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

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.

Epigenetic plasticity of hTERT gene promoter determines retinoid capacity to repress telomerase in maturation-resistant acute promyelocytic leukemia cells

“…In keeping, earlier studies showed that methylation of the hTERT promoter at the CTCF-binding site inhibited the binding of CTCF, thus derepressing hTERT in some human tumors. 16,17 Previous reports showed that a region between À578 and À300 bp upstream of the ATG initiation codon in the hTERT promoter, which corresponds to the domain identified in our study, 20,52 functions as a transcriptional silencer. This region includes MZF-2 and WT1 binding sites.…”

“…16,17 Thus, it was not surprising to observe constitutive binding of CTCF to this proximal region in the cells used in this study, as this region is unmethylated in both NB4-LR1 and NB4-LR1 SFD variants. However, through this hTERT regulation by retinoids in maturation-resistant APL cells A Azouz et al binding, CTCF has been described as a repressor of hTERT.…”

“…c-Myc/Max, as well as Sp1, positively regulates hTERT transcription, whereas Mad-1/Max has a negative regulatory effect. [11][12][13][14][15] In addition, a number of protein factors have been shown to negatively regulate hTERT; among them are the CCCTC-binding factor (CTCF), 16,17 the transcription activator protein 1 (AP-1), 18 the tumor suppressor, Menin, 19 the myeloid cell-specific zincfinger protein 2 (MZF-2), 20 and the Wilms' tumor protein (WT1). 21 Epigenetic control and overall chromatin structure have recently been shown to be also important parameters in hTERT gene regulation.…”

Preferential binding of the methyl-CpG binding domain protein 2 at methylated transcriptional start site regions

“…Firstly, this cell line expresses MBD2 to a level similar to that observed in many cancer cell lines. 28,29 Secondly, the fact that epigenetic studies have been performed on this cell line by independent laboratories 8,30 has enabled us to exploit previously published data for validation purposes. Our aim here was to investigate the relationship between promoter occupancy by MBD2, the DNA methylation profile and gene expression.…”

TERT promoter mutations in bladder cancer affect patient survival and disease recurrence through modification by a common polymorphism

“…The TERT promoter has been shown to be the most important element of telomerase expression because it harbors binding sites for numerous cellular transcriptional activators and repressors that directly or indirectly regulate the gene expression (4). In addition, the high GC content around the transcription start site of the TERT promoter confers epigenetic regulation through methylation and chromatin remodeling (5,6).…”