Our long-term goal is to determine the mechanisms of telomerase regulation during development. The hTERT gene, which encodes the limiting subunit of human telomerase, is primarily regulated at the level of transcription. It is highly expressed in pluripotent stem cells, but stringently repressed in most adult somatic cells. Despite intensive investigation in the past decade, mechanisms of its repression, including cis-regulatory elements and trans-acting factors remain to be elucidated. We previously reported that the endogenous hTERT locus was embedded in a condensed chromatin domain in many somatic cells, while such a domain did not exist in the less repressed mouse TERT gene. Consistent with the vital role of chromatin in its tight regulation, 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 hypothesize that 1) the interplay between distal elements and core promoter in their native chromatin context is important for hTERT repression;and 2) partial loss of this repression leads to hTERT transcription during cellular immortalization. To study the mechanisms of hTERT repression, we developed a novel technical platform, the recombinase-mediated BAC targeting or RMBT method, for targeted integration of single-copy BAC reporters into specified chromosomal sites. Using this technique, we demonstrated that chromosomal integration of a BAC construct containing the hTERT locus resulted in the establishment of a surrogate chromatin setting in which 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 hTERT repression in human fibroblasts. 2) Identify and characterize protein factors involved in hTERT repression in human fibroblasts. 3) Determine cis elements that confer humanized regulation of the mTERT gene in mESCs.

Public Health Relevance

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-related diseases. This grant application is proposed to determine the mechanisms of telomerase regulation in human somatic cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM071725-08
Application #
8324226
Study Section
Cellular Mechanisms in Aging and Development Study Section (CMAD)
Program Officer
Carter, Anthony D
Project Start
2004-08-01
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
8
Fiscal Year
2012
Total Cost
$313,558
Indirect Cost
$110,608
Name
Pennsylvania State University
Department
Physiology
Type
Schools of Medicine
DUNS #
129348186
City
Hershey
State
PA
Country
United States
Zip Code
17033
Zhao, Yuanjun; Cheng, De; Wang, Shuwen et al. (2014) Dual roles of c-Myc in the regulation of hTERT gene. Nucleic Acids Res 42:10385-98
Jia, Wenwen; Wang, Shuwen; Horner, James W et al. (2011) A BAC transgenic reporter recapitulates in vivo regulation of human telomerase reverse transcriptase in development and tumorigenesis. FASEB J 25:979-89
Wang, Shuwen; Hu, Chunguang; Zhu, Jiyue (2010) Distinct and temporal roles of nucleosomal remodeling and histone deacetylation in the repression of the hTERT gene. Mol Biol Cell 21:821-32
Mathew, Renjith; Jia, Wenwen; Sharma, Arati et al. (2010) Robust activation of the human but not mouse telomerase gene during the induction of pluripotency. FASEB J 24:2702-15
Wang, Shuwen; Zhao, Yuanjun; Leiby, Melanie A et al. (2009) Studying human telomerase gene transcription by a chromatinized reporter generated by recombinase-mediated targeting of a bacterial artificial chromosome. Nucleic Acids Res 37:e111
Wang, Shuwen; Zhao, Yuanjun; Hu, Chunguang et al. (2009) Differential repression of human and mouse TERT genes during cell differentiation. Nucleic Acids Res 37:2618-29
Wang, Shuwen; Zhao, Yuanjun; Leiby, Melanie et al. (2009) A new positive/negative selection scheme for precise BAC recombineering. Mol Biotechnol 42:110-6
Zhao, Yuanjun; Wang, Shuwen; Popova, Evgenya Y et al. (2009) Rearrangement of upstream sequences of the hTERT gene during cellular immortalization. Genes Chromosomes Cancer 48:963-74
Wang, Shuwen; Hu, Chunguang; Zhu, Jiyue (2007) Transcriptional silencing of a novel hTERT reporter locus during in vitro differentiation of mouse embryonic stem cells. Mol Biol Cell 18:669-77
Wang, Shuwen; Zhu, Jiyue (2004) The hTERT gene is embedded in a nuclease-resistant chromatin domain. J Biol Chem 279:55401-10