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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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Related in: MedlinePlus

Transient-expression assays to examine the role of HBZ and of AP-1 in the hTERT promoter. (A) Schematic diagram of the luciferase reporter plasmids containing various lengths of the hTERT promoter. The black squares indicate AP-1 responsive sites. The sequence of the proximal core promoter is located between -181 to +80. (B) Effect of HBZ and AP-1 on luciferase reporter constructs. HeLa cells were cotransfected with various lengths of the hTERT promoter plasmids (0.1 μg) and with HBZ- (0.2 to 0.8 μg), and/or c-Jun- (0.2 μg), and/or JunD (0.2 μg)-expression vectors. Luciferase activity was normalized to tk-luc activity and presented relative to cells transfected with the reporter plasmid alone. The values are those obtained in triplicate, from three different experiments. Error bars indicate standard deviations. Shown in the lower panel, a western blot analysis of HBZ and Jun protein levels in whole cell lysates of HeLa samples transfected with pGL3-378. The membrane was probed successively with a polyclonal anti-HBZ antibody, and a mouse anti-flag antibody. Actin was used as a loading control. (C) Transactivation of the hTERT promoter by HBZ and JunD in Jurkat cells. Cells were cotransfected with pGL3-378 reporter plasmid (4 μg), in combination with the indicated HBZ (2 μg) and/or JunD (2 μg)-expression vectors. Luciferase activity was normalized and presented as indicated in B. The values are those obtained in triplicate, from one representative experiment.
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Figure 1: Transient-expression assays to examine the role of HBZ and of AP-1 in the hTERT promoter. (A) Schematic diagram of the luciferase reporter plasmids containing various lengths of the hTERT promoter. The black squares indicate AP-1 responsive sites. The sequence of the proximal core promoter is located between -181 to +80. (B) Effect of HBZ and AP-1 on luciferase reporter constructs. HeLa cells were cotransfected with various lengths of the hTERT promoter plasmids (0.1 μg) and with HBZ- (0.2 to 0.8 μg), and/or c-Jun- (0.2 μg), and/or JunD (0.2 μg)-expression vectors. Luciferase activity was normalized to tk-luc activity and presented relative to cells transfected with the reporter plasmid alone. The values are those obtained in triplicate, from three different experiments. Error bars indicate standard deviations. Shown in the lower panel, a western blot analysis of HBZ and Jun protein levels in whole cell lysates of HeLa samples transfected with pGL3-378. The membrane was probed successively with a polyclonal anti-HBZ antibody, and a mouse anti-flag antibody. Actin was used as a loading control. (C) Transactivation of the hTERT promoter by HBZ and JunD in Jurkat cells. Cells were cotransfected with pGL3-378 reporter plasmid (4 μg), in combination with the indicated HBZ (2 μg) and/or JunD (2 μg)-expression vectors. Luciferase activity was normalized and presented as indicated in B. The values are those obtained in triplicate, from one representative experiment.

Mentions: To examine the role of HBZ in regulating the activity of the hTERT promoter, luciferase assays were performed with reporter plasmids containing various lengths of the 5' flanking sequence of the hTERT gene fused to the luciferase reporter gene (Fig 1A). The longest reporter pGL3-3300 contains 5 AP-1 binding sites; pGL3-2000 includes two of these sites, whereas the shortest construct pGL3-378 encompassing the proximal region is devoid of any AP-1 binding sequence. Each of these reporter plasmids was co-transfected in HeLa cells along with increasing amounts of an HBZ vector either alone or together with c-Jun or JunD expression plasmids. The expression levels of HBZ and Jun proteins were confirmed by Western blot analysis. Overexpression of HBZ with each of the three constructs did not exert any effect on the hTERT promoter activity (Fig 1B, lanes 2, 3, 4). Overexpression of c-Jun or JunD led to a small, but significant increase of this promoter activity. In presence of c-Jun, a 2-fold increase was observed with pGL3-3300, and a 3-fold increase with pGL3-2000 and pGL3-378. In presence of JunD, a 2-fold-increase was observed only with pGL3-378. Overexpression of HBZ with c-Jun resulted in a reduction of the hTERT promoter activity with the three reporter constructs (compare lanes 6, 7, 8 to lane 5). To note that the increased amounts of HBZ correlated with a decrease of c-Jun detected in cell lysates, as previously shown [12]. Intriguingly, overexpression of HBZ in the presence of JunD led to an increase of the hTERT promoter activity, which also correlated with the transfected amount of HBZ (compare lanes 10,11,12 to lane 9). Taken together, these observations show that HBZ expressed either with c-Jun or JunD is able to repress or enhance the hTERT promoter activity, respectively.


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)

Transient-expression assays to examine the role of HBZ and of AP-1 in the hTERT promoter. (A) Schematic diagram of the luciferase reporter plasmids containing various lengths of the hTERT promoter. The black squares indicate AP-1 responsive sites. The sequence of the proximal core promoter is located between -181 to +80. (B) Effect of HBZ and AP-1 on luciferase reporter constructs. HeLa cells were cotransfected with various lengths of the hTERT promoter plasmids (0.1 μg) and with HBZ- (0.2 to 0.8 μg), and/or c-Jun- (0.2 μg), and/or JunD (0.2 μg)-expression vectors. Luciferase activity was normalized to tk-luc activity and presented relative to cells transfected with the reporter plasmid alone. The values are those obtained in triplicate, from three different experiments. Error bars indicate standard deviations. Shown in the lower panel, a western blot analysis of HBZ and Jun protein levels in whole cell lysates of HeLa samples transfected with pGL3-378. The membrane was probed successively with a polyclonal anti-HBZ antibody, and a mouse anti-flag antibody. Actin was used as a loading control. (C) Transactivation of the hTERT promoter by HBZ and JunD in Jurkat cells. Cells were cotransfected with pGL3-378 reporter plasmid (4 μg), in combination with the indicated HBZ (2 μg) and/or JunD (2 μg)-expression vectors. Luciferase activity was normalized and presented as indicated in B. The values are those obtained in triplicate, from one representative experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 1: Transient-expression assays to examine the role of HBZ and of AP-1 in the hTERT promoter. (A) Schematic diagram of the luciferase reporter plasmids containing various lengths of the hTERT promoter. The black squares indicate AP-1 responsive sites. The sequence of the proximal core promoter is located between -181 to +80. (B) Effect of HBZ and AP-1 on luciferase reporter constructs. HeLa cells were cotransfected with various lengths of the hTERT promoter plasmids (0.1 μg) and with HBZ- (0.2 to 0.8 μg), and/or c-Jun- (0.2 μg), and/or JunD (0.2 μg)-expression vectors. Luciferase activity was normalized to tk-luc activity and presented relative to cells transfected with the reporter plasmid alone. The values are those obtained in triplicate, from three different experiments. Error bars indicate standard deviations. Shown in the lower panel, a western blot analysis of HBZ and Jun protein levels in whole cell lysates of HeLa samples transfected with pGL3-378. The membrane was probed successively with a polyclonal anti-HBZ antibody, and a mouse anti-flag antibody. Actin was used as a loading control. (C) Transactivation of the hTERT promoter by HBZ and JunD in Jurkat cells. Cells were cotransfected with pGL3-378 reporter plasmid (4 μg), in combination with the indicated HBZ (2 μg) and/or JunD (2 μg)-expression vectors. Luciferase activity was normalized and presented as indicated in B. The values are those obtained in triplicate, from one representative experiment.
Mentions: To examine the role of HBZ in regulating the activity of the hTERT promoter, luciferase assays were performed with reporter plasmids containing various lengths of the 5' flanking sequence of the hTERT gene fused to the luciferase reporter gene (Fig 1A). The longest reporter pGL3-3300 contains 5 AP-1 binding sites; pGL3-2000 includes two of these sites, whereas the shortest construct pGL3-378 encompassing the proximal region is devoid of any AP-1 binding sequence. Each of these reporter plasmids was co-transfected in HeLa cells along with increasing amounts of an HBZ vector either alone or together with c-Jun or JunD expression plasmids. The expression levels of HBZ and Jun proteins were confirmed by Western blot analysis. Overexpression of HBZ with each of the three constructs did not exert any effect on the hTERT promoter activity (Fig 1B, lanes 2, 3, 4). Overexpression of c-Jun or JunD led to a small, but significant increase of this promoter activity. In presence of c-Jun, a 2-fold increase was observed with pGL3-3300, and a 3-fold increase with pGL3-2000 and pGL3-378. In presence of JunD, a 2-fold-increase was observed only with pGL3-378. Overexpression of HBZ with c-Jun resulted in a reduction of the hTERT promoter activity with the three reporter constructs (compare lanes 6, 7, 8 to lane 5). To note that the increased amounts of HBZ correlated with a decrease of c-Jun detected in cell lysates, as previously shown [12]. Intriguingly, overexpression of HBZ in the presence of JunD led to an increase of the hTERT promoter activity, which also correlated with the transfected amount of HBZ (compare lanes 10,11,12 to lane 9). Taken together, these observations show that HBZ expressed either with c-Jun or JunD is able to repress or enhance the hTERT promoter activity, respectively.

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