Inhibitory role of transcription factor COUP-TFII in expression of hTERT in HeLa cells.
OBJECTIVE
To clone and identify the proteins involved in regulating the transcription of hTERT and study the role of genes in both hTERT transcription and telomerase activity.


METHODS
The full cDNA of COUP-TFII was cloned from HeLa cDNA library by hTERT promoter-based yeast one-hybrid assay and then in-frame inserted into His-tag fusion expression vector pEK318. The His-tag COUP-TFII fusion proteins were purified by Ni-NTA chromatography. The interaction of COUP-TFII with hTERT promoter in vitro was identified by electrophoretic mobility shift assay and Footprint. The role of COUP-TFII in both hTERT transcription and telomerase activity were probed through Luciferase reporter assay, Northern blot, and TRAP-PCR ELISA.


RESULTS
COUP-TFII could firmly bind to the downstream E-box and the other two binding sites in hTERT promoter. Luciferase reporter assay indicated COUP-TFII could suppress hTERT promoter activity and stable introduction of COUP-TFII into HeLa cells also decreased both endogenous hTERT transcription and telomerase activity.


CONCLUSIONS
The human COUP-TFII can firmly bind to hTERT promoter, and inhibit telomerase activity through decreasing hTERT transcription. It will greatly facilitate understanding of telomerase regulation in normal and cancer cells.
Wang
Qiang
Q
The Provincial Key Laboratory of Mechanisms of Development and Gene Control, Life Science School of Shandong University, Jinan 250100.
Bai
Zeng-liang
ZL
Xuan
Li
L
Hou
Lin
L
Zhang
Bo
B
D051837:COUP Transcription Factor II; D051836:COUP Transcription Factors; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000494:pharmacology*; D024721:E-Box Elements / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011987:Receptors, Steroid / Q000235:genetics; D019098:Telomerase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000494:pharmacology*; D014158:Transcription, Genetic; D015003:Yeasts / Q000235:genetics
D051837:COUP Transcription Factor II; D051836:COUP Transcription Factors; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D011987:Receptors, Steroid; D019098:Telomerase; D014157:Transcription Factors; C095569:NR2F2 protein, human