Our long-term goal is to determine the mechanisms of telomerase regulation during development. The hTERTgene, which encodes the limiting subunit of human telomerase, is primarily regulated at the level oftranscription. It is highly expressed in pluripotent stem cells, but stringently repressed in most adult somaticcells. Despite intensive investigation in the past decade, mechanisms of its repression, including cis-regulatoryelements and trans-acting factors remain to be elucidated. We previously reported that the endogenoushTERT locus was embedded in a condensed chromatin domain in many somatic cells, while such a domaindid not exist in the less repressed mouse TERT gene. Consistent with the vital role of chromatin in its tightregulation, we also found that an episomal hTERT locus in human fibroblasts was not subjected to repression,whereas a chromosomally integrated hTERT locus recapitulated its native regulation. Thus, we hypothesizethat 1) the interplay between distal elements and core promoter in their native chromatin context is importantfor hTERT repression; and 2) partial loss of this repression leads to hTERT transcription during cellularimmortalization. To study the mechanisms of hTERT repression, we developed a novel technical platform, therecombinase-mediated BAC targeting or RMBT method, for targeted integration of single-copy BAC reportersinto specified chromosomal sites. Using this technique, we demonstrated that chromosomal integration of aBAC construct containing the hTERT locus resulted in the establishment of a surrogate chromatin setting inwhich the hTERT promoter was tightly repressed and recapitulated its endogenous gene in human fibroblasts.In this application, we plan to pursue the following specific aims: 1) Delineate cis elements involved in hTERTrepression in human fibroblasts. 2) Identify and characterize protein factors involved in hTERT repression inhuman fibroblasts. 3) Determine cis elements that confer humanized regulation of the mTERT gene in mESCs.
Telomeres are ends of linear chromosomes and essential for long-term cell proliferation and survival.Telomerase; the enzyme that elongates telomeres; plays an important role in cancers and aging-relateddiseases. This grant application is proposed to determine the mechanisms of telomerase regulation in humansomatic cells.
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