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HTLV-1 HBZ cooperates with JunD to enhance transcription of the human telomerase reverse transcriptase gene (hTERT).

Kuhlmann AS, Villaudy J, Gazzolo L, Castellazzi M, Mesnard JM, Duc Dodon M - Retrovirology (2007)

Bottom Line: Interestingly, the -378/+1 proximal region, which does not contain any AP-1 site was found to be responsible for this activation.Finally, we provide evidence that HBZ/JunD heterodimers interact with Sp1 transcription factors and that activation of hTERT transcription by these heterodimers is mediated through GC-rich binding sites for Sp1 present in the proximal sequences of the hTERT promoter.These observations establish for the first time that HBZ by intervening in the re-activation of telomerase, may contribute to the development and maintenance of the leukemic process.

View Article: PubMed Central - HTML - PubMed

Affiliation: Virologie Humaine, INSERM-U758, 69364 Lyon Cedex 07, France. anne-sophie.kuhlmann@ens-lyon.fr

ABSTRACT

Background: Activation of telomerase is a critical and late event in tumor progression. Thus, in patients with adult-T cell leukaemia (ATL), an HTLV-1 (Human T cell Leukaemia virus type 1)-associated disease, leukemic cells display a high telomerase activity, mainly through transcriptional up-regulation of the human telomerase catalytic subunit (hTERT). The HBZ (HTLV-1 bZIP) protein coded by the minus strand of HTLV-1 genome and expressed in ATL cells has been shown to increase the transcriptional activity of JunD, an AP-1 protein. The presence of several AP-1 binding sites in the hTERT promoter led us to investigate whether HBZ regulates hTERT gene transcription.

Results: Here, we demonstrate using co-transfection assays that HBZ in association with JunD activates the hTERT promoter. Interestingly, the -378/+1 proximal region, which does not contain any AP-1 site was found to be responsible for this activation. Furthermore, an increase of hTERT transcripts was observed in cells co-expressing HBZ and JunD. Chromatin immunoprecipitation (ChIP) assays revealed that HBZ, and JunD coexist in the same DNA-protein complex at the proximal region of hTERT promoter. Finally, we provide evidence that HBZ/JunD heterodimers interact with Sp1 transcription factors and that activation of hTERT transcription by these heterodimers is mediated through GC-rich binding sites for Sp1 present in the proximal sequences of the hTERT promoter.

Conclusion: These observations establish for the first time that HBZ by intervening in the re-activation of telomerase, may contribute to the development and maintenance of the leukemic process.

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Functional interactions between HBZ, JunD and Sp1. (A) HeLa cells were cotransfected with the reporter construct 5XGal4-Luc (200 ng) consisting of five tandem Gal4-binding sites in the absence (-) or presence (100 ng) of expression vectors for Gal4-Sp1B (containing the B domain of Sp1), and/or HBZ and JunD. Luciferase activity was normalized to tk-luc activity. Results represent duplicate samples from three different experiments. (B) Western blot analysis of Sp1, HBZ and JunD protein levels in whole cell lysates of HeLa samples. The membrane was successively probed with rabbit polyclonal anti-Sp1, anti-HBZ sera, and mouse monoclonal anti-Flag and anti-actin antibodies.
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Figure 6: Functional interactions between HBZ, JunD and Sp1. (A) HeLa cells were cotransfected with the reporter construct 5XGal4-Luc (200 ng) consisting of five tandem Gal4-binding sites in the absence (-) or presence (100 ng) of expression vectors for Gal4-Sp1B (containing the B domain of Sp1), and/or HBZ and JunD. Luciferase activity was normalized to tk-luc activity. Results represent duplicate samples from three different experiments. (B) Western blot analysis of Sp1, HBZ and JunD protein levels in whole cell lysates of HeLa samples. The membrane was successively probed with rabbit polyclonal anti-Sp1, anti-HBZ sera, and mouse monoclonal anti-Flag and anti-actin antibodies.

Mentions: Additional experiments were next performed to establish functional interactions between JunD, HBZ and Sp1. Transient co-transfection experiments in HeLa cells showed that JunD transactivated a synthetic promoter consisting of five tandem high affinity binding sites for the yeast protein Gal4 upstream of a minimal TATA box (5XGal4-Luc) by 11-fold (Fig 6, compare lanes 3 and 2), when it was coexpressed with a Gal4-Sp1B chimeric protein consisting of the DNA binding domain of Gal4 fused to the B domain of Sp1, indicating that JunD and Sp1 are able to interact physically. Overexpression of HBZ in HeLa cells enhanced the JunD-mediated transactivation potential by 2.7-fold (compare lanes 3 and 4). Control experiments showed that the HBZ protein alone in the presence of Gal4-Sp1B had no significant effect on the activity of the 5XGal4 promoter (lane 2). These observations show that cooperative interactions between HBZ, JunD and Sp1 can transactivate promoters containing multiple Sp1-binding sites. Collectively, these results clearly underline that the synergistic transactivation of hTERT promoter by HBZ and JunD is Sp1-dependent.


HTLV-1 HBZ cooperates with JunD to enhance transcription of the human telomerase reverse transcriptase gene (hTERT).

Kuhlmann AS, Villaudy J, Gazzolo L, Castellazzi M, Mesnard JM, Duc Dodon M - Retrovirology (2007)

Functional interactions between HBZ, JunD and Sp1. (A) HeLa cells were cotransfected with the reporter construct 5XGal4-Luc (200 ng) consisting of five tandem Gal4-binding sites in the absence (-) or presence (100 ng) of expression vectors for Gal4-Sp1B (containing the B domain of Sp1), and/or HBZ and JunD. Luciferase activity was normalized to tk-luc activity. Results represent duplicate samples from three different experiments. (B) Western blot analysis of Sp1, HBZ and JunD protein levels in whole cell lysates of HeLa samples. The membrane was successively probed with rabbit polyclonal anti-Sp1, anti-HBZ sera, and mouse monoclonal anti-Flag and anti-actin antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2235888&req=5

Figure 6: Functional interactions between HBZ, JunD and Sp1. (A) HeLa cells were cotransfected with the reporter construct 5XGal4-Luc (200 ng) consisting of five tandem Gal4-binding sites in the absence (-) or presence (100 ng) of expression vectors for Gal4-Sp1B (containing the B domain of Sp1), and/or HBZ and JunD. Luciferase activity was normalized to tk-luc activity. Results represent duplicate samples from three different experiments. (B) Western blot analysis of Sp1, HBZ and JunD protein levels in whole cell lysates of HeLa samples. The membrane was successively probed with rabbit polyclonal anti-Sp1, anti-HBZ sera, and mouse monoclonal anti-Flag and anti-actin antibodies.
Mentions: Additional experiments were next performed to establish functional interactions between JunD, HBZ and Sp1. Transient co-transfection experiments in HeLa cells showed that JunD transactivated a synthetic promoter consisting of five tandem high affinity binding sites for the yeast protein Gal4 upstream of a minimal TATA box (5XGal4-Luc) by 11-fold (Fig 6, compare lanes 3 and 2), when it was coexpressed with a Gal4-Sp1B chimeric protein consisting of the DNA binding domain of Gal4 fused to the B domain of Sp1, indicating that JunD and Sp1 are able to interact physically. Overexpression of HBZ in HeLa cells enhanced the JunD-mediated transactivation potential by 2.7-fold (compare lanes 3 and 4). Control experiments showed that the HBZ protein alone in the presence of Gal4-Sp1B had no significant effect on the activity of the 5XGal4 promoter (lane 2). These observations show that cooperative interactions between HBZ, JunD and Sp1 can transactivate promoters containing multiple Sp1-binding sites. Collectively, these results clearly underline that the synergistic transactivation of hTERT promoter by HBZ and JunD is Sp1-dependent.

Bottom Line: Interestingly, the -378/+1 proximal region, which does not contain any AP-1 site was found to be responsible for this activation.Finally, we provide evidence that HBZ/JunD heterodimers interact with Sp1 transcription factors and that activation of hTERT transcription by these heterodimers is mediated through GC-rich binding sites for Sp1 present in the proximal sequences of the hTERT promoter.These observations establish for the first time that HBZ by intervening in the re-activation of telomerase, may contribute to the development and maintenance of the leukemic process.

View Article: PubMed Central - HTML - PubMed

Affiliation: Virologie Humaine, INSERM-U758, 69364 Lyon Cedex 07, France. anne-sophie.kuhlmann@ens-lyon.fr

ABSTRACT

Background: Activation of telomerase is a critical and late event in tumor progression. Thus, in patients with adult-T cell leukaemia (ATL), an HTLV-1 (Human T cell Leukaemia virus type 1)-associated disease, leukemic cells display a high telomerase activity, mainly through transcriptional up-regulation of the human telomerase catalytic subunit (hTERT). The HBZ (HTLV-1 bZIP) protein coded by the minus strand of HTLV-1 genome and expressed in ATL cells has been shown to increase the transcriptional activity of JunD, an AP-1 protein. The presence of several AP-1 binding sites in the hTERT promoter led us to investigate whether HBZ regulates hTERT gene transcription.

Results: Here, we demonstrate using co-transfection assays that HBZ in association with JunD activates the hTERT promoter. Interestingly, the -378/+1 proximal region, which does not contain any AP-1 site was found to be responsible for this activation. Furthermore, an increase of hTERT transcripts was observed in cells co-expressing HBZ and JunD. Chromatin immunoprecipitation (ChIP) assays revealed that HBZ, and JunD coexist in the same DNA-protein complex at the proximal region of hTERT promoter. Finally, we provide evidence that HBZ/JunD heterodimers interact with Sp1 transcription factors and that activation of hTERT transcription by these heterodimers is mediated through GC-rich binding sites for Sp1 present in the proximal sequences of the hTERT promoter.

Conclusion: These observations establish for the first time that HBZ by intervening in the re-activation of telomerase, may contribute to the development and maintenance of the leukemic process.

Show MeSH
Related in: MedlinePlus