Transcriptional and post-transcriptional regulation of telomerase reverse transcriptase (hTERT) expression : : The role of histone modification and alternative splicing

Sammanfattning: Telomerase, an RNA-dependent DNA polymerase responsible for telomeric DNA elongation, is silent in most normal differentiated human cells due to the tight repression of its catalytic unit, telomerase reverse transcriptase (hTERT), whereas constitutive hTERT expression and telomerase activation are prerequisites for cellular immortalization/malignant transformation. As reconfiguration of chromatin acts like a master on/off switch, determining whether particular genes are active or inactive, the project was specifically designed to first elucidate molecular strategies that integrate diverse signalling pathways at the hTERT chromatin and consequently result in telomerase activation or repression in normal and malignant human cells. Secondly, we address the role of alternative splicing in the regulation of hTERT mRNA expression. We identified the mitogen-activated protein kinase (MAPK) cascade-mediated histone H3 Ser10 phosphorylation to be a molecular link between proliferation and induction of hTERT expression. In normal human T lymphocytes and fibroblasts, growth or stress stimuli induce hTERT expression and/or telomerase activity that is preceded by phosphorylated histone H3 Ser10 at the hTERT promoter. Blockade of the MAPK-triggered H3 phosphorylation significantly abrogates hTERT induction and Ser10 phosphorylation at the promoter. These results define H3 phosphorylation as a key to hTERT transactivation induced by proliferation and reveal a fundamental mechanism for telomerase regulation in both normal human cells and transformed T cells (paper I). We found that histone H3 methyltransferase (HMT) SMYD3 directly activated transcription of the hTERT gene by binding to the hTERT core promoter and affecting tri-methylation of histone H3-K4 at the hTERT chromatin (paper II). In contrast to SMYD3, histone H3 demethylases (HDMs) LSD1 and RBP2 are shown to repress the hTERT gene. The inhibition of LSD1 function or expression up-regulated or de-repressed the hTERT expression. Similarly, RBP2 depletion led to the hTERT mRNA induction in normal human telomerase-deficient fibroblasts. During the differentiation of leukemic HL60 cells, both LSD1 and RBP2 were recruited to the hTERT promoter and demethylated the local H3-K4, which was accompanied by the repression of hTERT transcription. Taken together, LSD1 and RBP2 are required for the establishment of a stable repression state of the hTERT gene in human normal or differentiated malignant cells (papers III and IV). In addition, we found that human normal and malignant lymphoid cells, like other human cells, express splicing variants of hTERT mRNA and require transcriptional activation of the hTERT gene to acquire telomerase activity (paper V). Overall, the present findings provide significant insights into the regulatory mechanisms for hTERT transcription and telomerase expression, and may be implicated in manipulation of telomerase activity for anti-aging and anti-cancer therapeutic purposes.