The role of CTCF and DNA methylation in telomerase hTERT gene regulation

4321 Telomerase, which is involved in the regulation of the replicative life span by its maintaining telomere length, is activated in germ and stem cells, repressed in normal somatic cells and reactivated in a large majority of tumor cells. The expression of the catalytic subunit (hTERT) represents the limiting factor for telomerase activity. The hTERT promoter region is located in a CpG island and we observed that, in a majority of telomerase-positive cells, methylation of the hTERT promoter seems necessary for its transcription (Int J Cancer 101:335-341, 2002). This correlation is in stark contrast to the general model of regulation by DNA methylation. To understand how DNA methylation can lead to hTERT gene expression, several experiments were performed which showed that: 1) Strong activation of the hTERT minimal promoter is necessary but not sufficient to hTERT activation in vivo. 2) The strong inhibitory effect of the proximal exonic region was also investigated. We demonstrated that the inhibitory effect of this region is not promoter and cell dependent. 3) This inhibition is mainly due to the chromatin insulator protein CCCTC binding factor (CTCF), which binds in two sites in the hTERT proximal exonic region. ChiP experiments revealed that CTCF binds to the unmethylated hTERT exonic region (normal telomerase-negative cells) but not to the methylated one (telomerase-positive cells). 4) In transient or even stable transfection experiments, CTCF knockdown with siRNA resulted in a significant increasing activity of hTERT promoter in luciferase reporter constructs containing the hTERT promoter and the proximal exonic region. This indicates that activation of the unmethylated hTERT gene can be achieved by inhibition of CTCF. According to our data, a new model for hTERT gene regulation could be proposed. In telomerase-negative normal tissues and in some of telomerase-negative tumors, hypomethylation of the hTERT promoter allows CTCF to bind to the proximal exonic region and leads to hTERT transcription inhibition. In contrast, in telomerase-positive samples (normal or tumor tissues), hypermethylation of the hTERT gene prevents the methylation-sensitive CTCF inhibitor to bind and thus, lead to a weak but significant level of hTERT transcription.