Microenvironmental Regulation of Telomerase Isoforms in Human Embryonic Stem Cells

Abstract

Recent evidence points to extra-telomeric, noncanonical roles for telomerase in regulating stem cell function. In this study, human embryonic stem cells (hESCs) were cultured in 20% or 2% O 2 microenvironments for up to 5 days and evaluated for telomerase reverse transcriptase (TERT) expression and telomerase activity. Results showed increased cell survival and maintenance of the undifferentiated state with elevated levels of nuclear TERT in 2% O 2 -cultured hESCs despite no significant difference in telomerase activity compared with their high-O 2 -cultured counterparts. Pharmacological inhibition of telomerase activity using a synthetic tea catechin resulted in spontaneous hESC differentiation, while telomerase inhibition with a phosphorothioate oligonucleotide telomere mimic did not. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed variations in transcript levels of full-length and alternate splice variants of TERT in hESCs cultured under varying O 2 atmospheres. Steric-blocking of Da and Db hTERT splicing using morpholino oligonucleotides altered the hTERT splicing pattern and rapidly induced spontaneous hESC differentiation that appeared biased toward endomesodermal and neuroectodermal cell fates, respectively. Together, these results suggest that posttranscriptional regulation of TERT under varying O 2 microenvironments may help regulate hESC survival, selfrenewal, and differentiation capabilities through expression of extra-telomeric telomerase isoforms.

Human telomerase reverse transcriptase regulation by DNA methylation, transcription factor binding and alternative splicing (Review)

“…Splicing role of HIF-1 and hypoxia on hTERT. hypoxia is known to enhance hTERT expression through activation of the hTERT promoter in stem cells as well as in cancer (50,51). In hypoxic tumor conditions, hIF-1 is overexpressed and activates the hTERT promoter by binding at two sites between -165 and +51.…”

An intact putative mouse telomerase essential N‐terminal domain is necessary for proper telomere maintenance

“…Correlations of expression levels of alternatively spliced hTERT mRNAs relative to full‐length hTERT mRNA with cell proliferation, cell differentiation and cancer subtype have also been reported (Ulaner et al., ; Brenner et al., ; Fajkus et al., ; Hisatomi et al., ; Petrenko et al., ; Listerman et al., ). Relative levels of specific hTERT isoforms are also important for human embryonic stem (ES) cell self‐renewal and differentiation, with specific isoforms possibly enriching differentiation towards specific cell lineages (Radan et al., ).…”

The role of telomeres and telomerase reverse transcriptase isoforms in pluripotency induction and maintenance

“…In partial agreement, chemical inhibition of hTERT activity in hESCs leads to lowered pluripotency and increased differentiation, while curiously, similar blocking of telomerase activity through TERC steric inhibition has no effect suggesting an extra-telomeric role for TERT in maintaining self-renewal in the undifferentiated state. 85 In contrast, Sexton et al did not observe any reduction in pluripotency (as measured by sustained OCT3/4 expression only) after establishing stable hESC lines through zinc finger-mediated knockout of hTERT. 86 However, Sexton et al do observe a loss of selfrenewal and differentiation beginning around 75 days post TERT-knockout, when the cells reach a critical telomere length of 2-3kb.…”

“…138 However, Listerman et al and our group have recently shown that the b-variant transcript associates with polyribosomes, escapes NMD, and is likely translated. 24,85 Surprisingly, the b-deletion transcript not only survives degradation but is one of the most highly expressed TERT transcripts in various cell types including human embryonic stem cells. 24,128,132,147,151 A recent discovery suggests that the b-variant inhibits telomerase activity by sequestering TERC.…”