MDM2 negatively regulates the human telomerase RNA gene promoter.
BACKGROUND
We have previously demonstrated that NF-Y and Sp1 interact with the human telomerase RNA (hTR) promoter and play a central role in its regulation. We have also shown that pRB activates the hTR promoter, but the mechanism of pRb directed activation is unknown. It has recently been reported that pRB induces Sp1 activity by relieving inhibition mediated by mdm2. The aim was to investigate possible roles for mdm2 in hTR promoter regulation.


METHODS
Chromatin immunoprecipitation was used to determine binding of mdm2 to the hTR promoter. Transfection and luciferase assays were used to investigate mdm2 repression of the promoter activity and interaction with known transcriptional modulators.


RESULTS
Here we show using chromatin immunoprecipitation that mdm2 specifically binds the hTR promoter in vivo. Transient co-transfection experiments using an hTR promoter luciferase reporter construct show that hTR promoter activity is inhibited by over-expression of mdm2 in 5637 bladder carcinoma cells (p53 and pRB negative, low mdm2). Titration of mdm2 was able to antagonise activation of hTR promoter activity mediated by pRB or Sp1 over-expression, although in the presence of pRB, mdm2 could not repress promoter activity below basal levels. Using an Sp1 binding site mutation construct we showed that mdm2 repression did not absolutely require Sp1 binding sites in the hTR promoter, suggesting the possibility of pRB/Sp1 independent mechanisms of repression. Finally, we show that NF-Y mediated transactivation of the hTR promoter was also suppressed by mdm2 in a dose-dependent manner.


CONCLUSIONS
These studies suggest that mdm2 may inhibit the hTR promoter by multiple mechanisms. Mdm2 may directly repress activation by both pRB and Sp1, or activation by NF-Y. Furthermore, the ability of mdm2 to interact and interfere with components of the general transcription machinery might partly explain the general repressive effect seen here. Elucidation of new regulators affecting hTR basal promoter activity in cancer cells provides a basis for future studies aimed at improving our understanding of the differential hTR expression between normal and cancer cells.
Zhao
Jiangqin
J
Department of Cell Physiology and Pharmacology, Faculty of Medicine and Biological Sciences, University of Leicester, Leicester, LE1 9HN, UK. jz27@le.ac.uk <jz27@le.ac.uk>
Bilsland
Alan
A
Jackson
Katrina
K
Keith
W Nicol
WN
D023081:CCAAT-Binding Factor / Q000378:metabolism; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation / Q000379:methods; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D016160:Retinoblastoma Protein / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D023081:CCAAT-Binding Factor; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein; D016329:Sp1 Transcription Factor; C422952:telomerase RNA; D012313:RNA; D008156:Luciferases; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D019098:Telomerase