Telomerase reverse transcriptase promoter regulation during myogenic differentiation of human RD rhabdomyosarcoma cells.
During terminal differentiation of human and murine cells, telomerase activity and parallel transcription of telomerase reverse transcriptase (hTERT) are inhibited. In this study, we used in vitro and in vivo analyses to determine the role of hTERT promoter elements and associated factors during differentiation-induced inhibition of telomerase expression in RD, a human rhabdomyosarcoma cell line. Assay of telomerase enzyme activity, hTERT mRNA, and reporter gene assays confirmed that the hTERT promoter was silenced during 12-O-tetradecanoylphorbol-13-acetate-induced myogenic differentiation of telomerase-positive RD cells. Promoter deletion and mutation analyses revealed that two E-boxes and an AP-2 site present in a 320-bp region of the promoter were essential for the transcriptional activity of the hTERT gene. Electrophoretic mobility shift assays identified several factors that interact with this region of DNA, including the muscle-specific transcription factors Myf5, Myf6, and myogenin and the ubiquitously expressed factors Sp1 and AP-2. Ectopic expression of the E-box binding factors c-Myc and Mad did influence promoter activity in these cells; indeed, the presence of endogenous c-Myc protein was altered after differentiation. Our findings suggest that the acute regulation of hTERT transcription is primarily controlled by E-box elements, which bind a series of factors during the phased phenotypic changes occurring during the differentiation of RD human muscle cells.
Ma
Hongwen
H
UCSD Cancer Center, University of California, San Diego, La Jolla, CA 92093-0064, USA.
Urquidi
Virginia
V
Wong
Jeremy
J
Kleeman
Jeanine
J
Goodison
Steve
S
D001665:Binding Sites; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024721:E-Box Elements; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051741:Kruppel-Like Transcription Factors; D032342:Muscle Cells / Q000166:cytology*; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D012208:Rhabdomyosarcoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; C436492:KLF12 protein, human; D051741:Kruppel-Like Transcription Factors; D016271:Proto-Oncogene Proteins c-myc; D014157:Transcription Factors; D019098:Telomerase; D013755:Tetradecanoylphorbol Acetate