TERT distal promoter GC islands are critical for telomerase and together with DNMT3B silencing may serve as a senescence-inducing agent in gliomas

Abstract

Telomerase is reactivated in the majority of cancers. For instance, in gliomas, it is common that the TERT promoter is mutated. Research on telomere promoter GC islands have been focused primarily on proximal TERT promoter but little is known about the distal promoter. Therefore, in this study, we investigated the proximal and distal TERT promoter, in terms of DNA methylation. We did bisulfite sequencing in zebrafish tissue samples for the distal tert promoter. In the zebrafish brain tissues, we identified a hypomethylation site in the tert promoter, and found that this hypomethylation was associated with aging and shortened telomeres. Through site directed mutagenesis in glioma cell lines, we changed 10 GC spots individually, cloned into a reporter vector, and measured promoter activity. Finally, we silenced DNMT3B and measured telomerase activity along with vidaza and adriamycin treatments. Site directed mutagenesis of glioma cell lines revealed that each of the 10 GC spots are critical for telomerase activity. Changing GC to AT abolished promoter activity in all spots when transfected into glioma cell lines. Then, through silencing of DNMT3B, we observed a reduction in hTERT expression levels, while hTR remained the same, and a major increase in senescence-associated beta-galactosidase activity. Finally, we propose a model regarding the efficacy of two chemotherapeutic drugs, adriamycin and azacytidine, on gliomas. Here, we show that distal TERT promoter is critical; changing even one GC to AT abolishes TERT promoter activity. DNMT3B, a de novo methyltransferase, together with GC islands in distal TERT promoter plays an important role in regulation of telomerase expression and senescence.

Keywords:

• Telomere
• TERT promoter
• telomerase
• brain cancer
• DNA methylation

Acknowledgments

The authors thank Miguel Ferreiro and Şerif Şentürk for their invaluable suggestions and Arslan-Ergül Lab members; Elif Sena Temirci, Tuba Sena Oğurlu, and Elif Akış for their help in the revisions.

Ethical approval

Experiments are performed according to the rules determined by Bilkent University Animal Ethics Committee and approved with approval number of 2014/18.

Author contributions

Naz Serifoglu contributed to conceptualization, methodology, and writing—original draft. Begun Erbaba contributed to methodology and investigation. Michelle Adams contributed to investigation and writing—review and editing. Ayca Arslan-Ergul contributed to conceptualization, writing—original draft, and funding acquisition.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data are available in Mendeley: Arslan Ergül, Ayça (2021), “DNMT inhibition”, Mendeley Data, V1, doi: 10.17632/n387s8kpt5.1 and Arslan Ergül, Ayça (2022), “Serifoglu J neurogenet”, Mendeley Data, V1, doi: 10.17632/zvpzvjfzfr.1.

Additional information

Funding

This work was supported by The Scientific and Technological Research Council of Turkey 1001 grant [no: 114S548] and two Hacettepe University Scientific BAP grants [no: THD-2017–15492 and no: THD-2018–16904] to AA-E.