- Open Access
Inhibition of Tat activity by the HEXIM1 protein
© Fraldi et al; licensee BioMed Central Ltd. 2005
Received: 29 June 2005
Accepted: 02 July 2005
Published: 02 July 2005
The positive transcription elongation factor b (P-TEFb) composed by CDK9/CyclinT1 subunits is a dedicated co-factor of HIV transcriptional transactivator Tat protein. Transcription driven by the long terminal repeat (LTR) of HIV involves formation of a quaternary complex between P-TEFb, Tat and the TAR element. This recruitment is necessary to enhance the processivity of RNA Pol II from the HIV-1 5' LTR promoter. The activity of P-TEFb is regulated in vivo and in vitro by the HEXIM1/7SK snRNA ribonucleic-protein complex.
Here we report that Tat transactivation is effectively inhibited by co-expression of HEXIM1 or its paralog HEXIM2. HEXIM1 expression specifically represses transcription mediated by the direct activation of P-TEFb through artificial recruitment of GAL4-CycT1. Using appropriate HEXIM1 mutants we determined that effective Tat-inhibition entails the 7SK snRNA basic recognition motif as well as the C-terminus region required for interaction with cyclin T1. Enhanced expression of HEXIM1 protein modestly affects P-TEFb activity, suggesting that HEXIM1-mediated repression of Tat activity is not due to a global inhibition of cellular transcription.
These results point to a pivotal role of P-TEFb for Tat's optimal transcription activity and suggest that cellular proteins that regulate P-TEFb activity might exert profound effects on Tat function in vivo.
The positive transcription elongation factor b (P-TEFb) composed by CDK9/CyclinT1, has emerged as a significant co-factor of the HIV Tat protein. P-TEFb complex has been shown to associate with and phosphorylate the carboxyl-terminal domain (CTD) of RNA pol II, thereby enhancing elongation of transcription [1–3]. Tat protein binds an uracil containing bulge within the stem-loop secondary structure of the Tat-activated region (TAR-RNA) in HIV-1 transcripts [4–6]. Tat functions as an elongation factor and stabilizes the synthesis of full-length viral mRNAs by preventing premature termination by the TAR-RNA stem-loop. Physical and functional interactions between Tat and P-TEFb have been well documented [7, 8]. Tat binds to P-TEFb by direct interaction with the human cyclinT1, and the critical residues required for interaction have been delineated [9, 10]. The current model for recruitment of P-TEFb to the LTR, predicts the formation of the Tat-P-TEFb complex, which efficiently binds TAR, allowing CDK9 to phosphorylate the CTD of RNAPII, thereby, enhances processivity of the polymerase to produce full-length mRNAs [3, 7–10].
Like other CDKs, the P-TEFb activity is regulated by a dedicated inhibitor. Two different P-TEFb complexes exist in vivo [11, 12]. The active complex is composed of two subunits, the CDK9 and its regulatory partners cyclinT1 or T2. In addition, a larger inactive complex has been identified, which comprises of four subunits, CDK9, cyclinT1 or T2, the abundant small nuclear RNA 7SK and the HEXIM1 protein [13–17]. It has been recently shown that HEXIM1 has the inherent ability to associate with cyclin T1 and binding of 7SK snRNA turns the HEXIM1 into a P-TEFb inhibitor [15–17]. The relative presence of core and inactive P-TEFb complexes changes rapidly in vivo [11, 12]. Several stress-inducing agents trigger dissociation of the inactive P-TEFb complex and subsequent accumulation of kinase active P-TEFb . Thus, the 7SK-HEXIM1 ribonucleic complex represents a new type of CDK inhibitor that contributes to regulation of gene transcription. A further level of complexity of this system comes from the recent identification of HEXIM2, a HEXIM1 paralog, which regulates P-TEFb similarly as HEXIM1 through association with 7SK RNA [18, 19].
It has been showed that Tat binds exclusively to the active P-TEFb complex . Thus the presence of HEXIM1/7SK snRNA in P-TEFb complexes prevents Tat binding. Since the association between 7SK RNA/HEXIM1 and P-TEFb appears to compete with binding of Tat to cyclinT1, we have speculated that the TAR RNA/Tat system may compete with the cellular 7SK snRNA/HEXIM1 system in the recruitment of the active P-TEFb complex . Accordingly, it has been shown that over-expression of HEXIM1 represses Tat function [14, 17].
We show here that HEXIM1, or its paralog HEXIM2, inhibits Tat trans-activation of HIV-LTR driven gene expression, and more importantly, we demonstrated the role of the 7SK snRNA recognition motif as well as the binding to cyclin T1 as crucial elements for efficient Tat inhibition.
Tat activity is inhibited by HEXIM1
Definition of the HEXIM1 regulatory domains involved in repression
P-TEFb activity in the presence of enhanced expression of HEXIM1
Several lines of evidence have suggested that Tat function is largely dependent upon the physical and functional interaction with the cellular transcription factor P-TEFb. The recruitment of P-TEFb to the LTR, involves the formation of the Tat-P-TEFb complex which efficiently binds TAR, allowing CDK9 to phosphorylate the CTD of RNAPII, thereby, enhances processivity of the polymerase to produce full-length mRNAs [6–10]. Two different P-TEFb complexes exist in vivo. The core active P-TEFb comprises two subunits, the catalytic CDK9 and a regulatory partner cyclin T, and a larger inactive P-TEFb complex comprised by CDK9, cyclin T, HEXIM1 protein and the 7SK snRNA [11–17]. The relative presence of core and inactive P-TEFb complexes changes rapidly in vivo . We have previously shown that the presence of HEXIM1/7SK snRNA in P-TEFb complexes prevents Tat binding to P-TEFb . Since the association between 7SK RNA/HEXIM1 and P-TEFb competes with binding of Tat to cyclinT1, we have speculated that the TAR RNA/Tat system may compete with the cellular 7SK snRNA/HEXIM1 system . Accordingly, it has been shown that over-expression of HEXIM1 represses Tat function [14, 19] We show here that HEXIM1 inhibits Tat function, while expression of 7SK snRNA does not influence Tat activity. It is pertinent to note that 7SK RNA is an abundant snRNA , and it is unlikely that 7SK might be rate-limiting for the assembly of the inactive P-TEFb complex.
We have delineated important structural domains of HEXIM1 required for repression of Tat. First, we found that the C-terminal region is required for inhibition. Previous findings indicated that the C-terminal region of HEXIM1 is involved in binding with cyclinT1 as well as for homo and hetero-dimerization with HEXIM2 [15, 18, 19]. Second, point mutations in the evolutionarily conserved motif PYNT (aa 202–205) abolished inhibition. It has recently shown a critical role of threonine 205 in P-TEFb binding . Moreover, deletion mutants unable to bind P-TEFb failed to repress Tat (Figure 3). Therefore, it appears that HEXIM1 inhibition is strictly dependent upon the integrity of the protein to interact with P-TEFb. Third, a point mutant in the central part of HEXIM1 (KHRR motif aa 152–155) strongly affects Tat repression. Since this basic motif has been previously shown as the 7SK snRNA recognition motif , we conclude that interaction between HEXIM1 and 7SK snRNA is required for Tat repression. Collectively, these findings strongly suggested that HEXIM1-mediated inhibition of Tat required the formation of the P-TEFb/HEXIM1/7SK complex.
We determined that enhanced expression of HEXIM1 resulted in a modest inhibition (2-fold) of P-TEFb activity in vivo. Thus, HEXIM1-mediated inhibition of Tat activity is unlikely due to a global inhibition of P-TEFb activity. Moreover, we found that basal transcription from the LTR sequences was largely unaffected by over-expression of HEXIM1. Finally, ectopic expression of this protein does not have significant effects on TBP-mediated basal transcription. Thus, it appears that P-TEFb is specifically required for Tat-dependent HIV LTR transcription. Our results differ somewhat from those obtained in the Zhou lab who found that exogenous expression of HEXIM1 affects both basal as well as Tat-induced transcription . These apparent discrepancies are possible due to different transfection conditions in which the relative amounts of the over-expressed exogenous proteins are likely different. We found that Tat expression which is under the control of SV40 promoter remains largely unaffected by co-expression of HEXIM. Our findings suggest a dedicated role of P-TEFb in Tat activity. Recent studies point to a specific role of P-TEFb for certain promoters. It has recently found that P-TEFb is recruited to the IL-8 but not to the IkBα promoter , and it also represses transcription of regulators such as the nuclear receptor coactivator, PGC-1, in cardiac myocytes . The specific HEXIM-mediated inhibition of Tat activity underlines the pivotal role of P-TEFb in the HIV LTR transcription.
The repression exerted by the HEXIM1 protein is likely the results of a competition between Tat and HEXIM1 in binding the P-TEFb. Since Tat binds only to the active P-TEFb complex, it has been suggested that Tat might trap the active form of P-TEFb as the PTEFb/7SK RNA/HEXIM1 complex appears to undergo continuous formation and disruption in vivo. In this scenario over expression of HEXIM1 may counteract the binding of Tat to P-TEFb, through a competitive association between the ectopic expressed HEXIM1 and P-TEFb. Accordingly, we found that exogenous expression of HEXIM1 results in a small but detectable reduction in Tat-bound- P-TEFb. Our co-immunoprecipitation results are consistent with recent findings showing a mutually exclusive interaction of HEXIM1 and Tat with cyclinT1 using recombinant purified proteins . Because Tat and HEXIM1 interact with the cyclin-box region of cyclinT1, it is plausible if not likely, that the mutually exclusive interaction of these two molecules with cyclinT1 is due to binding to the same domain or to a sterical hindrance. However, these studies have been performed in vitro in the absence of 7SK snRNA.
The results reported here along with previous findings strongly suggest the crucial role of 7SK in the interaction between HEXIM1 and cyclinT1. In fact, HEXIM1 ILAA mutant does not associate with 7SK in vivo and in vitro, and co-immuprecipitation of cyclinT1 and 7SK RNA was markedly reduced with ILAA mutant compared to wild type . Finally, as shown here ILAA mutant failed to repress Tat activity, suggesting an important role of HEXIM1/7SK interaction in Tat inhibition. Thus, association between HEXIM1 and 7SK snRNA appears an important determinant for Tat inhibition. Future in vitro and in vivo interaction studies, in the presence of 7SK snRNA may be instrumental to elucidate the role of 7SK/HEXIM1 complex in Tat activity.
The studies described in this provides further support to the pivotal role of P-TEFb for the optimal transcription Tat activity and highlight the importance of the P-TEFb cellular co-factors HEXIM1/7SK snRNA complex in Tat activity.
Tissue culture and transfections
Human 293 and rodent CHO cells were grown at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (Gibco, Life Technologies). Subconfluent cell cultures were transfected cell cultures were transfected by a liposome method (LipofectAMINE reagent; Life Technologies, Inc.) in 2 cm/dish in multiwells, using 100 ng of reporter DNA and different amounts of activator plasmid DNA as indicated in the text and 20 ng of Renilla luciferase expression plasmid (pRL-CMV, Promega) for normalization of transfections efficiencies. Cells were harvested 48 h after DNA transfections, and cellular extracts were assayed for luciferase activity using Dual-Luciferase Reporter assay (Promega) according to the manufacturer's instructions. The experimental reporter luciferase activity was normalized to transfection efficiency as measured by the activity deriving from pRL-CMV.
The G5HIV-Luc contained the HIV-1 LTR sequences from -83 to +82 of LTR driven the Luc gene with 5 GAL4 DNA-binding sites inserted at -83. The pSV-Tat, GAL4-TBP, GAL4-CycT1, have been described . 7SK snRNA plasmid was kindly provided by S. Murphy . All Flag-taggeted HEXIM1 and HEXIM2 expression vectors were constructed by insertion of the corresponding cDNA regions into the EcoRV site of p3xFlag-CMV10 vector (Clontech). Description of the deletion and point HEXIM1 mutants have been described previously . Full description of the expression vectors used in this work is available upon request.
Western blotting and antibodies
Cells were lysed in ice-chilled buffer A (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 200 mM NaCl, 0.2 mM EDTA), supplemented with 1 mM dithiothreitol, 40 U/ml of RNasin (Promega), protease inhibitor cocktail (P-8340; Sigma), and 0.5 % Nonidet P-40. Lysates were vortexed and incubated for 20 min on ice and clarified by centrifugations. Western blottings were performed using the following antibodies: the rabbit polyclonal anti-HEXIM1 (C4) has been previously described (6); anti-FLAG M2 Monoclonal Antibody (Sigma), goat polyclonal anti-CycT1 (T-18), rabbit polyclonal anti-CDK9 (H-169) from Santa Cruz, anti-Tat (NIH AIDS Research Reagent Program). Binding was visualized by enhanced chemiluminescence (ECL-plus Kit, Amersham Biosciences).
Co-immunoprecipitation and kinase assay
293 cells were transfected with pSV-Tat in the presence or absence of F:HEXIM1 and cell extracts were prepared at 48 hrs after transfection. CycT1 was immunopurified from cell extracts (1 mg) using anti-CycT1 (H-245) (sc-10750, Santa Cruz). Input, immunoprecipited and flow through materials were used in western blottings using anti-cycT1, anti-HEXIM1 and anti-Tat, respectively. For kinase assays 293 cells were transfected with F:HEXIM1 and after 48 hr P-TEFb complex was immunopurified from cell extracts (1 mg) using anti-CycT1 (H-245) (sc-10750, Santa Cruz) as previously described [13, 15]. Briefly, whole cell extracts from mock and F:HEXIM1 transfected 293 cells were used in immunoprecipitations together with 40μl of slurry beads (protein G-Sepharose 4 Fast Flow, Amersham Biosciences) pre-adsorbed with anti-CycT1 and the interactions were carried out in buffer A for one hour at 4°C on a wheel. After extensive washes one half of the immunopurified materials was used in western blotting to ensure the presence of equal amounts of CDK9, HEXIM1 and CycT1, respectively. The remaining material was suspended and stirred at room temperature and split in two equal aliquots. One of the aliquot was treated with 10U of RNase A for 15 min at 30°C. Samples treated or not with RNase were stirred at room temperature for three minutes in 65 μl of buffer A containing [γ-32P]ATP (0,1 μCi/μl), 40 mM ATP, 0,1 μg/ml (YSPTSPS)4 peptide CTD4 (6, 8) and RNasin (40 U/ml). Aliquots (20 μl) of the suspension were mixed with SDS-PAGE loading buffer at intervals of three minutes to stop the reaction. The phosphorylated CTD4 substrate was separated on a 15% SDS-PAGE and visualized by radiography. Incorporation of [32P] into CTD peptide was quantified on a Bio-Rad phosphoimager.
We thank S. Murphy for 7SK snRNA plasmid. This work was supported by grants from Istituto Superiore di Sanità Programma Nazionale di Ricerca AIDS and from Italian Association for Cancer Research (AIRC) (L.L.), from Association pour la Recherche sur le Cancer, Agence Nationale de Recherche sur le SIDA (O.B.), and from the Galileo Italy-France exchange program (G.N.).
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