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regulates human immunodeficiency virus 1 gene expression through its association with cdk9
1 Department of Neuroscience, Center for Neurovirology, Temple University School of Medicine, 1900 N 12th Street 015-96, Philadelphia, PA 19122, USA
2 Section of Microbiology, Department of Biomedical Sciences, Center of Excellence for Biotechnology Development and Biodiversity Research, Sassari, Italy
3 Department of Biology, College of Science and Technology, Temple University School of Medicine, 1900 N 12th Street 015-96, Philadelphia, PA 19122, USA
Correspondence
Bassel E. Sawaya
sawaya{at}temple.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, NF-
B) factors. The HIV-1 Tat protein recruits the human positive transcription elongation factor P-TEFb, consisting of cdk9 and cyclin T1, to the HIV-1 transactivation response (TAR) region. In the absence of TAR, Tat activates the HIV-1 long terminal repeat (LTR) through its association with several cellular factors including C/EBP. C/EBP is a member of the CCAAT/enhancer-binding protein family of transcription factors and has been shown to be a critical transcriptional regulator of HIV-1 LTR. We examined whether Tat–C/EBP association requires the presence of the P-TEFb complex. Using immunoprecipitation followed by Western blot, we demonstrated that C/EBP–cyclin T1 association requires the presence of cdk9. Further, due to its instability, cdk9 was unable to physically interact with C/EBP in the absence of cyclin T1 or Tat. Using kinase assays, we demonstrated that cdk9, but not a cdk9 dominant-negative mutant (cdk9-dn), phosphorylates C/EBP. Our functional data show that co-transfection of C/EBP and cdk9 leads to an increase in HIV-1 gene expression when compared to C/EBP alone. Addition of C/EBP homologous protein (CHOP) inhibits C/EBP transcriptional activity in the presence and absence of cdk9 and causes a delay in HIV-1 replication in T-cells. Together, our data suggest that Tat–C/EBP association is mediated through cdk9, and that phosphorylated C/EBP may influence AIDS progression by increasing expression of HIV-1 genes. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In addition to its association with the P-TEFb complex, Tat is able to physically and functionally associate with several cellular transcription factors including p300, P/CAF and C/EBP (Brady & Kashanchi, 2005; Peruzzi, 2006).
The P-TEFb complex is composed of two subunits, cyclin T1 and its associated kinase, cdk9 (Yu et al., 2006). Cyclin T1 was first identified as a Tat-binding protein that mediates cooperative binding of the Tat–cyclin T1 complex to the transactivation response (TAR) region and in turn recruits cdk9 to RNA polymerase II (Garriga & Graña, 2004; Ghafouri et al., 2006). Cyclin T1 contains several domains including the N-terminal domain, a putative coiled-coil motif, a His-rich motif and a carboxy-terminal PEST sequence (Wei et al., 1998). Cyclin T1–Tat interaction requires the entire cyclin box and a key cysteine residue (C261) within the TRM (Tat–TAR recognition motif) (Garber et al., 1998). This residue is essential for zinc-dependent HIV-Tat binding. Upregulation of cyclin T1 correlates with hyperphosphorylation of RNA polymerase II and HIV replication in stimulated peripheral blood lymphocytes (Liou et al., 2004; Marshall & Graña, 2006).
The 42 kDa protein, cdk9, like many other cyclin-dependent kinases (CDKs), was identified during a cDNA screening intended to isolate novel regulators of the mammalian cell cycle (Graña et al., 1994). As no cyclin partner or cell cycle function was demonstrated at that time, cdk9 was temporarily designated PITALRE for its PSTAIRE-like sequence, a conserved motif found in CDC2 and related kinases (Pines, 1994; Jeffrey et al., 1995). cdk9 was shown to phosphorylate itself, as well as a variety of substrates and to be associated with various proteins including TRAF2 (MacLachlan et al., 1998), B-Myb (De Falco et al., 2000), the 7SK small nuclear RNA (snRNA) (Nguyen et al., 2001), pRb (Simone et al., 2002), gp130 (Falco et al., 2002), Pch1/Pct1 (Pei et al., 2003) and p53 (Claudio et al., 2006). cdk9 also phosphorylates the molecular chaperone Hsp70 or a kinase-specific chaperone complex, Hsp90–Cdc37, to form two separate chaperone–cdk9 complexes (Zhang et al., 2004). Depletion of cdk9 blocks Tat-dependent transactivation, indicating that cdk9 is a subunit of the cellular cofactor required for Tat to stimulate transcription (Zhu et al., 1997). Thus, cdk9 and cyclin T1, in addition to Tat, are key regulators of HIV-1 gene expression. In the nucleus, not every cyclin T1–cdk9 heterodimer displays P-TEFb transcriptional activity. In fact, about half of the heterodimers in HeLa cells are sequestered in a complex with the 7SK snRNA and the HEXIM1 protein (Chen et al., 2004; Yik et al., 2004). Whereas 7SK serves as a molecular scaffold to mediate the interaction of HEXIM1 with cyclin T1–cdk9, HEXIM1 inhibits the cdk9 kinase activity and prevents P-TEFb from binding to transcriptional templates (Michels et al., 2004). HEXIM1 was also shown to inhibit Tat transcriptional activity (Fraldi et al., 2005).
C/EBPs comprise a family of basic region leucine zipper (bZIP) transcription factors that, as a prerequisite for DNA binding through their basic regions, dimerize through their adjacent C-terminal leucine zippers to form homodimers or heterodimers with other C/EBP family members (Ramji & Foka, 2002). The C/EBP gene is transcribed into a single 1.4 kb mRNA (Cao et al., 1991; Descombes & Schibler, 1991). At the protein level, however, multiple C/EBP isoforms, varying in size from 14 to 38 kDa, have been reported (Descombes & Schibler, 1991). The C/EBP isoforms include two full-length LAP (liver-enriched activator protein) isoforms (38 and 34 kDa) and two truncated 14 and 21 kDa LIP (liver-enriched inhibitory protein) isoforms (Descombes & Schibler, 1991). Another member of the C/EBP family is called CHOP (C/EBP-homologous protein) and acts in most, but not all, circumstances as a dominant-negative inhibitor of DNA binding when it is heterodimerized to another C/EBP partner (Ron & Habener, 1992). While the C/EBPs are well conserved through their bZIP regions, they exhibit more limited conservation in their N-terminal activation and regulatory domains (Sebastian & Johnson, 2006). The functional importance of amino-terminal determinants in C/EBP-mediated transcriptional regulation is supported by the fact that C/EBP153–297 (LIP), a truncated form of C/EBP, can function as a repressor of C/EBP transcriptional activity (Ruocco et al., 1996; Kowenz-Leutz et al., 1994). C/EBP
, and 
commonly function as strong transcriptional activators, while C/EBP
and 
(CHOP) generally act to inhibit C/EBP transcriptional activators (Lekstrom-Himes & Xanthopoulos, 1998; Gao et al., 2002). C/EBP-binding sites have been identified in the promoter regions of numerous genes, including HIV-1 LTR (Henderson et al., 1995), the genes for interleukin 6 (IL-6; Akira et al., 1990), tumour necrosis factor alpha (TNF-; Pope et al., 1994) and monocyte chemoattractant protein-1(MCP-1; Sekine et al., 2002). Moreover, the activity of C/EBP is influenced by various inflammatory stimuli, including lipopolysaccharide (An et al., 1996), IL-6 (Poli et al., 1990) and TNF- (Yin et al., 1996).
We previously demonstrated the ability of HIV-Tat and C/EBP to physically and functionally associate (Coyle-Rink et al., 2002; Abraham et al., 2005). In this study, we examined whether the interplay between Tat and C/EBP is dependent on the Tat partners, cyclin T1 and cdk9.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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and CHOP expression plasmids were previously described (Sawaya et al., 2000; Abraham et al., 2005; Claudio et al., 2006). pGEX-2T-cyclin T1 (wild-type and mutants), glutathione S-transferase (GST)–cdk9 and GST–C/EBP were previously described (Abraham et al., 2005).
Cell culture and transfection assays.
The human U-87 MG glioblastoma cell line was maintained in Dulbecco's modified Eagle's medium+10 % FBS. Cells were transfected with 0.5 µg reporter plasmid [HIV-LTR-luc (full-length or mutant)] or co-transfected with 1.0 µg various expression cDNAs (Amini et al., 2004). The amount of DNA used for each transfection was normalized with pcDNA3 vector plasmids. Each transfection was repeated multiple times with different plasmid preparations. Cell extracts were prepared 48 h after transfection and luciferase or chloramphenicol acetyl transferase (CAT) assays were performed as previously described (Amini et al., 2002).
Overexpression and purification of recombinant proteins.
GST–cdk9, GST–C/EBP and GST–cyclin T1 fusion proteins were expressed and purified as previously described (Rossi et al., 2006). The integrity and purity of the GST fusion proteins were analysed by SDS-PAGE followed by Coomassie blue staining. Radiolabelled cdk9 and C/EBP full-length and deletion mutant proteins were synthesized using the TNT Coupled Wheat Germ Extract system according to the manufacturer's recommendations (Promega).
In vitro protein–protein interactions (GST pull-down assay).
Four microlitres of 35S-labelled and in vitro-translated (IVT) C/EBP T1 was incubated with 5 µg GST or GST–cyclin T1 fusion proteins coupled to glutathione–Sepharose beads in 300 µl lysis buffer 150 (LB-150) for 2 h at 4 °C with continuous rocking. After incubation, beads were pelleted and washed five times with LB-150 buffer. The same procedure was used with labelled and translated cdk9, when incubated with GST and GST–C/EBP. Bound proteins were eluted with Laemmli sample buffer, heated to 95 °C for 5 min and separated by SDS-PAGE.
Immunoprecipitation and Western blotting.
U-87MG cells were transfected with 5 µg HIV-1 Tat, cyclin T1, cdk9 and C/EBP expression plasmids using various combinations. Forty-eight hours post-transfection, 300 µg cell extract was immunoprecipitated with antibodies as indicated. Western blot analysis was performed as described previously (Claudio et al., 2006) using anti-cdk9, anti-cyclin T1 or anti-C/EBP antibody. Anti-Grb2 antibody was used as a control for equal protein loading.
Kinase assays.
Kinase assays were performed essentially as previously described (Claudio et al., 2006) with 100 ng substrate and 0.5 µg each [35S]IVT-cdk9, [35S]IVT-cdk9-dn or 500 µg whole-cell extracts prepared from U-87MG in 10 µl reaction mixture containing 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 100 mg BSA ml–1, 50 mM ATP and 2.5 µCi (92.5 kBq) of [-32P]ATP. Reactions were incubated at 37 °C for 30 min, stopped with SDS loading buffer, run on an SDS-PAGE (10 % gel), fixed, stained and then allowed to dry before autoradiography.
Infection of cells.
The human macrophage U937 cell line (maintained in RPMI+10 % FBS), in the exponential phase of growth was infected with the JR-FL strain of HIV-1 as follows. Fifty nanograms of p24-containing virus stock was added to every 106 cells. Cells were incubated with virus stock in a small volume (200 µl) of serum-free medium for 2 h at 37 °C. The cells were then washed twice with PBS and fresh medium containing 2 % FBS was added (5x105 cells ml–1). The cells were transfected with pcDNA3-CHOP 24 h pre-HIV-1 infection. Cells were collected every alternate day for preparation of cell extracts.
p24 ELISA.
p24 antigen-capture ELISA for tissue culture supernatants was performed as described by the manufacturer (Beckman Coulter). Each sample was assayed over a 104-fold range of dilution to ensure quantification was based on an absorbance value within the linear range of the standards.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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(Wei et al., 1998; Coyle-Rink et al., 2002) provides us with the rationale to examine whether addition of cyclin T1 or cdk9 affects Tat–C/EBP association. First, we investigated whether C/EBP associates with cyclin T1 in the absence of Tat and cdk9. U-87MG cells were transfected with plasmids encoding C/EBP or cyclin T1 (as indicated). As shown in Fig. 1(a), non-transfected (lane 1) or C/EBP-transfected cells (lane 4) were used as controls to mark the positions of C/EBP as well as the efficiency of transfection (endogenous vs overexpressed, respectively). Proteins were immunoprecipitated with a polyclonal anti-cyclin T1 antibody (lanes 2 and 5) or with non-immune rabbit serum (NIS) (lanes 3 and 6), followed by Western blot analysis using anti-C/EBP antibody. This experiment demonstrated the ability of endogenous (lane 2) and exogenous C/EBP (lane 5) to interact with endogenous cyclin T1. Specificity of cyclin T1–C/EBP interaction was confirmed using NIS. Interestingly, immunoprecipitation/Western blot analysis performed with extracts prepared from C/EBP-transfected cells revealed that cyclin T1 associates with several C/EBP isoforms (45 and 38 kDa). Note that C/EBP migrates around 45 kDa as previously described (Roman et al., 1990).
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antibody (Fig. 1b
, lanes 2 and 5) or with NIS (lanes 3 and 6), followed by Western blot analysis using anti-cyclin T1 antibody. Non-transfected (lane 1) or cyclin T1-transfected cells (lane 4) were used as negative and positive controls, respectively. Specificity of C/EBP–cyclin T1 interaction was confirmed using NIS (lanes 3 and 6). Cyclin T1 was co-immunoprecipitated by anti-C/EBP antibody (lanes 2 and 5). These experiments demonstrated the ability of endogenous and exogenous cyclin T1 to interact with endogenous and exogenous C/EBP in the absence of Tat.
Cyclin T1 binding to C/EBP was also tested by GST pull-down assays using IVT-35S-labelled C/EBP. Various C-terminal cyclin T1 deletion mutants were fused to GST and incubated with purified recombinant C/EBP, Tat or C/EBP+Tat (Fig. 1d). Labelled proteins were incubated with GST or GST–cyclin T1 fusion proteins coupled to glutathione–Sepharose beads. As shown in Fig. 1(c), deletion of the C-terminal regions of cyclin T1 up to residue 300 did not affect its ability to bind to C/EBP. However, deletion of residues 300–250 abrogated the interaction of C/EBP with cyclin T1 (Fig. 1d). Interestingly, HIV-1 Tat protein was previously shown to interact with the same domain within cyclin T1 (Sawaya et al., 2000). Cyclin T1–Tat interaction requires the entire cyclin box and a key cysteine residue (C261) (Garber et al., 1998). In the light of this observation, the experiment was repeated using labelled Tat and/or C/EBP. Data obtained from incubation of labelled Tat protein with GST or GST–cyclin T1 full-length and deletion mutants corroborate the previously described data. Interestingly, formation of the C/EBP–cyclin T1 complex was not affected by the addition of Tat. These results led us to suggest that C/EBP–Tat–cyclin T1 co-exist in the same protein complex. Fig. 1(e) illustrates the size of GST (lane 2) and GST–cyclin T1 deletion mutants (lanes 3–8).
Next, we examined the ability of cyclin T1 to associate with C/EBP in the absence of the cyclin T1 partner, cdk9. It should be noted that cdk9 and cyclin T1, in addition to Tat, are key regulators of transcription of HIV-1 gene expression. U-87MG cells were transfected with C/EBP expression plasmid. Twenty-four hours later, total cell protein was isolated from these cells as well as untransfected controls and 500 µg was incubated with anti-cdk9 antibody. After continuous rotation for 12 h, 20 µl Sepharose A beads were added to the mixture and incubated for another 2 h. The beads were then removed by microcentrifugation and the cdk9-depleted extracts (supernatant) were subjected to Western blot analysis. Fifty micrograms of protein isolated from the Sepharose beads or from the supernatant was subjected to Western blot analysis to verify that the antibodies had immunodepleted cdk9. As shown in Fig. 2(a), depletion of cdk9 was complete in the supernatant of extracts incubated with anti-cdk9 antibody but not with NIS (compare lane 3 to lane 2). Extracts prepared from non-transfected or cdk9-transfected cells were used as negative and positive controls, respectively (lanes 1 and 4).
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to physically associate with cyclin T1 in the presence and absence of cdk9. Three hundred micrograms of extracts prepared from non-transfected or C/EBP-transfected cells was incubated with anti-cyclin T1 antibody (Fig. 2b, lanes 2 and 5) or with NIS (lanes 3 and 6). Extracts prepared from cdk9-depleted extracts were also immunoprecipitated with anti-cyclin T1 antibody (lane 8). As a control, 50 µg extract prepared from non-transfected, C/EBP-transfected or cdk9-depleted cells was subjected to Western blot analysis using C/EBP antibody (lanes 1, 4 and 7). As shown in Fig. 2(b), endogenous, as well as overexpressed, C/EBP physically associates with endogenous cyclin T (lanes 2 and 5). Interestingly, C/EBP lost its ability to interact with cyclin T1 in the absence of cdk9 (compare lane 8 to lanes 2 and 5). These results led us to conclude that cdk9 associates with C/EBP and therefore C/EBP was depleted with cdk9. Note that C/EBP was not detected in cdk9-depleted extracts (lane 7), which may be due to the association of C/EBP with cdk9.
Reciprocally, the cells were immunoprecipitated with anti-C/EBP antibody and subjected to Western blot analysis using anti-cyclin T1 antibody. As shown in Fig. 2(c), endogenous and overexpressed C/EBP remain associated with cyclin T1 (lanes 2 and 5) in the presence of cdk9 but not in its absence (lane 8). Interestingly, cyclin T1 was not detected in cdk9-depleted extracts (lane 7). The observed associations are specific because no bands were observed when the immunoprecipitations were performed using NIS (lanes 3 and 6). These results led us to conclude that association of C/EBP with cyclin T1 is cdk9-dependent.
To further demonstrate the specificity of these interactions, and to confirm the dependence of C/EBP–cyclin T1 association on the presence of cdk9, similar experiments were performed using non-transfected and cdk9-transfected U-87MG cells where cyclin T1 was depleted. As shown in Fig. 2(d), non-transfected (lane 1) and cdk9-transfected cells (lane 4) were used as negative and positive controls, respectively. Specificity of C/EBP–cdk9 interaction was shown with NIS (lanes 3 and 6). The C/EBP was specifically co-immunoprecipitated by anti-cdk9 antibody (lanes 2 and 5). Extracts prepared from cyclin T1-depleted extracts were also immunoprecipitated with anti-C/EBP antibody (lane 8). As a control, 50 µg extract prepared from cyclin T1-depleted cells was subjected to Western blot analysis using cdk9 antibody (lane 7). Interestingly, cdk9 lost its ability to interact with C/EBP in the absence of cyclin T1 (compare lane 8 to lanes 2 and 5). These results can be explained by the fact that cdk9 is not stable in the absence of cyclin T1 (Garriga & Graña, 2004).
Next, we sought to examine the physical association between C/EBP and cdk9 in the presence of Tat and in the absence of cyclin T1 using non-transfected and cdk9-transfected U-87MG cells. Note that Tat expression plasmid was transfected in all the samples. As shown in Fig. 2(e), extracts prepared from non-transfected (lane 1) and cdk9-transfected cells (lane 4) were used as negative and positive controls, respectively. Specificity of C/EBP–cdk9 interaction was shown with NIS (lanes 3 and 6). The C/EBP was specifically co-immunoprecipitated by anti-cdk9 antibody (lanes 2 and 5). Extracts prepared from cyclin T1-depleted extracts were also immunoprecipitated with anti-C/EBP antibody (lane 8). As a control, 50 µg extract prepared from cyclin T1-depleted cells was subjected to Western blot analysis using cdk9 antibody (lane 7). Surprisingly, cdk9 remained associated with C/EBP in the absence of cyclin T1 (compare lane 8 to lanes 2 and 5). These results led us to conclude that Tat helped the stabilization of cdk9 in the absence of cyclin T1 and therefore allowed cdk9–C/EBP association.
To further investigate the dependence of C/EBP–Tat association on the presence of cdk9, immunoprecipitation/Western blot assays were performed using cdk9-depleted extracts. U-87MG cells were transfected with Tat expression plasmid. Twenty-four hours post-transfection, the cells were washed and cellular proteins were prepared from non- or Tat-transfected cells. As shown in Fig. 2(f), extracts prepared from non-transfected cells were used as negative controls (lane 1). Specificity of C/EBP–Tat interaction was shown using NIS (lanes 3 and 5). C/EBP was specifically co-immunoprecipitated by anti-Tat antibody (lane 2). Extracts prepared from cdk9-depleted extracts were also immunoprecipitated with anti-Tat antibody (lane 4). Interestingly, Tat lost its ability to associate with C/EBP in the absence of cdk9 (compare lanes 2 and 4). These results confirmed our hypothesis regarding the ability of Tat to help the stabilization of cdk9 in the absence of cyclin T1 observed in Fig. 2(e).
Note that physical association between cyclin T1 and C/EBP was also studied in the presence of Tat and absence of cdk9. Our results demonstrated the inability of cyclin T1 to associate with C/EBP in the absence of cdk9, regardless of the presence or absence of Tat (data not shown).
C/EBP–cdk9 association was also tested in vitro by GST pull-down assays using IVT-35S-labelled cdk9 or IVT-35S-labelled C/EBP. Labelled proteins were incubated with GST, GST–C/EBP or GST–cdk9 fusion protein coupled to glutathione–Sepharose beads. As shown in Fig. 3(a), C/EBP was retained by the GST–cdk9 fusion protein but not by GST alone (compare lanes 2 and 3). Reciprocally, 35S-labelled cdk9 was incubated with GST or GST–C/EBP coupled to beads. As shown in Fig. 3(b), cdk9 was retained by GST–C/EBP but not by GST alone (compare lanes 2 and 3). Fig. 3(c) illustrates the sizes of GST (lane 2), GST–cdk9 (lane 3) and GST–C/EBP (lane 4), respectively.
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–cdk9 association leads to C/EBP phosphorylation. Further, it has been shown that phosphorylation of C/EBP contributes to cell survival and inhibition of pro-caspases, which leads to C/EBP induction (Buck et al., 2001). Kinase assays were performed using 300 µg cell proteins prepared from U-87MG cells and incubated with GST or GST–C/EBP. A kinase assay was performed as described in Methods. As shown in Fig. 3(d), endogenous cdk9 phosphorylates endogenous C/EBP and GST–C/EBP (lane 2) but not GST (lane 1).
The specificity of C/EBP phosphorylation by cdk9 was further confirmed using a cdk9 dominant-negative mutant (cdk9-dn) (Graña et al., 1994) using IVT-cdk9 proteins. cdk9 or cdk9-dn was incubated with GST or with GST–C/EBP and the kinase assay was performed. As shown in Fig. 3(e), cdk9 phosphorylates GST–C/EBP (lane 2) but not GST alone (lane 1). IVT-cdk9-dn was unable to phosphorylate GST (lane 3) or GST–C/EBP (lane 4). These results demonstrated the ability of endogenous, immunoprecipitated and pure cdk9 to phosphorylate C/EBP; they also confirmed the specificity of this phosphorylation.
Phosphorylation of C/EBP by cdk9 was functionally studied using transient transfection assays. In addition, it was previously demonstrated that CHOP forms heterodimers with C/EBP and inhibits its DNA-binding activity and its function as a transcriptional regulator (Shirakawa et al., 2006). U-87MG cells were transfected with 0.5 µg HIV-LTR reporter plasmid alone or in the presence of 1.0 µg Tat, cdk9, cdk9-dn, C/EBP or CHOP expression plasmids using various combinations. The amount of DNA used for each transfection was normalized with pCDNA3 or pCEP-4 vector plasmids. Forty-eight hours after transfection, the cells were washed and processed for luciferase assay. As shown in Fig. 4(a), HIV-1 LTR was activated by C/EBP, cdk9 and Tat expression plasmids, respectively (compare lanes 3, 5 and 11 to lane 1). As expected, addition of CHOP or cdk9-dn did not appreciably affect HIV-1 gene expression (compare lanes 2 and 8 to lane 1). Activation of HIV-LTR by C/EBP was further enhanced in the presence of cdk9 (lane 6), Tat (lane 12) or cdk9+Tat (lane 14) expression plasmids. This effect decreased when C/EBP was co-transfected with CHOP (lane 4), cdk9-dn (lane 9) or Tat+cdk9-dn (lane 16) expression plasmids. Therefore, we concluded that phosphorylation of C/EBP by cdk9 is important for C/EBP to perform its transcriptional activity. Note that the cells were transfected in triplicate in which one dish was used to verify the efficiency of transfection, which was examined by Western blot analysis as shown in Fig. 4(b). Grb2 was used to verify the equal loading of cellular proteins.
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to activate HIV-1 LTR through its direct binding to the region spanning nt –115 to –107 (Lee et al., 2001; Ruocco et al., 1996) prompted us to study its effect on HIV-1 LTR in the absence of this region (–105 to +66). U-87MG cells were transfected with 0.5 µg HIV-LTR (full-length and mutant) reporter plasmids alone or in the presence of 1.0 µg Tat or C/EBP expression plasmids. The amount of DNA used for each transfection was normalized with pCDNA3 vector plasmid. Forty-eight hours after transfection, the cells were washed and processed for CAT assay. As shown in Fig. 4(c), C/EBP activates full-length (–456 to +66) (lane 2) and mutant (–105 to +66) (lane 5) HIV-1 LTR by approx. 11- and seven-fold, respectively. As a positive control, the cells were transfected with Tat expression plasmid (lanes 3 and 6). These results further confirm our observation regarding the functional and physical interplay between C/EBP and cdk9. Our results also corroborate previously published results where transcription from HIV-1 LTRs lacking C/EBP sites was shown to be induced following addition of C/EBP (Henderson et al., 1995).
Finally, we confirmed a role for C/EBP in HIV-1 transcription and replication, using a replication assay. U937 cells were infected with HIVJR-FL after being transfected with 2.5 µg CHOP expression plasmid. Empty pcDNA3 vector was used as a negative control (Fig. 4b). The progression of infection was visually observed by the increase in syncytium formation in the infected cells. The cells and the supernatants were collected every 48 h. At the indicated times, using ELISA assays against p24, we observed that inhibition of C/EBP activity by CHOP leads to a delay in HIV-1 replication in these cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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activates several cellular and viral genes including the HIV-1 LTR and that C/EBP is necessary for HIV-1 replication in monocytes (Henderson et al., 1995; Henderson & Calame, 1997). C/EBP does not influence HIV-1 infection, but can affect HIV-1 replication by regulating virus transcription in part through its association with viral proteins and the recruitment of co-activators to LTR (Lee et al., 2001). Based on these findings, we, as well as others, were able to demonstrate the ability of C/EBP to physically and functionally interact with HIV-1 Tat and Vpr proteins (Coyle-Rink et al., 2002; Hogan et al., 2003). Here, we investigated the relationship between C/EBP and Tat's partners, cyclin T1 and cdk9. We demonstrated the ability of C/EBP to interact with cyclin T1 in the presence of cdk9 but not in its absence. We also showed that C/EBP physically and functionally associates with cdk9, which in turn has the ability to phosphorylate C/EBP.
The HIV-1 C/EBP binding site overlaps with a consensus sequence (E box), which is a binding site for proteins of the B class of basic helix–loop–helix leucine zipper (b-HLH-Zip) family (Dang et al., 1992). Members of this family include c-Myc, Max, Mad, TFE3, TFEB and Mxi1 (Murre et al., 1989; Blackwood & Eisenman, 1991; Ayer et al., 1993), which form homo- and heteromultimers and are potentially able to associate with C/EBP or nuclear factor (NF)-B transcription factors. These factors play a positive role in the regulation of HIV-1 promoter. The HIV-1 LTR contains at least two C/EBP sites (491–483 and 300–292), which could positively contribute to promoter activation (Zhang et al., 1995), suggesting that C/EBP sites can function as positive or negative regulators of gene expression depending on their location relative to the TATA box of the LTR. The biological relevance of the HIV-1 C/EBP binding site is suggested by the fact that the sequence appears to be conserved in primary isolates from AIDS patients over several years (Michael et al., 1994). C/EBP was also shown to impact HIV-1 replication through the recruitment of co-activators to the LTR (Lee et al., 2001). This latest observation is in accord with our earlier data, where we demonstrated that depletion of cdk9 prevents association between C/EBP and Tat. Further, it has been shown that the depletion of cdk9 blocks Tat-dependent transactivation and Tat-associated kinase activity, indicating that cdk9 is a subunit of a cellular cofactor and a co-activator required for Tat to stimulate viral transcription (Zhu et al., 1997).
The interplay between Tat and C/EBP is not limited to HIV-1 LTR. We previously reported the ability of both proteins to affect MCP-1 gene expression (Abraham et al., 2005). Other studies have also previously described such interplay in regulating the IL-6 promoter (Spooner et al., 2007). Tat was shown to activate IL-6 gene expression through its cooperation with C/EBP transcription factors. In this study, Tat increased the nuclear levels of C/EBP transcription factors by inducing post-translational modifications of C/EBP factors through the activation of specific kinases, which phosphorylate C/EBP and render it more transcriptionally active (Nakajima et al., 1993). The above data are consistent with our findings where we demonstrated the ability of cdk9 to phosphorylate C/EBP and enhance its transcriptional ability. These data suggest that Tat may promote HIV-1 gene expression by upregulating the cellular levels of transcription factors acting on the viral LTR. The association of Tat with phosphorylated C/EBP on the IL-6 promoter increases the DNA-binding activity of C/EBP dimers by enhancing their affinity for the target DNA. In this regard, the identity of the specific phosphorylated residue within C/EBP and whether phosphorylation of C/EBP by cdk9 leads to an increase of C/EBP-binding activity remain to be clarified.
Our results also show that CHOP, a member of the C/EBP family, which was known as an inhibitor of C/EBP, can function as an inhibitor of HIV-1 gene expression and replication. In this regard, one may suggest that interaction of C/EBP with CHOP might prevent cdk9–C/EBP association. Finally, it is possible that cdk9 facilitates cross-communication of C/EBP with Tat or cellular transcription initiation factors. This observation is in accord with studies describing the ability of C/EBP to interact with components of the basal transcriptional machinery such as the TATA-binding protein and transcription factor IIB (Nerlov & Ziff, 1995) and other transcription factors, including Nf-B and the cAMP response element-binding protein (Hohaus et al., 1995; Westendorf et al., 1998). These proteins may cooperate in the recruitment of co-activators to the HIV-1 LTR in cells.
In summary, we demonstrated that C/EBP is necessary for establishing HIV-1 infection of T cells and influencing HIV-1 gene expression through its interaction with cdk9. Further, our data also suggest that phosphorylated C/EBP may affect the expression of inflammatory cytokines and, if so, could have an indirect influence on HIV-1 disease progression. Finally, our results provide insight into the general mechanisms by which C/EBP regulates HIV-1 transcription through the recruitment of Tat activators to the LTR (e.g. cdk9).
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ACKNOWLEDGEMENTS |
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Akira, S., Isshiki, H., Sugita, T., Tanabe, O., Kinoshita, S., Nishio, Y., Nakajima, T., Hirano, T. & Kishimoto, T. (1990). A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J 9, 1897–1906.[Medline]
Amini, S., Clavo, A., Nadraga, Y., Giordano, A., Khalili, K. & Sawaya, B. E. (2002). Interplay between cdk9 and NF-kappaB factors determines the level of HIV-1 gene transcription in astrocytic cells. Oncogene 21, 5797–5803.[CrossRef][Medline]
Amini, S., Saunders, M., Kelley, K., Khalili, K. & Sawaya, B. E. (2004). Interplay between HIV-1 Vpr and Sp1 modulates p21(WAF1) gene expression in human astrocytes. J Biol Chem 279, 46046–46056.
An, M. R., Hsieh, C. C., Reisner, P. D., Rabek, J. P., Scott, S. G., Kuninger, D. T. & Papaconstantinou, J. (1996). Evidence for posttranscriptional regulation of C/EBPalpha and C/EBPbeta isoform expression during the lipopolysaccharide-mediated acute-phase response. Mol Cell Biol 16, 2295–2306.[Abstract]
Ayer, D. E., Kretzner, L. & Eisenman, R. N. (1993). Mad: a heterodimeric partner for Max that antagonizes Myc transcriptional activity. Cell 72, 211–222.[CrossRef][Medline]
Blackwood, E. M. & Eisenman, N. R. (1991). Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science 251, 1211–1217.
Brady, J. & Kashanchi, F. (2005). Tat gets the ‘green’ light on transcription initiation. Retrovirology 2, 69.[CrossRef][Medline]
Buck, M., Poli, V., Hunter, T. & Chojkier, M. (2001). C/EBPbeta phosphorylation by RSK creates a functional XEXD caspase inhibitory box critical for cell survival. Mol Cell 8, 807–816.[CrossRef][Medline]
Cao, Z., Umek, R. M. & McKnight, S. L. (1991). Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev 5, 1538–1552.
Chen, R., Yang, Z. & Zhou, Q. (2004). Phosphorylated positive transcription elongation factor b (P-TEFb) is tagged for inhibition through association with 7SK snRNA. J Biol Chem 279, 4153–4160.
Claudio, P. P., Cui, J., Ghafouri, M., Mariano, C., White, M. K., Safak, M., Sheffield, J. B., Giordano, A., Khalili, K. & other authors (2006). Cdk9 phosphorylates p53 on serine 392 independently of CKII. J Cell Physiol 208, 602–612.[CrossRef][Medline]
Coyle-Rink, J., Sweet, T., Abraham, S., Sawaya, B., Batuman, O., Khalili, K. & Amini, S. (2002). Interaction between TGFbeta signaling proteins and C/EBP controls basal and Tat-mediated transcription of HIV-1 LTR in astrocytes. Virology 299, 240–247.[CrossRef][Medline]
Dang, C. V., Dolde, C., Gillison, M. C. & Kato, G. J. (1992). Discrimination between related DNA sites by a single amino acid residue of Myc-related basic-helix-loop-helix proteins. Proc Natl Acad Sci U S A 89, 599–602.
De Falco, G., Bagella, L., Claudio, P. P., De Luca, A., Fu, Y., Calabretta, B., Sala, A. & Giordano, A. (2000). Physical interaction between CDK9 and B-Myb results in suppression of B-Myb gene autoregulation. Oncogene 19, 373–379.[CrossRef][Medline]
Descombes, P. & Schibler, U. (1991). A liver-enriched transcriptional activator protein, LAP, and transcriptional inhibitory protein, LIP, are translated from the same mRNA. Cell 67, 569–579.[CrossRef][Medline]
Falco, G. D., Neri, L. M., Falco, M. D., Bellan, C., Yu, Z., Luca, A. D., Leoncini, L. & Giordano, A. (2002). Cdk9, a member of the cdc2-like family of kinases, binds to gp130, the receptor of the IL-6 family of cytokines. Oncogene 21, 7464–7470.[CrossRef][Medline]
Fraldi, A., Varrone, F., Napolitano, G., Michels, A. A., Majello, B., Bensaude, O. & Lania, L. (2005). Inhibition of Tat activity by the HEXIM1 protein. Retrovirology 2, 42.[CrossRef][Medline]
Gao, H., Parkin, S., Johnson, P. F. & Schwartz, R. C. (2002). C/EBP gamma has a stimulatory role on the IL-6 and IL-8 promoters. J Biol Chem 277, 38827–38837.
Garber, M. E., Wei, P., KewalRamani, V. N., Mayall, T. P., Herrmann, C. H., Rice, A. P., Littman, D. R. & Jones, K. A. (1998). The interaction between HIV-1 Tat and human cyclin T1 requires zinc and a critical cysteine residue that is not conserved in the murine CycT1 protein. Genes Dev 12, 3512–3527.
Garriga, J. & Graña, X. (2004). Cellular control of gene expression by T-type cyclin/CDK9 complexes. Gene 337, 15–23.[CrossRef][Medline]
Ghafouri, M., Amini, S., Khalili, K. & Sawaya, B. E. (2006). HIV-1 associated dementia: symptoms and causes. Retrovirology 3, 28.[CrossRef][Medline]
Graña, X., De Luca, A., Sang, N., Fu, Y., Claudio, P. P., Rosenblatt, J., Morgan, D. O. & Giordano, A. (1994). PITALRE, a nuclear CDC2-related protein kinase that phosphorylates the retinoblastoma protein in vitro. Proc Natl Acad Sci U S A 91, 3834–3838.
Henderson, A. J. & Calame, K. L. (1997). CCAAT/enhancer binding protein (C/EBP) sites are required for HIV-1 replication in primary macrophages but not CD4(+) T cells. Proc Natl Acad Sci U S A 94, 8714–8719.
Henderson, A. J., Zou, X. & Calame, K. L. (1995). C/EBP proteins activate transcription from the human immunodeficiency virus type 1 long terminal repeat in macrophages/monocytes. J Virol 69, 5337–5344.[Abstract]
Hogan, T. H., Nonnemacher, M. R., Krebs, F. C., Henderson, A. & Wigdahl, B. (2003). HIV-1 Vpr binding to HIV-1 LTR C/EBP cis-acting elements and adjacent regions is sequence-specific. Biomed Pharmacother 57, 41–48.[Medline]
Hohaus, S., Petrovick, M. S., Voso, M. T., Sun, Z., Zhang, D. E. & Tenen, D. G. (1995). PU.1 (Spi-1) and C/EBP alpha regulate expression of the granulocyte-macrophage colony-stimulating factor receptor alpha gene. Mol Cell Biol 15, 5830–5845.[Abstract]
Jeffrey, P. D., Russo, A. A., Polyak, K., Gibbs, E., Hurwitz, J., Massague, J. & Pavletich, N. P. (1995). Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex. Nature 376, 313–320.[CrossRef][Medline]
Kowenz-Leutz, E., Twamley, G., Ansieau, S. & Leutz, A. (1994). Novel mechanism of C/EBP beta (NF-M) transcriptional control: activation through derepression. Genes Dev 8, 2781–2791.
Lee, E. S., Zhou, H. & Henderson, A. J. (2001). Endothelial cells enhance human immunodeficiency virus type 1 replication in macrophages through a C/EBP-dependent mechanism. J Virol 75, 9703–9712.
Lekstrom-Himes, J. & Xanthopoulos, K. G. (1998). Biological role of the CCAAT/enhancer-binding protein family of transcription factors. J Biol Chem 273, 28545–28548.
Liou, L. Y., Herrmann, C. H. & Rice, A. P. (2004). Human immunodeficiency virus type 1 infection induces cyclin T1 expression in macrophages. J Virol 78, 8114–8119.
MacLachlan, T. K., Sang, N., De Luca, A., Puri, P. L., Levrero, M. & Giordano, A. (1998). Binding of CDK9 to TRAF2. J Cell Biochem 71, 467–478.[CrossRef][Medline]
Marshall, R. M. & Graña, X. (2006). Mechanisms controlling CDK9 activity. Front Biosci 11, 2598–2613.[Medline]
Michael, N. L., D'Arcy, L., Ehrenberg, P. K. & Redfield, R. R. (1994). Naturally occurring genotypes of the human immunodeficiency virus type 1 long terminal repeat display a wide range of basal and Tat-induced transcriptional activities. J Virol 68, 3163–3174.
Michels, A. A., Fraldi, A., Li, Q., Adamson, T. E., Bonnet, F., Nguyen, V. T., Sedore, S. C., Price, J. P., Price, D. H. & other authors (2004). Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/cyclin T) inhibitor. EMBO J 23, 2608–2619.[CrossRef][Medline]
Murre, C., McCaw, P. S., Vaessin, H., Caudy, M., Jan, L. Y., Jan, Y. N., Cabrera, C. V., Buskin, J. N., Hauschka, S. D. & other authors (1989). Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell 58, 537–544.[CrossRef][Medline]
Nakajima, T., Kinoshita, S., Sasagawa, T., Sasaki, K., Naruto, M., Kishimoto, T. & Akira, S. (1993). Phosphorylation at threonine-235 by a ras-dependent mitogen-activated protein kinase cascade is essential for transcription factor NF-IL6. Proc Natl Acad Sci U S A 90, 2207–2211.
Nerlov, C. & Ziff, E. B. (1995). CCAAT/enhancer binding protein-alpha amino acid motifs with dual TBP and TFIIB binding ability co-operate to activate transcription in both yeast and mammalian cells. EMBO J 14, 4318–4328.[Medline]
Nguyen, V. T., Kiss, T., Michels, A. A. & Bensaude, O. (2001). 7SK small nuclear RNA binds to and inhibits the activity of CDK9/cyclin T complexes. Nature 414, 322–325.[CrossRef][Medline]
Pei, Y., Schwer, B. & Shuman, S. (2003). Interactions between fission yeast Cdk9, its cyclin partner Pch1, and mRNA capping enzyme Pct1 suggest an elongation checkpoint for mRNA quality control. J Biol Chem 278, 7180–7188.
Peruzzi, F. (2006). The multiple functions of HIV-1 Tat: proliferation versus apoptosis. Front Biosci 11, 708–717.[CrossRef][Medline]
Peterlin, B. M. & Price, D. H. (2006). Controlling the elongation phase of transcription with P-TEFb. Mol Cell 23, 297–305.[CrossRef][Medline]
Pines, J. (1994). The cell cycle kinases. Semin Cancer Biol 5, 305–313.[Medline]
Poli, V., Mancini, F. P. & Cortese, R. (1990). IL-6DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to C/EBP. Cell 63, 643–653.[CrossRef][Medline]
Pope, R. M., Leutz, A. & Ness, S. A. (1994). C/EBP beta regulation of the tumor necrosis factor alpha gene. J Clin Invest 94, 1449–1455.[Medline]
Ramji, D. P. & Foka, P. (2002). CCAAT/enhancer-binding proteins: structure, function and regulation. Biochem J 365, 561–575.[Medline]
Roman, C., Platero, J. S., Shuman, J. & Calame, K. (1990). Ig/EBP-1: a ubiquitously expressed immunoglobulin enhancer binding protein that is similar to C/EBP and heterodimerizes with C/EBP. Genes Dev 4, 1404–1415.
Ron, D. & Habener, J. F. (1992). CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription. Genes Dev 6, 439–453.
Rossi, A., Mukerjee, R., Ferrante, P., Khalili, K., Amini, S. & Sawaya, B. E. (2006). Human immunodeficiency virus type 1 Tat prevents dephosphorylation of Sp1 by TCF-4 in astrocytes. J Gen Virol 87, 1613–1623.
Ruocco, M. R., Chen, X., Ambrosino, C., Dragonetti, E., Liu, W., Mallardo, M., De Falco, G., Palmieri, C., Franzoso, G. & other authors (1996). Regulation of HIV-1 long terminal repeats by interaction of C/EBP(NF-IL6) and NF-kappaB/Rel transcription factors. J Biol Chem 271, 22479–22486.
Sawaya, B. E., Khalili, K., Gordon, J., Taube, R. & Amini, S. (2000). Cooperative interaction between HIV-1 regulatory proteins Tat and Vpr modulates transcription of the viral genome. J Biol Chem 275, 35209–35214.
Sebastian, T. & Johnson, P. F. (2006). Stop and go: anti-proliferative and mitogenic functions of the transcription factor C/EBPbeta. Cell Cycle 5, 953–957.[Medline]
Sekine, O., Nishio, Y., Egawa, K., Nakamura, T., Maegawa, H. & Kashiwagi, A. (2002). Insulin activates CCAAT/enhancer binding proteins and proinflammatory gene expression through the phosphatidylinositol 3-kinase pathway in vascular smooth muscle cells. J Biol Chem 277, 36631–36639.
Shirakawa, K., Maeda, S., Gotoh, T., Hayashi, M., Shinomiya, K., Ehata, S., Nishimura, R., Mori, M., Onozaki, K. & other authors (2006). CCAAT/enhancer-binding protein homologous protein (CHOP) regulates osteoblast differentiation. Mol Cell Biol 26, 6105–6116.
Simone, C., Stiegler, P., Bagella, L., Pucci, B., Bellan, C., De Falco, G., De Luca, A., Guanti, G., Puri, P. L. & Giordano, A. (2002). Activation of MyoD-dependent transcription by cdk9/cyclin T2. Oncogene 21, 4137–4148.[CrossRef][Medline]
Spooner, C. J., Guo, X., Johnson, P. F. & Schwartz, R. C. (2007). Differential roles of C/EBP beta regulatory domains in specifying MCP-1 and IL-6 transcription. Mol Immunol 44, 1384–1392.[CrossRef][Medline]
Wei, P., Garber, M. E., Fang, S. M., Fischer, W. H. & Jones, K. A. (1998). A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell 92, 451–462.[CrossRef][Medline]
Westendorf, J. J., Yamamoto, C. M., Lenny, N., Downing, J. R., Selsted, M. E. & Hiebert, S. W. (1998). The t(8;21) fusion product, AML-1-ETO, associates with C/EBP-alpha, inhibits C/EBP-alpha-dependent transcription, and blocks granulocytic differentiation. Mol Cell Biol 18, 322–333.
Yik, J. H., Chen, R., Pezda, A. C., Samford, C. S. & Zhou, Q. (2004). A human immunodeficiency virus type 1 Tat-like arginine-rich RNA-binding domain is essential for HEXIM1 to inhibit RNA polymerase II transcription through 7SK snRNA-mediated inactivation of P-TEFb. Mol Cell Biol 24, 5094–5105.
Yin, M., Yang, S. Q., Lin, H. Z., Lane, M. D., Chatterjee, S. & Diehl, A. M. (1996). Tumor necrosis factor alpha promotes nuclear localization of cytokine-inducible CCAAT/enhancer binding protein isoforms in hepatocytes. J Biol Chem 271, 17974–17978.
Yu, W., Wang, Y., Shaw, C. A., Qin, X. F. & Rice, A. P. (2006). Induction of the HIV-1 Tat co-factor cyclin T1 during monocyte differentiation is required for the regulated expression of a large portion of cellular mRNAs. Retrovirology 3, 32.[CrossRef][Medline]
