Studying human telomerase gene transcription by a chromatinized reporter generated by recombinase-mediated targeting of a bacterial artificial chromosome.
The endogenous human telomerase reverse transcriptase (hTERT) gene is repressed in somatic cells. To study the mechanisms of its repression, we developed a strategy of retrovirus-directed Cre recombinase-mediated BAC targeting, or RMBT, to generate single-copy integrations of BAC at pre-engineered chromosomal sites. This technique involved retroviral transduction of acceptor loci, containing an HSV thymidine kinase marker, and subsequent integration of BAC constructs into the acceptor sites, utilizing the loxP and lox511 sites present in the vector backbones. The BAC reporter, with a Renilla luciferase cassette inserted downstream of the hTERT promoter, was retrofitted with a puromycin marker. Through puromycin selection and ganciclovir counter-selection, a targeting efficiency of over 50% was achieved. We demonstrated that the activity and chromatin structures of the hTERT promoter in chromosomally integrated BAC reporter recapitulated its endogenous counterpart of the host cells. Therefore, we have established a genetically amendable platform to study chromatin and epigenetic regulation of the hTERT gene. The highly efficient and versatile RMBT technique has general applicability for studying largely unexplored chromatin-dependent mechanisms of promoter regulation of various genes.
Wang
Shuwen
S
Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
Zhao
Yuanjun
Y
Leiby
Melanie A
MA
Zhu
Jiyue
J
D002460:Cell Line; D002843:Chromatin / Q000737:chemistry; D022202:Chromosomes, Artificial, Bacterial; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors; D015971:Gene Expression Regulation, Enzymologic; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D006801:Humans; D019426:Integrases / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012190:Retroviridae / Q000235:genetics; D019098:Telomerase / Q000235:genetics*; D014158:Transcription, Genetic
D002843:Chromatin; D004248:DNA (Cytosine-5-)-Methyltransferases; C045073:Cre recombinase; D019426:Integrases; C509186:TERT protein, human; D019098:Telomerase