| The distinctive features of telomerase and telomere biology between human and mouse result from their species-specific regulation. These differences contribute to the distinct characteristics of aging and cancer development in mice and humans. To investigate the fundamental mechanisms of these biological processes, I have studied the developmental silencing of human and mouse telomerases in transgenic models and their activation during cancer formation in a mouse breast cancer model and nuclear reprogramming in induced pluripotent stem (iPS) cells. The results of my thesis research demonstrated that the hTERT gene, which encodes the catalytic subunit of human telomerase, was transcriptionally silenced during embryonic development and dramatically activated in breast cancer cells. It was also strongly activated during the induction of pluripotency. On the other hand, the mouse counterpart, the mTERT gene, was expressed at high levels in the majority of adult tissues and somatic cells. Because telomere elongation is crucial for self-renewal of human stem cells and replicative aging of their differentiated progeny, these findings have important implications in the generation and applications of iPS cells and their application in regenerative medicine.My thesis is organized into the following three parts.1. In vivo regulation of human telomerase reverse transcriptase in mouse development and tumorigenesis.To explore hTERT gene regulation in vivo, we engineered mice with a 158-kb transgenic BAC spanning the hTERT locus with a Renilla luciferase (Rluc) cassette downstream of its promoter. Analysis of multiple founder lines revealed that the Rluc expression profile from the transgenic hTERT reporter (hTERT-Rluc) locus reproduced that of the native hTERT gene in all tissues and organs examined. Both the hTERT-Rluc and the endogenous mTERT locus were expressed in early embryonic stages and their mRNA levels progressively decreased throughout embryonic and postnatal development. Whereas hTERT-Rluc transcription was much lower than the endogenous mTERT expression in most organs, it increased significantly during postnatal development of thymus, testis, and ovary. Additionally, the transcription of hTERT-Rluc, but surprisingly not the endogenous mTERT gene, increased during Wntl-induced mammary tumorigenesis, allowing the monitoring of tumor development via non-invasive bioluminescent imaging. Collectively, our results demonstrate that the hTERT-Rluc system recapitulates well the developmental regulation of the hTERT gene in a chromosomal position-independent manner and serves as legitimate model to explore telomerase regulation in the development of normal and neoplastic tissues in vivo. This study provides strong evidence that genetic sequence determines the species-specific developmental regulation of the TERT gene and that mouse epigenetic and transcription machineries could faithfully regulate hTERT transcription.2. Telomerase activation in induced pluripotent stem cells.To study telomerase activation during reprogramming,3 classes of embryonic stem cell (ESC)-like clones were isolated from mouse fibroblasts containing a transgenic hTERT reporter. In order to analyze the telomerase activation during reprogramming, we compared these cell lines with hiPS cell lines. Interestingly, whereas the endogenous mTERT gene expression was only moderately increased during reprogramming, hTERT increased dramatically. Our study mainly focused on regulation of telomerase in hiPS cells. The hTERT and telomerase activity were variable among different iPS clones during reprogramming. Only in cells with the highest telomerase were telomeres restored to the lengths in hES cells. Telomere elongation required more extensive proliferation of reprogrammed cells. Telomerase activity and telomere length decreased dramatically when reprogrammed cells were differentiated into fibroblast like cells in embryoid bodies. Our data demonstrated that the hTERT promoter was strongly activated in discrete steps, revealing a critical difference in human and mouse cell reprogramming. Because telomere elongation is crucial for self-renewal of hPSCs and replicative aging of their differentiated progeny, these findings have important implications in the generation and applications of iPS cells.3. The functions of telomerase subunits TERT and TERC in human and mouse fibroblasts.Telomerase synthesizes telomeric sequences and its core enzyme is composed of a reverse transcriptase, known as TERT, and an RNA template, known as TERC. Although both TERT and TERC were essential for telomerase activity, their contributions to telomerase regulation in somatic cells remained unknown. Therefore, mouse and human TERT, TERC, or both subunits were introduced into NHF (normal human fibroblast) and MEF (mouse embryonic fibroblast) cells by retroviral transduction. qRT-PCR analysis was used to determine the exogenous gene expression in infected cells. Telomerase activity and telomere lengths were determined by TRAP assay and Southern blot analysis of telomeric. restriction fragments in infected cells. As expected, we were able to reconstitute telomerase activity in NHF cells by ectopic expression of hTERT. When we overexpressed hTERT and hTERC in NHF cells, both telomerase activity and telomere length increased dramatically. Introduction of human or mouse TERT, TERC, or both genes in MEFs resulted in higher telomerase activities. However, telomerase was more active and processive in NHF cells overexpressing hTERT/hTERC than cells overexpressing hTERT/mTERC, suggesting that species-specific determinants of telomerase activity and processivity was conferred by the TERC component. Although telomerase activity in NHF cells expressing mTERT/hTERC was same as those expressing hTERT/hTERC, the former cells had shorter telomeres, indicating that the hybrid telomerase formed by mTERT/hTERC was functionally defective in vivo. Our data suggest that both TERT and TERC subunits contributed to species-specific regulation of telomerase activity.
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