Grainyhead-like 2 Enhances the Human Telomerase Reverse Transcriptase Gene Expression by Inhibiting DNA Methylation at the 5'-CpG Island in Normal Human Keratinocytes

We recently identified Grainyhead-like 2 (GRHL2) as a novel transcription factor that binds to and regulates the activity of the human telomerase reverse transcriptase (hTERT) gene promoter. In this study, we investigated the biological functions of GRHL2 and the molecular mechanism underlying hTERT gene regulation by GRHL2. Retroviral transduction of GRHL2 in normal human keratinocytes (NHK) led to a significant extension of replicative life span, whereas GRHL2 knockdown notably repressed telomerase activity and cell proliferation. Using promoter magnetic precipitation coupled with Western blotting, we confirmed the binding of GRHL2 to the hTERT promoter and mapped the minimal binding region at -53 to -13 of the promoter. Furthermore, mutation analysis revealed the three nucleotides from -21 to -19 to be critical for GRHL2 binding. Because hTERT expression is regulated in part by DNA methylation, we determined the effects of GRHL2 on the methylation status of the hTERT promoter. Senescent NHK exhibited hypermethylation of the CpG island, which occurred with the loss of hTERT expression. On the contrary, the promoter remained hypomethylated in GRHL2-transduced NHK, irrespective of cell proliferation status. Also, knockdown of endogenous GRHL2 led to hypermethylation of the promoter. These results indicate that GRHL2 regulates the hTERT expression through an epigenetic mechanism and controls the cellular life span.