A real-time view of the TAR:Tat:P-TEFb complex at HIV-1 transcription sites
- Dorothée Molle1,
- Paolo Maiuri2,
- Stéphanie Boireau1,
- Edouard Bertrand1,
- Anna Knezevich2,
- Alessandro Marcello†2 and
- Eugenia Basyuk†1Email author
© Molle et al; licensee BioMed Central Ltd. 2007
Received: 04 May 2007
Accepted: 30 May 2007
Published: 30 May 2007
HIV-1 transcription is tightly regulated: silent in long-term latency and highly active in acutely-infected cells. Transcription is activated by the viral protein Tat, which recruits the elongation factor P-TEFb by binding the TAR sequence present in nascent HIV-1 RNAs. In this study, we analyzed the dynamic of the TAR:Tat:P-TEFb complex in living cells, by performing FRAP experiments at HIV-1 transcription sites. Our results indicate that a large fraction of Tat present at these sites is recruited by Cyclin T1. We found that in the presence of Tat, Cdk9 remained bound to nascent HIV-1 RNAs for 71s. In contrast, when transcription was activated by PMA/ionomycin, in the absence of Tat, Cdk9 turned-over rapidly and resided on the HIV-1 promoter for only 11s. Thus, the mechanism of trans-activation determines the residency time of P-TEFb at the HIV-1 gene, possibly explaining why Tat is such a potent transcriptional activator. In addition, we observed that Tat occupied HIV-1 transcription sites for 55s, suggesting that the TAR:Tat:P-TEFb complex dissociates from the polymerase following transcription initiation, and undergoes subsequent cycles of association/dissociation.
The human immunodeficiency virus type 1 (HIV-1) virus can have latent and acute phases. Latent viruses remain in infected organisms for a long time, and this prevents viral clearance by anti-retroviral agents. The control of HIV-1 latency is highly dependent upon transcriptional regulation: acutely-infected cells synthesize high levels of virus, while latently-infected cells transcribe little or no viral RNAs. HIV-1 transcription requires cellular co-factors, but it is highly activated by the viral protein Tat (for review, see [1, 2]). In latent cells that do not express Tat, polymerases initiating at the HIV-1 promoter are poorly processive and do not transcribe the entire viral genome. However, extra-cellular signals can drive latent cells into acute phase by stimulating the HIV-1 promoter. Indeed, this induces the production of small amounts of Tat, which then initiates a positive feedback loop leading to full transcriptional activation .
Tat activates transcription by recruiting the active form of the positive transcription elongation factor P-TEFb to the HIV-1 promoter . P-TEFb is composed of a complex between Cyclin T1 (CycT1) and the kinase Cdk9, and Tat directly binds CycT1 (see  for a review). Tat also binds TAR (trans-activation-responsive region), an RNA element present at the 5' end of all HIV-1 transcripts, and this induces the formation of a ternary complex on nascent RNAs, consisting of TAR, Tat, and P-TEFb. When incorporated in this complex, Cdk9 phosphorylates several components of the transcription machinery, including the C-terminal domain (CTD) of the large sub-unit of RNA polymerase II (RNAPII), and elongation factors DSIF and NELF . This transforms RNAPII into a highly processive enzyme, which can transcribe the entire viral genome.
P-TEFb is not the only partner of Tat. In particular, Tat has also been shown to interact and recruit the histone acetyl-transferases p300 and PCAF, which can modify chromatin at the provirus integration site [1, 2]. Moreover, Tat itself can be acetylated at Lysines 28 and 50, and these modifications have been shown to regulate its interactions with P-TEFb/TAR and PCAF [6–9].
While Tat and its various partners have been the subject of many studies, how these complexes behave in vivo is still a matter of debate. Indeed, several models currently exist. It has been proposed that P-TEFb dissociates from the HIV-1 gene following transcription initiation, while Tat and PCAF become transferred to the elongating polymerase . In contrast, other models suggest that P-TEFb remains associated with Tat in the elongating complex [9, 10]. To discriminate between these possibilities, we developed an assay to analyze the dynamic of the Tat:P-TEFb complex, directly in living cells and at HIV-1 transcription sites.
To evaluate the dynamic properties of the TAR:Tat:P-TEFb complex, we performed experiments in live cells. We identified HIV-1 transcription sites with a yellow or red fluorescent variant of MS2, and performed photobleaching experiments (FRAP) on CFP and GFP-tagged versions of Tat and Cdk9 (see Additional file 1). When Tat or Cdk9 were bleached in the nucleoplasm of U2OS cells, the fluorescence recovered quickly, indicating that these proteins diffused rapidly through the nucleoplasm (Figure 1B and 1C). We then bleached Tat and Cdk9 at HIV-1 transcription sites, to analyze the dynamic properties of the complexes formed on nascent RNAs. The turn-over of Tat was slow, as complete fluorescence recovery took nearly three minutes (Figure 1B and Additional file 2). In the case of Cdk9, we observed two contrasting situations depending on the mode of activation of the HIV-1 promoter (Figure 1C). In the presence of Tat, Cdk9 recovery was also slow and took several minutes to go to completion (see Additional file 3). In contrast, when HIV-1 transcription was activated by PMA/ionomycin, Cdk9 was highly dynamic, and recovery was complete within seconds.
Kinetic parameters of the fitted FRAP curves.
Cdk9 (with Tat)
Cdk9 (with PMA/ionomycin)
Our data show that Tat and P-TEFb remained bound to nascent RNAs for about a minute. If HIV-1 transcription proceeds with the previously described rate of 2 Kb/min, then elongation through our reporter RNA would last more than 2 minutes . This raises the possibility that the TAR:Tat:P-TEFb complex could be dissociated from the polymerase before the gene is completely transcribed. Following dissociation, the fate of Tat and Cdk9 is likely to differ, as shown by their significant difference in τd (Table 1). It is remarkable that Tat and Cdk9 have very similar dynamics. This supports the idea that they remain together in the elongating complex, rather then Tat being transferred to the polymerase while P-TEFb dissociating from it. Since chromatin immunoprecipitation data have shown that Tat and P-TEFb are present with elongating polymerases all along the gene , we suggest that Tat and P-TEFb could undergo constant association and dissociation cycles with TAR and the elongating polymerase.
Altogether, our data show that the TAR:Tat:P-TEFb complex is remodeled during HIV-1 transcription. This work opens an opportunity to study the kinetic properties of factors involved in HIV-1 transcription, and could also be extended to the analysis of the contribution of post-translational modifications to the dynamics of the Tat:P-TEFb complex.
human immunodeficiency virus
RNA polymerase II
C-terminal domain of RNAPII
fluorescence recovery after photobleaching
We thank O. Bensaude for the gift of anti-Hexim antibodies. This work was supported by NOE EURASNET. Support to A.M. was from EC STREP consortium 012182, from a HFSP Young Investigators Grant, and from the AIDS project of the ISS of Italy. E.B. was supported by a fellowship from l'ANRS.
- Marcello A, Zoppe M, Giacca M: Multiple modes of transcriptional regulation by the HIV-1 Tat transactivator. IUBMB Life. 2001, 51: 175-181.View ArticlePubMedGoogle Scholar
- Jeang KT, Xiao H, Rich HA: Multifaceted activities of the HIV-1 transactivator of transcription, Tat. J Biol Chem. 1999, 274: 28837-28840. 10.1074/jbc.274.41.28837.View ArticlePubMedGoogle Scholar
- Weinberger LS, Burnett JC, Toettcher JE, Arkin AP, Schaffer DV: Stochastic gene expression in a lentiviral positive-feedback loop: HIV-1 Tat fluctuations drive phenotypic diversity. Cell. 2005, 122: 169-182. 10.1016/j.cell.2005.06.006.View ArticlePubMedGoogle Scholar
- Wei P, Garber ME, Fang SM, Fischer WH, Jones KA: 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. 1998, 92: 451-462. 10.1016/S0092-8674(00)80939-3.View ArticlePubMedGoogle Scholar
- Peterlin PM, Price DH: Controlling the elongation phase of transcription with P-TEFb. Mol Cell. 2006, 23: 297-305. 10.1016/j.molcel.2006.06.014.View ArticlePubMedGoogle Scholar
- Ott M, Schnolzer M, Garnica J, Fischle W, Emiliani S, Rackwitz HR, Verdin E: Acetylation of the HIV-1 Tat protein by p300 is important for its transcriptional activity. Curr Biol. 1999, 9: 1489-1492. 10.1016/S0960-9822(00)80120-7.View ArticlePubMedGoogle Scholar
- Kiernan ER, Vanhulle C, Schiltz L, Adam L, Xiao H, Maudoux F, Calomme C, Burny A, Nakatani Y, Jeang KT, et al: HIV-1 tat transcriptional activity is regulated by acetylation. Embo J. 1999, 18: 6106-6118. 10.1093/emboj/18.21.6106.PubMed CentralView ArticlePubMedGoogle Scholar
- Kaehlcke K, Dorr A, Hetzer-Egger C, Kiermer V, Henklein P, Schnoelzer M, Loret E, Cole PA, Verdin E, Ott M: Acetylation of Tat defines a cyclinT1-independent step in HIV transactivation. Mol Cell. 2003, 12: 167-176. 10.1016/S1097-2765(03)00245-4.View ArticlePubMedGoogle Scholar
- Bres V, Tagami H, Peloponese JM, Loret E, Jeang KT, Nakatani Y, Emiliani S, Benkirane S, Kiernan RE: Differential acetylation of Tat coordinates its interaction with the co-activators cyclin T1 and PCAF. Embo J. 2002, 21: 6811-6819. 10.1093/emboj/cdf669.PubMed CentralView ArticlePubMedGoogle Scholar
- Bres V, Gomes N, Pickle L, Jones K: A human splicing factor, SKIP, associates with P-TEFb and enhances transcription elongation by HIV-1 Tat. Genes Dev. 2005, 19: 1211-26. 10.1101/gad.1291705.PubMed CentralView ArticlePubMedGoogle Scholar
- Fusco D, Accornero N, Lavoie B, Shenoy S, Blanchard J, Singer R, Bertrand E: Single mRNA molecules demonstrate probabilistic movement in living mammalian cells. Curr Biol. 2003, 13: 161-7. 10.1016/S0960-9822(02)01436-7.View ArticlePubMedGoogle Scholar
- Chene I du, Basyuk E, Lin Y, Triboulet R, Knezevich R, Chable-Bessia C, Mettling C, Baillat V, Reynes J, Corbeau P, et al: Suv39H1 and HP1gamma are responsible for chromatin-mediated HIV-1 transcriptional silencing and post-integration latency. EMBO J. 2007, 26: 424-35. 10.1038/sj.emboj.7601517.PubMed CentralView ArticlePubMedGoogle Scholar
- Michels AA, Nguyen VT, Fraldi A, Labas V, Edwards M, Bonnet F, Lania L, Bensaude O: MAQ1 and 7SK RNA interact with CDK9/cyclin T complexes in a transcription-dependent manner. Mol Cell Biol. 2003, 23:Google Scholar
- Sprague BL, Pego RL, Stavreva DA, McNally JG: Analysis of binding reactions by fluorescence recovery after photobleaching. Biophys J. 2004, 86: 3473-3495. 10.1529/biophysj.103.026765.PubMed CentralView ArticlePubMedGoogle Scholar
- Yao J, Munson K, Webb W, Lis J: Dynamics of heat shock factor association with native gene loci in living cells. Nature. 2006, 442: 1050-3. 10.1038/nature05025.View ArticlePubMedGoogle Scholar
- Tennyson CN, Klamut HJ, Worton RG: The human dystrophin gene requires 16 hours to be transcribed and is cotranscriptionally spliced. Nat Genet. 1995, 9: 184-190. 10.1038/ng0295-184.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.