Abstract
BackgroundWe 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.MethodsChromatin 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.ResultsHere 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.ConclusionsThese 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.
Highlights
We have previously demonstrated that NF-Y and Sp1 interact with the human telomerase RNA promoter and play a central role in its regulation
The activity of the human telomerase RNA (hTR) promoter is controlled by NF-Y, Sp1 and Sp3 in bladder cancer cells in vitro and we have recently shown that an Sp1 site mutation in the hTR promoter detected in a blood sample taken from a paroxysmal nocturnal haemoglobinuria (PNH) patient can alter core promoter activity in vitro, raising the possibility that mutation might affect hTR gene transcription in hematopoietic cells in vivo [12,13,14]
We investigated regulation of hTR reporter constructs by Sp1, pRb, NF-Y and mdm2 and performed chromatin immunoprecipitation (ChIP) assays to determine whether mdm2 plays a role in hTR regulation in the p53 and pRb negative bladder cancer cell line 5637 which expresses relatively low levels of mdm2 [16]
Summary
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. Several groups have recently reported transcriptionally targeted cancer gene therapy strategies based on the differential activities of hTR and hTERT promoters between normal and cancer cells [7,8,9,10,11]. The activity of the hTR promoter is controlled by NF-Y, Sp1 and Sp3 in bladder cancer cells in vitro and we have recently shown that an Sp1 site mutation in the hTR promoter detected in a blood sample taken from a paroxysmal nocturnal haemoglobinuria (PNH) patient can alter core promoter activity in vitro, raising the possibility that mutation might affect hTR gene transcription in hematopoietic cells in vivo [12,13,14]. Several other known transcriptional regulators, including the retinoblastoma protein pRB, are able to affect hTR transcription in the experimental setting of over-expression [12,13]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
