The long term goal of this research is to understand the mechanisms of telomerase regulation in stem cells, which we know very little about at present, to facilitate the development of therapies to treat cancer and diseases associated with defects in telomerase and/or telomere maintenance. In recent studies, we have identified hypoxia inducible factor 1 alpha (Hif11) as one transcription factor that, via regulation of levels of telomerase reverse transcriptase (Tert), is essential for maintenance of functional levels of telomerase and telomere reserve in murine embryonic stem (mES) cell lines. In the following study, we now propose a number of specific hypotheses to more closely examine the potential development of Hif11 as a target to treat specific diseases associated with defects in telomerase and/or accelerated telomere loss, as well as certain types of tumor cells. To accomplish this, we propose 3 specific aims. In the first specific aim, we will more closely assess the mechanism of Hif11-mediated regulation of telomerase in embryonic stem cells. In this specific aim, we will determine whether Hif11 directly associates with the Tert gene promoter, and also test possible other indirect mechanisms of Hif11-mediated effects on Tert and telomerase. For the second specific aim, we will test the hypothesis that transient activation Hif11 in stem cells will induce repair of shortened telomeres via a mechanism involving activation of telomerase. In this specific aim, we will utilize the Tert Het (heterozygous) strain to assess whether expression of a mutant Hif11 transgene, which is constitutively stable, in pluripotent stem cells in the blastocyst effects telomerase activity. The Tert Het mouse strain has short, 'human-like', telomeres, due to telomerase haploinsufficiency induced by the loss of 1 functional copy of the Tert gene, and therefore is well suited for analysis of potential effects on telomere length. We will also test whether altered expression of Hif11 can repair telomeres in ES cells established from Tert Het mice or Tert knock out mice. This analysis in Tert deficient ES cells will allow us to determine whether Hif11 could have telomerase- independent effects on telomeres. The third specific aim is designed to test the hypothesis that the crucial role for Hif11 in maintenance of functional levels of telomerase activity in mES cell lines translates to pluripotent stem cells in vivo, as well as human ES cells and human tumor cell lines that constitutively express Hif11. The results from these studies could potentially provide impetus for the development of therapeutic strategies to activate or inhibit telomerase, via the targeting of Hif11, in (i), stem and/or progenitor cells to treat diseases, such as pulmonary fibrosis and anemia, associated with defects in telomerase, and conditions, such accelerated telomere loss in bone marrow transplant recipients and in T lymphocytes from AIDS patients, and (ii), in tumor cells that persist or thrive in hypoxic environments and depend on telomerase for long term viability and proliferative capacity.
Telomerase is essential for long term survival of all proliferating cells in the body, including stem cells. It is also essential for the long term survival of most types cancer cells. In this study, we assess the role of a newly discovered regulator of telomerase in mouse embryonic stem cells, Hif11, in the regulation of telomerase in stem cells during development and in adult organisms, using suitable strains of mice. We also explore the potential role of Hif11 in regulating telomerase in human embryonic stem cells as well as cancer cell lines known to express Hif11. The results from this study could potentially identify Hif11 as a new target to treat diseases associated with telomerase defects or accelerated telomere shortening, such as pulmonary fibrosis, AIDS and cancer.
