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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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Proposed model of hTERT transcriptional regulation. In telomerase-negative somatic cells, CTCF binds to unmethylated CTCF binding site and inhibits hTERT transcription. Full methylation of the hTERT minimal promoter and exon1 in some telomerase-negative tumor cells cannot lead to hTERT transcription. A majority of the tumor CTCF sites within the hTERT gene are methylated, thus preventing CTCF binding. Partial demethylation of the hTERT promoter region with the formation of an active transcriptional complex can lead to hTERT transcription and telomerase activity. CTS stands for CTCF target sites. The hatched box represents the region A showed as unmethylated in cancer cell lines and tumor tissues. Question marks represent the two regions surrounding the region A, and that might have a strong influence on the hTERT promoter activity, most probably methylation-sensitive binding sites to transcription factors. Empty squares represent unmethylated CpG sites, and solid squares represent methylated CpG sites.
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Figure 5: Proposed model of hTERT transcriptional regulation. In telomerase-negative somatic cells, CTCF binds to unmethylated CTCF binding site and inhibits hTERT transcription. Full methylation of the hTERT minimal promoter and exon1 in some telomerase-negative tumor cells cannot lead to hTERT transcription. A majority of the tumor CTCF sites within the hTERT gene are methylated, thus preventing CTCF binding. Partial demethylation of the hTERT promoter region with the formation of an active transcriptional complex can lead to hTERT transcription and telomerase activity. CTS stands for CTCF target sites. The hatched box represents the region A showed as unmethylated in cancer cell lines and tumor tissues. Question marks represent the two regions surrounding the region A, and that might have a strong influence on the hTERT promoter activity, most probably methylation-sensitive binding sites to transcription factors. Empty squares represent unmethylated CpG sites, and solid squares represent methylated CpG sites.

Mentions: Methylation cassette experiments shows that the expression levels of the construct with partially methylated hTERT minimal promoter and methylated exon 1 in transient transfection is very close to the levels of the endogenous hTERT in human cancers showing similar methylation pattern. The methylation of the exon1 of hTERT seems to be sufficient to allow the transcription from the minimal promoter, and does no more exert its inhibitory effect as seen in umethylated constructs. Moreover, a CTCF site located at the beginning of exon 2 also plays an important role in the downregulation of the constructs with unmethylated hTERT (18). However, as previously mentioned, the presence of an intron between the two CTCF sites complicates the interpretation of the transfection assays (22). Nevertheless, in our proposed model for the transcriptional regulation of the hTERT gene (Figure 5), we took the effect of the two CTCF sites into account.Figure 5.


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)

Proposed model of hTERT transcriptional regulation. In telomerase-negative somatic cells, CTCF binds to unmethylated CTCF binding site and inhibits hTERT transcription. Full methylation of the hTERT minimal promoter and exon1 in some telomerase-negative tumor cells cannot lead to hTERT transcription. A majority of the tumor CTCF sites within the hTERT gene are methylated, thus preventing CTCF binding. Partial demethylation of the hTERT promoter region with the formation of an active transcriptional complex can lead to hTERT transcription and telomerase activity. CTS stands for CTCF target sites. The hatched box represents the region A showed as unmethylated in cancer cell lines and tumor tissues. Question marks represent the two regions surrounding the region A, and that might have a strong influence on the hTERT promoter activity, most probably methylation-sensitive binding sites to transcription factors. Empty squares represent unmethylated CpG sites, and solid squares represent methylated CpG sites.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Figure 5: Proposed model of hTERT transcriptional regulation. In telomerase-negative somatic cells, CTCF binds to unmethylated CTCF binding site and inhibits hTERT transcription. Full methylation of the hTERT minimal promoter and exon1 in some telomerase-negative tumor cells cannot lead to hTERT transcription. A majority of the tumor CTCF sites within the hTERT gene are methylated, thus preventing CTCF binding. Partial demethylation of the hTERT promoter region with the formation of an active transcriptional complex can lead to hTERT transcription and telomerase activity. CTS stands for CTCF target sites. The hatched box represents the region A showed as unmethylated in cancer cell lines and tumor tissues. Question marks represent the two regions surrounding the region A, and that might have a strong influence on the hTERT promoter activity, most probably methylation-sensitive binding sites to transcription factors. Empty squares represent unmethylated CpG sites, and solid squares represent methylated CpG sites.
Mentions: Methylation cassette experiments shows that the expression levels of the construct with partially methylated hTERT minimal promoter and methylated exon 1 in transient transfection is very close to the levels of the endogenous hTERT in human cancers showing similar methylation pattern. The methylation of the exon1 of hTERT seems to be sufficient to allow the transcription from the minimal promoter, and does no more exert its inhibitory effect as seen in umethylated constructs. Moreover, a CTCF site located at the beginning of exon 2 also plays an important role in the downregulation of the constructs with unmethylated hTERT (18). However, as previously mentioned, the presence of an intron between the two CTCF sites complicates the interpretation of the transfection assays (22). Nevertheless, in our proposed model for the transcriptional regulation of the hTERT gene (Figure 5), we took the effect of the two CTCF sites into account.Figure 5.

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.

Show MeSH
Related in: MedlinePlus