Determining the role of epigenetics in telomerase regulation

Abstract

Telomeres are guanine-rich 5'-TTAGGG-3' repeats found at the ends of human chromosomes that protect chromosomes from degradation and also provide stabilization. Telomerase macromolecule is the primary enzyme responsible for telomere elongation. Telomerase has a critical role in cell proliferation, tumourigenesis, and therapy resistance by modifying many signalling pathways. The human telomerase reverse transcriptase enzyme (hTERT) is responsible for encoding the catalytic subunit of telomerase. It is demonstrated that TERT promoter mutations might increase telomerase activation, but regulation of the telomerase gene is incompletely understood with epigenetic, transcriptional, and posttranscriptional modifications all combined. Epigenetic alterations are just as significant as genetic mutations in a normal cell's transformation into cancer. Understanding the epigenetic molecular mechanism behind telomerase regulation and the responsible genes holds significant prospects for cancer treatment. Pluripotent stem cells provide a valuable tool to better understand this mechanism as telomerase activity is downregulated in stem cells during their differentiation. The link between telomerase activity and differentiation may provide an excellent tool for studying the epigenetic regulation of telomerase.
Physiological oxygen (physoxia) microenvironments affect several cellular aspects, including proliferation, metabolic activity, stemness, and differentiation. This environment also plays a critical role in TERT gene regulation and epigenetic changes such as histone modifications and DNA methylation. The role of oxygen tension on driving promoter modifications of the TERT gene in cancer and pluripotent stem cells (PSC) is poorly understood either in vitro or in vivo. Therefore, further research is required to determine the association between telomerase, epigenetics, and physiological oxygen microenvironment.
Here, various cancer cell lines and hESCs (embryonic stem cells) were studied to determine the role of low oxygen in the epigenetic regulation of telomerase and epigenetic associated genes, including TERT, DNMTs, HDACs, and differentiation markers for stem cells. This study design enabled an exploration of differences in molecular mechanisms of TERT regulation in different cell types. To understand physoxia effects, cells were cultured in an atmospheric oxygen incubator, 2% O2 -Pre-gassed media (pre-conditioned to 2% O2 in a HypoxyCool unit) in a 2% O2 incubator and an oxygen controlled chamber to provide a fully defined 2% O2 environment (tri-gas workstation). In this study, we investigated the effect of different oxygen tensions on proliferation, telomerase activity, TERT gene, promoter methylation and telomere length association with stem cell differentiation.
Reduced oxygen culture increased the proliferation rate, metabolic activity and stemness of ESCs. qRT-PCR data showed that EBs express three germ layer differentiation markers during extensive culture while retaining pluripotency and telomerase activity beyond the levels observed with monolayer differentiated cells. Monolayer and 3D differentiated cells displayed slower onset of differentiation in physoxia. Also, downregulated TERT expression was correlated to telomerase activity during differentiation with a higher telomerase enzyme activity in physoxia.A significant increase in promoter methylation was noted during the differentiation of ESCs and EBs correlated to decreased telomerase expression. Further, DNMT3B inhibition with nanaomycin A decreased methylation on the promoter and increased TERT expression, associated with increased enzyme activity in stem cells and cancer cell models. However, DNMT1 inhibitor (decitabine) treated cells had no consistent results in cancer cells. Additionally, CHIP data showed a considerable decrease in DNMT3B antibody binding to TERT promoter regions after nanaomycin A treatment compared to untreated cells.
Overall, our data for the first time demonstrated that DNMT3B binding to TERT promoter increases methylation in differentiated stem cells and cancer cells using CHIP qPCR. TERT promoter displayed an oxygen-sensitive methylation pattern associated with the DNMT3B enzyme activity in monolayer and 3D differentiated ESCs. Together these data suggest that DNMT3B inhibition positively regulates TERT gene expression and telomerase activity in stem cells and cancer cells.