Regulation of Cellular Immortalization and Steady-State Levels of the Telomerase Reverse Transcriptase through Its Carboxy-Terminal Domain

Abstract

Telomerase maintains cell viability and chromosomal stability through the addition of telomere repeats to chromosome ends. The reactivation of telomerase through the upregulation of TERT, the telomerase protein subunit, is an important step during cancer development, yet TERT protein function and regulation remain incompletely understood. Despite its close sequence similarity to human TERT (hTERT), we find that mouse TERT (mTERT) does not immortalize primary human fibroblasts. Here we exploit these differences in activity to understand TERT protein function by creating chimeric mouse-human TERT proteins. Through the analysis of these chimeric TERT proteins, we find that sequences in the human carboxy-terminal domain are critical for telomere maintenance in human fibroblasts. The substitution of the human carboxy-terminal sequences into the mouse TERT protein is sufficient to confer immortalization and maintenance of telomere length and function. Strikingly, we find that hTERT protein accumulates to markedly higher levels than does mTERT protein and that the sequences governing this difference in protein regulation also reside in the carboxy-terminal domain. These elevated protein levels, which are characteristic of hTERT, are necessary but not sufficient for telomere maintenance because stabilized mTERT mutants cannot immortalize human cells. Thus, the TERT carboxy terminus contains sequences that regulate TERT protein levels and determinants that are required for productive action on telomere ends.

Supplemental material for this article may be found at http://mcb.asm.org/.

We thank J. Shay for the hTERT cDNA, R. Weinberg for the BJT fibroblasts, K. Collins for the hTERC expression plasmids, and P. Lansdorp for the TFL-Telo V.2.1 software. We thank L. Attardi, J. Sage, and members of the Artandi laboratory for critical reading of the manuscript.

E.J.M. was supported by PHS training grant CA09302 from the NCI. J.C. is supported by a Samsung Fellowship. A.S.V. was supported by Medical Scientist Training Program grant GM07365. This work was supported by NIH RO1 CA111691, CA109088, KO8 CA082176, the Rita Allen Foundation and the V Foundation.