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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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Up-regulation of the hTERT gene transcription in cells overexpressing HBZ and JunD. HeLa cells were cotransfected with expression plasmids for HBZ and JunD and incubated for 48 hours. (A) Western blot analysis of cell lysates using appropriate antibodies; lane 1, mock transfected; lane 2, JunD-transfected; lane 3, HBZ-transfected; lane 4, HBZ/JunD-transfected. (B) RT-PCR analysis of mRNA extracted from transfected cells. The RNAs were isolated and reverse transcribed. PCR was performed using primers specific for hTERT and actin. (C) Real-time PCR quantification of hTERT mRNA expression from cells transfected with indicated plasmids was performed as described in "Materials and methods". The expression level in mock-transfected cells was defined as 1.0. Experimental variations are indicated by error bars. (D) Recruitment of HBZ and JunD to the hTERT proximal promoter by chromatin immunoprecipitation assay (ChIP) in HeLa cells overexpressing HBZ and JunD. PCR results from IP reactions using preimmune rabbit serum (IgG) and antibodies against HBZ and JunD are shown. Each panel shows amplification of 0.4% of the total input chromatin (input). Purified DNA was analyzed by PCR using primers spanning the hTERT proximal promoter (upper panel) or the hTERT distal promoter (lower panel). DNA size standards are indicated. Data are shown for one representative experiment from three independent assays.
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Figure 3: Up-regulation of the hTERT gene transcription in cells overexpressing HBZ and JunD. HeLa cells were cotransfected with expression plasmids for HBZ and JunD and incubated for 48 hours. (A) Western blot analysis of cell lysates using appropriate antibodies; lane 1, mock transfected; lane 2, JunD-transfected; lane 3, HBZ-transfected; lane 4, HBZ/JunD-transfected. (B) RT-PCR analysis of mRNA extracted from transfected cells. The RNAs were isolated and reverse transcribed. PCR was performed using primers specific for hTERT and actin. (C) Real-time PCR quantification of hTERT mRNA expression from cells transfected with indicated plasmids was performed as described in "Materials and methods". The expression level in mock-transfected cells was defined as 1.0. Experimental variations are indicated by error bars. (D) Recruitment of HBZ and JunD to the hTERT proximal promoter by chromatin immunoprecipitation assay (ChIP) in HeLa cells overexpressing HBZ and JunD. PCR results from IP reactions using preimmune rabbit serum (IgG) and antibodies against HBZ and JunD are shown. Each panel shows amplification of 0.4% of the total input chromatin (input). Purified DNA was analyzed by PCR using primers spanning the hTERT proximal promoter (upper panel) or the hTERT distal promoter (lower panel). DNA size standards are indicated. Data are shown for one representative experiment from three independent assays.

Mentions: The above results propose for the first time that HBZ, in cooperation with JunD, activates the transcription of the hTERT gene. To examine the effect of HBZ on hTERT transcription, HeLa cells were co-transfected either with JunD and HBZ expression vectors or a blank vector. A Western blotting analysis, performed 48 hours later, confirmed the expression of both HBZ and JunD proteins (Fig 3A). The level of hTERT transcription evaluated by RT-PCR analysis showed a slight increase of hTERT transcripts, when HBZ and JunD were overexpressed (Fig 3B). Likewise, a quantitative analysis of hTERT mRNAs using real-time PCR revealed a significant 1.8-fold increase (P < 0.05) of hTERT mRNAs in cells co-expressing HBZ and JunD (Fig 3C, lane 6). Such an increase was not observed in cells expressing either JunD alone (lane 2) or HBZ alone (lane 3) or JunD together with one of the mutated forms of HBZ (lanes 7 and 8). These findings demonstrate that HBZ acts synergistically with JunD to increase the transcription of the hTERT gene.


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)

Up-regulation of the hTERT gene transcription in cells overexpressing HBZ and JunD. HeLa cells were cotransfected with expression plasmids for HBZ and JunD and incubated for 48 hours. (A) Western blot analysis of cell lysates using appropriate antibodies; lane 1, mock transfected; lane 2, JunD-transfected; lane 3, HBZ-transfected; lane 4, HBZ/JunD-transfected. (B) RT-PCR analysis of mRNA extracted from transfected cells. The RNAs were isolated and reverse transcribed. PCR was performed using primers specific for hTERT and actin. (C) Real-time PCR quantification of hTERT mRNA expression from cells transfected with indicated plasmids was performed as described in "Materials and methods". The expression level in mock-transfected cells was defined as 1.0. Experimental variations are indicated by error bars. (D) Recruitment of HBZ and JunD to the hTERT proximal promoter by chromatin immunoprecipitation assay (ChIP) in HeLa cells overexpressing HBZ and JunD. PCR results from IP reactions using preimmune rabbit serum (IgG) and antibodies against HBZ and JunD are shown. Each panel shows amplification of 0.4% of the total input chromatin (input). Purified DNA was analyzed by PCR using primers spanning the hTERT proximal promoter (upper panel) or the hTERT distal promoter (lower panel). DNA size standards are indicated. Data are shown for one representative experiment from three independent assays.
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Figure 3: Up-regulation of the hTERT gene transcription in cells overexpressing HBZ and JunD. HeLa cells were cotransfected with expression plasmids for HBZ and JunD and incubated for 48 hours. (A) Western blot analysis of cell lysates using appropriate antibodies; lane 1, mock transfected; lane 2, JunD-transfected; lane 3, HBZ-transfected; lane 4, HBZ/JunD-transfected. (B) RT-PCR analysis of mRNA extracted from transfected cells. The RNAs were isolated and reverse transcribed. PCR was performed using primers specific for hTERT and actin. (C) Real-time PCR quantification of hTERT mRNA expression from cells transfected with indicated plasmids was performed as described in "Materials and methods". The expression level in mock-transfected cells was defined as 1.0. Experimental variations are indicated by error bars. (D) Recruitment of HBZ and JunD to the hTERT proximal promoter by chromatin immunoprecipitation assay (ChIP) in HeLa cells overexpressing HBZ and JunD. PCR results from IP reactions using preimmune rabbit serum (IgG) and antibodies against HBZ and JunD are shown. Each panel shows amplification of 0.4% of the total input chromatin (input). Purified DNA was analyzed by PCR using primers spanning the hTERT proximal promoter (upper panel) or the hTERT distal promoter (lower panel). DNA size standards are indicated. Data are shown for one representative experiment from three independent assays.
Mentions: The above results propose for the first time that HBZ, in cooperation with JunD, activates the transcription of the hTERT gene. To examine the effect of HBZ on hTERT transcription, HeLa cells were co-transfected either with JunD and HBZ expression vectors or a blank vector. A Western blotting analysis, performed 48 hours later, confirmed the expression of both HBZ and JunD proteins (Fig 3A). The level of hTERT transcription evaluated by RT-PCR analysis showed a slight increase of hTERT transcripts, when HBZ and JunD were overexpressed (Fig 3B). Likewise, a quantitative analysis of hTERT mRNAs using real-time PCR revealed a significant 1.8-fold increase (P < 0.05) of hTERT mRNAs in cells co-expressing HBZ and JunD (Fig 3C, lane 6). Such an increase was not observed in cells expressing either JunD alone (lane 2) or HBZ alone (lane 3) or JunD together with one of the mutated forms of HBZ (lanes 7 and 8). These findings demonstrate that HBZ acts synergistically with JunD to increase the transcription of the hTERT gene.

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