAAF1 mRNA in control and PAAF1 knockdown cells was quantitated by Q-RT-PCR and normalized to the amount of GAPDH mRNA in the same samples (right panel).

hyperphosphorylates RNAPII CTD, among other substrates, to alleviate transcriptional pausing and promote transcriptional elongation. Overexpression and RNA interference experiments showed that 19S components and PAAF1 strongly coactivate Tat-mediated transcription. Furthermore, ChIP analysis revealed that these coactivators are highly recruited by Tat but that 19S components remain largely associated with the promoter-proximal region and do not appear to travel with Tat and elongating RNAPII in the coding region. As APIS has been shown to promote transcriptional elongation at the Gal1-10 promoter (Ferdous et al., 2001), we wondered if the 19-like complex might be involved in resolving paused transcriptional elongation complexes at the HIV-1 LTR. Q-RT-PCR using different primer pairs can be used to distinguish paused versus elongated transcripts. Thus, paused (TAR), early elongated, and midelongated transcripts were amplified using specific primer pairs (Figure 6A). The amount of TAR in control cells (Sc) was equivalent in the presence or absence of Tat (Figure 6B), as expected, since Tat does not influence the rate of transcriptional initiation

but instead promotes transcriptional elongation (Taube et al., 1999). Analysis of early elongated and midelongated transcripts in control cells showed an increase relative to TAR, consistent with the activity of Tat in enhancing transcriptional elongation. Quantitative analyses of transcripts synthesized in cells in which expression of a 19S (Rpt1) or 20S (20S a4) component was knocked down (Figure 6B, lower right panel), or in cells mock-treated or treated with proteasome inhibitor MG132 (Figure 6C), were performed. The quantity of early and midelongated transcripts was significantly diminished in Rpt1 knockdown cells (Figure 6B). In 20S a4 knockdown cells, on the other hand, the quantity of elongated transcripts was increased by approximately 2-fold. Table S1 summarizes the relative efficiencies of initiation (R.E.I.) and elongation (R.E.E.) of transcription, together with the luciferase reporter gene activity in each experiment. R.E.I. was determined by normalizing the amount of TAR transcript in each sample to that of GST-treated cells transfected with control siRNA (Sc + GST) or GST-treated cells without MG132 (MG132 mock + GST), which were given an arbitrary value of 1.

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Figure 6. Knockdown of Proteasome Subunits and Inhibition of Proteasome Activity Affect Different Stages of HIV-1 Transcription (A) Schematic diagram showing the locations of the different primers used in Q-RT-PCR analysis. HIV-1 promoter is shown as a gray line, and luciferase reporter gene is shown as a white box. The RNA transcript is shown as a black line. TAR RNA hairpin is indicated at the 50 end of the transcript. The location of the RNAPII pause site is indicated. Forward and reverse primers used to amplify TAR sequences are located within TAR; to amplify early elongated transcripts, a forward primer corresponding to the stem sequence of TAR was used together with a reverse primer corresponding to the 50 end of luciferase; midelongated transcripts were amplified by using forward and reverse primers within luciferase. (B) Knockdown of a 19S or 20S subunit affects transcriptional elongation. HeLa-LTR-luc cells were transfected with control siRNA or siRNAs specific for a 19S subunit (Rpt1) or a 20S subunit (20S a4), then treated with GST or GST-Tat as indicated. Total RNA was isolated, and reverse transcripts were analyzed by Q-PCR using the primer pairs indicated on the figure. Values were normalized to the quantity of GAPDH in each sample. Knockdown of Rpt1 and 20S a4 was verified by western blot of cell extracts using the antibodies indicated as shown in the top panel. (C) Inhibition of proteasome activity increases initiation of transcription. HeLa-LTR-luc cells were treated with MG132 or carrier in the presence or absence of GST or GST-Tat, as indicated on the figure. Total RNA was purified, reverse transcribed, and analyzed by Q-PCR using the primer pairs indicated. Values were normalized to the quantity of GAPDH in each sample.

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R.E.E. was determined by calculating the ratio of elongated transcripts to paused transcripts normalized to the value for Sc + GST or MG132 mock + GST. Rpt1 knockdown cells showed a significant defect in transcriptional elongation (Table S1, R.E.E. for early elongated transcripts of 1.36 compared to 10 for control cells in the presence of Tat). Ablation of 20S a4 lead to slight increases in both initiation and elongation, depending on the presence or absence of Tat. Thus, the increased luciferase activity may be due to effects on both initiation and elongation of transcription. Transcript analysis of cells mock treated or treated with proteasome inhibitor MG132 showed that amounts of TAR and elongated transcripts were significantly upregulated in MG132-treated cells (Figure 6C). Since R.E.I. was increased by 27-fold whereas R.E.E. was not significantly affected (Table S1), these data indicate that MG132 increases basal transcription by specifically increasing initiation of transcription from the promoter. Because 19S components, such as Rpt1, appeared to be required for efficient transcriptional elongation, we next analyzed clearance of the transcription elongation complex from the promoter region. To this end, recruitment of the positive transcription elongation factor (PTEFb) and RNAPII to promoter-proximal and coding regions was analyzed by ChIP in control and Rpt1 knockdown cells. Knockdown of Rpt1 expression was verified by western blot of cell extracts (Figure 7A, lower right panel, and Figure 7B, far right panel), and, as expected, Rpt1 was not recruited to the promoter-proximal region in the presence of Tat in Rpt1 knockdown cells (Figure 7A, bottom left panel). Recruitment of GST-Tat, RNAPII, and Cdk9 to the promoter-proximal region was equivalent in Rpt1 knockdown cells compared to controls (Figures 7A and 7B, left panels). In contrast, association of both RNAPII and Cdk9 with the coding region was reduced in the presence of Tat in cells lacking Rpt1 (Figures 7A and 7B, right panels). These data are consistent with a defect in clearance of the paused transcriptional elongation complex from the promoter in the absence of a functional 19S subcomplex. DISCUSSION Our study has provided evidence that the proteasome, or subunits thereof, can both positively and negatively regulate transcription from the same promoter at different stages of transcription and that the switch is mediated by transcription factor. By using the well-studied Tat/PTEFb-inducible HIV-1 promoter, our data suggest a model in which proteasome activity negatively regulates basal transcription in the absence of Tat. In support of this, 19S and 20S subunits of the proteasome are associated with the promoter and coding regions, and treatment of cells with proteasome inhibitor or knockdown of a 20S component enhanced basal transcription. In the presence of Tat, proteasome function is switched from a proteolytic to nonproteolytic mode in which a 19S-like complex is

required for Tat-mediated transcriptional elongation. We show that Tat recruits a coactivator, PAAF1, that is known to promote proteasome disassembly. The abundance of the 19S-like complex was enhanced in cells expressing both Tat and PAAF1. A role for 19S in transcription was demonstrated by showing that PAAF1 and 19S components strongly coactivated Tat-dependent transcription and were recruited to the promoter-proximal region in the presence of Tat. Finally, analysis of transcripts from the HIV-1 promoter suggests that the 19S-like complex may facilitate clearance of paused transcriptional elongation complexes from the promoter. The function of proteasome-mediated protein degradation in the regulation of transcription has been aptly described as a conundrum (Lipford and Deshaies, 2003). In some cases, ubiquitylation and degradation of activators increase transcription, whereas in other cases transcriptional repression is observed (Kinyamu et al., 2005). At the HIV-1 promoter, inhibition of proteasome-dependent proteolysis significantly enhanced basal transcription, which was due to a significant upregulation of initiation of transcription. The inference from this finding is that one or more factors required for transcription initiation are presumably degraded by proteasome. Potential candidates could be RNAPII and/or PCAF, which have been reported to be ubiquitylated and degraded (Jin et al., 2004; Somesh et al., 2005). However, it is possible that components of the general transcription machinery might also be regulated by proteolytic degradation. Negative regulation of basal HIV-1 transcription may have important implications for the control of viral latency that is regulated in many cases at the level of viral transcription (Lin et al., 2003). 19S has previously been proposed to play a nonproteolytic role in transcription. 19S RP has been shown to be recruited by monoubiquitination of H2B (Ezhkova and Tansey, 2004) and to subsequently recruit SAGA acetyltransferase complex (Lee et al., 2005), suggesting a role in initiation of transcription at yeast promoters. On the other hand, other studies in yeast have indicated a role for the APIS complex in transcriptional elongation (Ferdous et al., 2001; Gonzalez et al., 2002). Recruitment of 19S RP and APIS have been proposed to occur via ubiquitylation of H2B or activator, respectively (Ezhkova and Tansey, 2004; Ferdous et al., 2002; Gonzalez et al., 2002). Indeed, Rpt5, Rpn1, and Rpn10 subunits of 19S have been shown to interact with ubiquitin (Pickart and Cohen, 2004). On the HIV-1 promoter, we observed that, while proteasome is present at low amounts in the absence of Tat, significant additional recruitment of 19S was observed in the presence of Tat. Since Tat has been shown to be ubiquitylated, which subsequently enhances HIV-1 transcription (Bres et al., 2003), it is tempting to speculate that Tat ubiquitylation may be implicated in recruiting additional 19S to the HIV-1 promoter. Indeed, we have observed that fusion of ubiquitin to Tat greatly enhances its interaction with 19S subunits (I.L. and R.E.K., unpublished data).

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Figure 7. 19S Subunits Are Required for Clearance of RNAPII and Cdk9 from the HIV-1 Promoter (A) HeLa-LTR-luc cells were transfected with control or Rpt1-specific siRNA and treated with GST or GST-Tat as indicated on the figure. Samples were analyzed by ChIP using anti-GST, anti-RNAP II, or anti-Rpt1 antibodies as indicated. Promoter-proximal and coding region sequences were amplified by Q-PCR from immunoprecipitates. An aliquot of cell extract was analyzed by western blot using anti-Rpt1 and anti-tubulin antibodies (bottom right panel). (B) Experiment was performed as described in (A) except that cells were transfected with pHA-CDK9 and pCyclinT1 and samples were immunoprecipitated by using anti-HA antibody.

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Although ubiquitin may be the signal that recruits 19S to promoters, an unexplained element in the models proposed for 19S function is how 19S is recruited independently of 20S. We have described a mechanism for the specific recruitment of 19S. We show that the transactivator, Tat, interacts with a cellular coactivator, PAAF1, which promotes proteasome disassembly. Recruitment of PAAF1 provides Tat with a mechanism to inhibit proteasome through disassembly and to simultaneously liberate 19S. We showed that the abundance of a 19S-like complex is increased in cells expressing both Tat and PAAF1, and the specific recruitment of 19S, independent of 20S, to the promoter by Tat is dependent on PAAF1. It will be interesting to determine whether PAAF1, or a functionally related protein, is recruited to other promoters, particularly those at which 19S has been shown to enhance transcription. Interestingly, ChIP analysis suggests that Tat-dependent recruitment of PAAF1 does not appear to affect proteasome that is already localized on the promoter. Instead, Tat may be recruited to TAR RNA as part of a PAAF1/19S complex. Consistent with this, Tat does not significantly enhance transcription initiation as observed in cells treated with MG132, suggesting that proteasome acting on transcription initiation may remain functional, even in the presence of Tat. In this case, proteasome may regulate transcription through both proteolytic (initiation) and nonproteolytic (19S; elongation) mechanisms simultaneously on the same promoter. Although the complex observed in the presence of Tat and PAAF1 appears to resemble 19S, it is biochemically distinguishable from 19S. Further analysis will be required to determine its exact composition. If the 19S-like complex, 19S RP, and APIS are discernible complexes, perhaps obtained by successive dissociation of 19S, it will be interesting to determine what regulates this process and whether these complexes perform different functions. In this respect, we noted that, although several 19S components were recruited specifically to the promoter-proximal region, presumably as a 19S-like complex, only Rpt5 crosslinked to the coding region in the presence of Tat. The same result was obtained in cells transfected with a plasmid expressing Flag-Rpt5 and immunoprecipitated using anti-Flag, suggesting that the signal is specific for Rpt5 (I.L. and R.E.K., unpublished data). It is possible that Tat interacts directly with Rpt5, since Rpt5 was first identified as a Tat binding protein (Nelbock et al., 1990). Thus, Rpt5, through its interaction with Tat, may be associated with coding region sequences independently of 19S. It has been reported that 20S subunits, absent from coding region sequences at certain yeast promoters, become associated with sequences at the 30 ends of genes (Gillette et al., 2004), leading to the suggestion that a proteolytic function of 26S proteasome may be required for the termination of transcription. In this respect, it is possible that Rpt5 associated with Tat in the coding region may act as a platform for the reassociation of 19S and 20S subunits at the 30 end of HIV-1 DNA sequences. Alternatively,

Rpt5 may perform additional functions independent of its role in 19S. Despite the mounting evidence that 19S or 19S subcomplexes are required for transcription, it is still unclear how these complexes function to enhance discrete stages of the transcription cycle. In the case of 19S RP and APIS, the ATP-dependent chaperonin activity of the complexes has been proposed to remodel the SAGA complex and transcription elongation complex, respectively (Ferdous et al., 2001; Gonzalez et al., 2002; Lee et al., 2005). Our data would be consistent with a role for 19S in remodeling the transcription elongation complex at HIV-1 DNA sequences since a significant defect in clearance of the transcription elongation complex from the promoter was observed in Rpt1 knockdown cells. However, ChIP analysis revealed that 19S showed a somewhat different localization on the HIV-1 promoter to that observed for APIS in yeast. While APIS was detected throughout the coding region at yeast genes, 19S localized specifically to a promoter-proximal site of the LTR, which may be the paused transcription elongation complex. Indeed, this pattern is similar to that described for the elongation factor, TFIIF (Sims et al., 2004), that is believed to specifically associate with elongation complexes that have paused near the promoter. TFIIF, in concert with TFIIS, facilitates transcriptional elongation by preventing stalled RNAPII from becoming arrested. In the absence of TFIIF, paused elongation complexes are more likely to decay into arrested complexes. This function of TFIIF depends on an ATPdependent DNA helicase function provided by TFIIH. It has been proposed that 19S ATPases possess ATPasedependent RNA and DNA helicase activity in addition to chaperonin activity. Thus, an alternative hypothesis for the function of 19S in enhancing transcription elongation may be that the complex provides ATP-dependent helicase activity to assist TFIIF/TFIIS in preventing RNAPII that has paused near the promoter at TAR from becoming arrested and blocking transcription. In vitro studies using mutant ATPases may be required to determine the precise function of 19S in transcriptional elongation. EXPERIMENTAL PROCEDURES Nuclear Extracts All steps were performed at 4 C with cold buffers. Cells were washed in PBS and then in hypotonic buffer (HB; 10 mM Tris HCl [pH 7.4], 20 mM KCl, and 1.5 mM MgCl2). Cell pellets were resuspended in HB and placed on ice for 10 min to swell. Cells were homogenized by using a dounce homogenizer and B type pestle, and cell disruption was monitored microscopically. Cells were then centrifuged at 3900 rpm for 15 min. Supernatants, corresponding to cytoplasmic extracts, were removed, and pellets were washed once with HB. Nuclear pellets were resuspended in a half volume of low-salt buffer (Tris HCl 20 mM [pH 7.4], 20 mM KCl, 10% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 10 mM b mercaptoethanol, and 0.2 mM PMSF). A half volume of highsalt buffer (low-salt buffer + 1.2 M KCl) was added dropwise. Proteins were extracted by stirring on ice for 30 min. Samples were clarified by centrifugation at 14,000 3 g for 30 min to pellet nuclear debris. Supernatants, corresponding to nuclear extracts, were harvested and protein concentrations were quantified by Bradford reagent (Pierce).

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Gel Filtration Chromatography Nuclear extract (100 mg) was resolved on a Superose 6 (3.2/30) gel filtration column (Smart system, GE Healthcare) in Nuclear Buffer (20 mM Tris HCl [pH 7.4], 400 mM KCl, 10% glycerol, 1.5 mM MgCl2, and 0.2 mM EDTA). Thirty fractions of 50 mL were collected and analyzed by in vitro peptidase assay, SDS/PAGE, and native PAGE followed by western blot where indicated. Molecular weight markers for gel filtration chromatography (Sigma Aldrich) were analyzed under the same conditions. Quantitative RT-PCR Total RNA was extracted from a sample of GST- or GST-Tat-treated cells by using mirVana (Ambion) or NucleoSpin RNA Kit (Machery Nagel, Duren, Germany) and reverse transcribed using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). RT products were amplified by quantitative PCR (Bio-Rad) by using the oligonucleotides described in Supplemental Experimental Procedures. Additional experimental procedures can be found in the Supplemental Data. Supplemental Data Supplemental Data include five figures, one table, Supplemental Experimental Procedures, and Supplemental References and can be found with this article online at http://www.molecule.org/cgi/content/ full/25/3/369/DC1/. ACKNOWLEDGMENTS We wish to thank J.-B. Yoon, Y. Shaul, and V. Baldin for plasmids; K.-T. Jeang for HeLa-LTR-luc cells; and M. Mechali for his support. Grants from the Human Frontier Science Program (HFSP) (Young Investigator Program), European Union (EU012182), and Agence Nationale de Recherches sur le Sida (ANRS) to M.B.; Sidaction to O.C.; and ANRS and Sidaction to R.E.K. supported this work. L.K.L. was supported by ANRS and Sidaction, C.C.-B. by HFSP, and I.L. by HFSP and EU012182. Received: July 31, 2006 Revised: October 25, 2006 Accepted: December 20, 2006 Published: February 8, 2007 REFERENCES Auld, K.L., Brown, C.R., Casolari, J.M., Komili, S., and Silver, P.A. (2006). Genomic association of the proteasome demonstrates overlapping gene regulatory activity with transcription factor substrates. Mol. Cell 21, 861–871. Bres, V., Kiernan, R.E., Linares, L.K., Chable-Bessia, C., Plechakova, O., Treand, C., Emiliani, S., Peloponese, J.M., Jeang, K.T., Coux, O., et al. (2003). A non-proteolytic role for ubiquitin in Tat-mediated transactivation of the HIV-1 promoter. Nat. Cell Biol. 5, 754–761. Dembla-Rajpal, N., Seipelt, R., Wang, Q., and Rymond, B.C. (2004). Proteasome inhibition alters the transcription of multiple yeast genes. Biochim. Biophys. Acta 1680, 34–45. Deroo, B.J., and Archer, T.K. (2002). Proteasome inhibitors reduce luciferase and beta-galactosidase activity in tissue culture cells. J. Biol. Chem. 277, 20120–20123.

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