Limits...
Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene.

Renaud S, Loukinov D, Abdullaev Z, Guilleret I, Bosman FT, Lobanenkov V, Benhattar J - Nucleic Acids Res. (2007)

Bottom Line: Although complete hTERT promoter methylation was associated with full transcriptional repression, detailed mapping showed that, in telomerase-positive cells, not all the CpG sites were methylated, especially in the promoter region.This study underlines the dual role of DNA methylation in hTERT transcriptional regulation.In our model, hTERT methylation prevents binding of the CTCF repressor, but partial hypomethylation of the core promoter is necessary for hTERT expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland.

ABSTRACT
Expression of hTERT is the major limiting factor for telomerase activity. We previously showed that methylation of the hTERT promoter is necessary for its transcription and that CTCF can repress hTERT transcription by binding to the first exon. In this study, we used electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) to show that CTCF does not bind the methylated first exon of hTERT. Treatment of telomerase-positive cells with 5-azadC led to a strong demethylation of hTERT 5'-regulatory region, reactivation of CTCF binding and downregulation of hTERT. Although complete hTERT promoter methylation was associated with full transcriptional repression, detailed mapping showed that, in telomerase-positive cells, not all the CpG sites were methylated, especially in the promoter region. Using a methylation cassette assay, selective demethylation of 110 bp within the core promoter significantly increased hTERT transcriptional activity. This study underlines the dual role of DNA methylation in hTERT transcriptional regulation. In our model, hTERT methylation prevents binding of the CTCF repressor, but partial hypomethylation of the core promoter is necessary for hTERT expression.

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Methylation-sensitive binding of CTCF to the first exon of hTERT. (A)In vivo binding of CTCF to the first exon of hTERT in telomerase-positive and negative cell lines was analyzed by ChIP assay using anti-CTCF antibody. PCR coamplification of the test fragments (hTERT and H19) using as template DNA input fraction and DNA recovered from immunoprecipitated fractions bound by the anti-CTCF antibody. (B) 5′-end-labeled control (F1) and SssI methylase-treated (F1-met) fragments were digested with methylation-sensitive BstUI and analyzed on polyacrylamide gels to verify efficiency of in vitro methylation. (C) Control unmethylated (F1) or SssI-methylated (F1-met) fragments were analyzed by gel-shift assay (EMSA). F, free probe; B, CTCF-bound probe. (D) Representation of the hTERT sequence cloned into the pTERT-297/ex2/FRT. Arrows represent the localization of the primers used for hTERT methylation analysis of stable transfectant. (E) The methylation status of the stable transfectants was verified by MS-SSCA. Unmethylated and fully methylated controls were obtained from plasmids used for stable transfection. UT and MT represent, respectively, the unmethylated and methylated plasmids stably transfected into HeLa cells, and after 30 population doublings. (F) Binding of CTCF to the first exon of hTERT in stably transfected cell line was analyzed by ChIP assay using anti-CTCF antibody.
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Figure 1: Methylation-sensitive binding of CTCF to the first exon of hTERT. (A)In vivo binding of CTCF to the first exon of hTERT in telomerase-positive and negative cell lines was analyzed by ChIP assay using anti-CTCF antibody. PCR coamplification of the test fragments (hTERT and H19) using as template DNA input fraction and DNA recovered from immunoprecipitated fractions bound by the anti-CTCF antibody. (B) 5′-end-labeled control (F1) and SssI methylase-treated (F1-met) fragments were digested with methylation-sensitive BstUI and analyzed on polyacrylamide gels to verify efficiency of in vitro methylation. (C) Control unmethylated (F1) or SssI-methylated (F1-met) fragments were analyzed by gel-shift assay (EMSA). F, free probe; B, CTCF-bound probe. (D) Representation of the hTERT sequence cloned into the pTERT-297/ex2/FRT. Arrows represent the localization of the primers used for hTERT methylation analysis of stable transfectant. (E) The methylation status of the stable transfectants was verified by MS-SSCA. Unmethylated and fully methylated controls were obtained from plasmids used for stable transfection. UT and MT represent, respectively, the unmethylated and methylated plasmids stably transfected into HeLa cells, and after 30 population doublings. (F) Binding of CTCF to the first exon of hTERT in stably transfected cell line was analyzed by ChIP assay using anti-CTCF antibody.

Mentions: To evaluate binding of CTCF to sequences within the first exon of hTERT, ChIP assays were performed on telomerase-positive tumor cell lines (HeLa, SW480), normal telomerase-negative cells (BJ) and telomerase-positive cells immortalized by hTERT transfection (HLF/hTERT). CTCF binding to the ICR of H19 gene was used as a positive control for the efficiency of the experimental protocol. PCR reactions were performed under conditions in which the negative control samples (no antibody) always showed negligible levels of background amplification (data not shown). ChIP experiments were performed in a way that the positive control, H19, was coamplified with the hTERT exon 1 in a single reaction and PCR fragments were resolved on the same gel. Results show that CTCF bound to the first exon of the endogenous hTERT gene of hTERT-negative BJ and HLF/hTERT cells but not of the tumor cell lines tested (Figure 1A). These results support our previous data (18) and are more convincing because positive controls and experimental fragments were amplified in the same reaction.Figure 1.


Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene.

Renaud S, Loukinov D, Abdullaev Z, Guilleret I, Bosman FT, Lobanenkov V, Benhattar J - Nucleic Acids Res. (2007)

Methylation-sensitive binding of CTCF to the first exon of hTERT. (A)In vivo binding of CTCF to the first exon of hTERT in telomerase-positive and negative cell lines was analyzed by ChIP assay using anti-CTCF antibody. PCR coamplification of the test fragments (hTERT and H19) using as template DNA input fraction and DNA recovered from immunoprecipitated fractions bound by the anti-CTCF antibody. (B) 5′-end-labeled control (F1) and SssI methylase-treated (F1-met) fragments were digested with methylation-sensitive BstUI and analyzed on polyacrylamide gels to verify efficiency of in vitro methylation. (C) Control unmethylated (F1) or SssI-methylated (F1-met) fragments were analyzed by gel-shift assay (EMSA). F, free probe; B, CTCF-bound probe. (D) Representation of the hTERT sequence cloned into the pTERT-297/ex2/FRT. Arrows represent the localization of the primers used for hTERT methylation analysis of stable transfectant. (E) The methylation status of the stable transfectants was verified by MS-SSCA. Unmethylated and fully methylated controls were obtained from plasmids used for stable transfection. UT and MT represent, respectively, the unmethylated and methylated plasmids stably transfected into HeLa cells, and after 30 population doublings. (F) Binding of CTCF to the first exon of hTERT in stably transfected cell line was analyzed by ChIP assay using anti-CTCF antibody.
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Related In: Results  -  Collection

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Figure 1: Methylation-sensitive binding of CTCF to the first exon of hTERT. (A)In vivo binding of CTCF to the first exon of hTERT in telomerase-positive and negative cell lines was analyzed by ChIP assay using anti-CTCF antibody. PCR coamplification of the test fragments (hTERT and H19) using as template DNA input fraction and DNA recovered from immunoprecipitated fractions bound by the anti-CTCF antibody. (B) 5′-end-labeled control (F1) and SssI methylase-treated (F1-met) fragments were digested with methylation-sensitive BstUI and analyzed on polyacrylamide gels to verify efficiency of in vitro methylation. (C) Control unmethylated (F1) or SssI-methylated (F1-met) fragments were analyzed by gel-shift assay (EMSA). F, free probe; B, CTCF-bound probe. (D) Representation of the hTERT sequence cloned into the pTERT-297/ex2/FRT. Arrows represent the localization of the primers used for hTERT methylation analysis of stable transfectant. (E) The methylation status of the stable transfectants was verified by MS-SSCA. Unmethylated and fully methylated controls were obtained from plasmids used for stable transfection. UT and MT represent, respectively, the unmethylated and methylated plasmids stably transfected into HeLa cells, and after 30 population doublings. (F) Binding of CTCF to the first exon of hTERT in stably transfected cell line was analyzed by ChIP assay using anti-CTCF antibody.
Mentions: To evaluate binding of CTCF to sequences within the first exon of hTERT, ChIP assays were performed on telomerase-positive tumor cell lines (HeLa, SW480), normal telomerase-negative cells (BJ) and telomerase-positive cells immortalized by hTERT transfection (HLF/hTERT). CTCF binding to the ICR of H19 gene was used as a positive control for the efficiency of the experimental protocol. PCR reactions were performed under conditions in which the negative control samples (no antibody) always showed negligible levels of background amplification (data not shown). ChIP experiments were performed in a way that the positive control, H19, was coamplified with the hTERT exon 1 in a single reaction and PCR fragments were resolved on the same gel. Results show that CTCF bound to the first exon of the endogenous hTERT gene of hTERT-negative BJ and HLF/hTERT cells but not of the tumor cell lines tested (Figure 1A). These results support our previous data (18) and are more convincing because positive controls and experimental fragments were amplified in the same reaction.Figure 1.

Bottom Line: Although complete hTERT promoter methylation was associated with full transcriptional repression, detailed mapping showed that, in telomerase-positive cells, not all the CpG sites were methylated, especially in the promoter region.This study underlines the dual role of DNA methylation in hTERT transcriptional regulation.In our model, hTERT methylation prevents binding of the CTCF repressor, but partial hypomethylation of the core promoter is necessary for hTERT expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland.

ABSTRACT
Expression of hTERT is the major limiting factor for telomerase activity. We previously showed that methylation of the hTERT promoter is necessary for its transcription and that CTCF can repress hTERT transcription by binding to the first exon. In this study, we used electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) to show that CTCF does not bind the methylated first exon of hTERT. Treatment of telomerase-positive cells with 5-azadC led to a strong demethylation of hTERT 5'-regulatory region, reactivation of CTCF binding and downregulation of hTERT. Although complete hTERT promoter methylation was associated with full transcriptional repression, detailed mapping showed that, in telomerase-positive cells, not all the CpG sites were methylated, especially in the promoter region. Using a methylation cassette assay, selective demethylation of 110 bp within the core promoter significantly increased hTERT transcriptional activity. This study underlines the dual role of DNA methylation in hTERT transcriptional regulation. In our model, hTERT methylation prevents binding of the CTCF repressor, but partial hypomethylation of the core promoter is necessary for hTERT expression.

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