This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cellular immortality requires the complete replication of essential genetic elements at the ends of chromosomes called telomeres, a process that is accomplished by the enzymatic complex telomerase. Our long-term goal is to elucidate the mechanisms governing telomerase regulation in vivo, which in turn could facilitate our understanding of cancer, aging and infertility and may lead to therapies to treat these ailments. The specific hypothesis is that transcriptional inhibitors of the telomerase reverse transcriptase (TERT) gene locus play a key role in the activation or suppression of telomerase activity in vivo. This hypothesis is based on the following observations: 1) TERT functions as the catalytic component of telomerase and is essential for telomerase activity, 2) telomerase and TERT expression are suppressed during development and over-expression of TERT in somatic cells in vitro and in vivo is sufficient to re-activate telomerase, 3) a number of transcription factors have now been identified which associate with the TERT promoter and effect the suppression of TERT expression in cultured cells under physiological conditions. Based on these observations, we plan to search for candidate TERT inhibitors in cultured murine stem cells and assess the role of these inhibitors, along with other recently identified candidate TERT inhibitors, in the regulation of TERT expression in vivo using murine primordial stem cells (PGCs) as a model system. Murine PGCs provide an excellent model system to study telomerase regulation because they are well characterized, readily isolated, are an essentially homogeneous population of cells, and undergo a suppression telomerase activity during embryonic development.
The specific aims are: 1) Analyze TERT expression and the effect of TERT over-expression on telomerase activity in murine PGCs. We will assess the level of expression of murine TERT (mTERT) mRNA and protein using real time RT-PCR, in situ hybridization, and immunohistochemistry, and develop a transgenic mouse strain that over-expresses mTERT in PGCs to assess whether mTERT upregulation is sufficient to restore telomerase activity in post-mitotic PGCs. 2) Analyze the role of candidate TERT inhibitors in the regulation of TERT expression in murine PGCs. We will assess the expression of the candidate TERT inhibitors Mad1, SIP-1 and Menin in PGCs during development, assess whether siRNA-mediated knock-down expression of these genes effects mTERT and telomerase activity in PGCs, and analyze mTERT expression and telomerase activity in PGCs from mouse strains in which these candidate TERT inhibitors have been knocked out. 3) Search for new candidate TERT inhibitors using an enhanced-retroviral-mutagen based genetic screen, and test their role in TERT regulation in murine PGCs. We will use an enhanced retrovival mutagenic vector to screen for new transcriptional inhibitors of the mTERT gene in murine embryonic stem cells stably transfected with a mTERT-GFP reporter construct. Any candidate mTERT inhibitor thereby identified will be tested for its role in the regulation of mTERT expression in PGCs in vivo using siRNA technology.
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