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.

Show MeSH

Related in: MedlinePlus

Genomic bisulfite sequencing of hTERT promoter and 5′ exon region (−200 to +100 nucleotide bases around the ATG translational start site). After PCR amplification of bisulfite-modified DNA and cloning into pGEM-T vector, 12 clones of telomerase-positive cell lines (HeLa and SW480), 8 clones of endogenously telomerase-negative cells (BJ and HLF/hTERT) and 8 clones from telomerase-positive tumor tissues (breast, bladder and cervix) (Figure 2) were analyzed by sequencing. Clones of each are shown on the figure. Each square represents one CpG site. Filled squares: methylated; open squares: unmethylated. Region A: in this region (−80 to −165 bp), multiple CpG sites were found to be unmethylated in tumors tissues and cell lines. Region B: localization of the CTCF binding site within the first exon of the hTERT gene, most of the CpG sites were found to be methylated in telomerase-positive tumor tissues and cell lines. Percentages of methylated CG are indicated for regions A and B.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1851636&req=5

Figure 2: Genomic bisulfite sequencing of hTERT promoter and 5′ exon region (−200 to +100 nucleotide bases around the ATG translational start site). After PCR amplification of bisulfite-modified DNA and cloning into pGEM-T vector, 12 clones of telomerase-positive cell lines (HeLa and SW480), 8 clones of endogenously telomerase-negative cells (BJ and HLF/hTERT) and 8 clones from telomerase-positive tumor tissues (breast, bladder and cervix) (Figure 2) were analyzed by sequencing. Clones of each are shown on the figure. Each square represents one CpG site. Filled squares: methylated; open squares: unmethylated. Region A: in this region (−80 to −165 bp), multiple CpG sites were found to be unmethylated in tumors tissues and cell lines. Region B: localization of the CTCF binding site within the first exon of the hTERT gene, most of the CpG sites were found to be methylated in telomerase-positive tumor tissues and cell lines. Percentages of methylated CG are indicated for regions A and B.

Mentions: As methylation of CpG dinucleotides within the CTCF recognition sequence can influence its binding (32), we examined the methylation status of CpG sites within the hTERT minimal promoter and the first exon sequences from −200 to +100 from the ATG using bisulfite modification, PCR amplification, and subsequent cloning and sequencing of the PCR products. Clones from two tumor cell lines, HeLa and SW480, two normal cell lines, BJ and HLF/hTERT, as well as tumor tissues from breast, bladder and cervical cancer were sequenced and are presented in Figure 2. In tumor tissues and tumor cell lines, almost all CpGs between −100 and +100, including the CTCF binding site (Region B) were methylated (75 to 100% methylated). By comparison, most of the CpGs from approximately −165 to −100 (Region A) were unmethylated (0 to 55% methylated). In BJ and HLF/hTERT, very few CpGs in the entire region were methylated (3 to 23%). These findings suggested that methylation of CpGs in Region B might inhibit CTCF binding while leaving unexplained the selective hypomethylation of Region A.Figure 2.


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)

Genomic bisulfite sequencing of hTERT promoter and 5′ exon region (−200 to +100 nucleotide bases around the ATG translational start site). After PCR amplification of bisulfite-modified DNA and cloning into pGEM-T vector, 12 clones of telomerase-positive cell lines (HeLa and SW480), 8 clones of endogenously telomerase-negative cells (BJ and HLF/hTERT) and 8 clones from telomerase-positive tumor tissues (breast, bladder and cervix) (Figure 2) were analyzed by sequencing. Clones of each are shown on the figure. Each square represents one CpG site. Filled squares: methylated; open squares: unmethylated. Region A: in this region (−80 to −165 bp), multiple CpG sites were found to be unmethylated in tumors tissues and cell lines. Region B: localization of the CTCF binding site within the first exon of the hTERT gene, most of the CpG sites were found to be methylated in telomerase-positive tumor tissues and cell lines. Percentages of methylated CG are indicated for regions A and B.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851636&req=5

Figure 2: Genomic bisulfite sequencing of hTERT promoter and 5′ exon region (−200 to +100 nucleotide bases around the ATG translational start site). After PCR amplification of bisulfite-modified DNA and cloning into pGEM-T vector, 12 clones of telomerase-positive cell lines (HeLa and SW480), 8 clones of endogenously telomerase-negative cells (BJ and HLF/hTERT) and 8 clones from telomerase-positive tumor tissues (breast, bladder and cervix) (Figure 2) were analyzed by sequencing. Clones of each are shown on the figure. Each square represents one CpG site. Filled squares: methylated; open squares: unmethylated. Region A: in this region (−80 to −165 bp), multiple CpG sites were found to be unmethylated in tumors tissues and cell lines. Region B: localization of the CTCF binding site within the first exon of the hTERT gene, most of the CpG sites were found to be methylated in telomerase-positive tumor tissues and cell lines. Percentages of methylated CG are indicated for regions A and B.
Mentions: As methylation of CpG dinucleotides within the CTCF recognition sequence can influence its binding (32), we examined the methylation status of CpG sites within the hTERT minimal promoter and the first exon sequences from −200 to +100 from the ATG using bisulfite modification, PCR amplification, and subsequent cloning and sequencing of the PCR products. Clones from two tumor cell lines, HeLa and SW480, two normal cell lines, BJ and HLF/hTERT, as well as tumor tissues from breast, bladder and cervical cancer were sequenced and are presented in Figure 2. In tumor tissues and tumor cell lines, almost all CpGs between −100 and +100, including the CTCF binding site (Region B) were methylated (75 to 100% methylated). By comparison, most of the CpGs from approximately −165 to −100 (Region A) were unmethylated (0 to 55% methylated). In BJ and HLF/hTERT, very few CpGs in the entire region were methylated (3 to 23%). These findings suggested that methylation of CpGs in Region B might inhibit CTCF binding while leaving unexplained the selective hypomethylation of Region A.Figure 2.

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