The Identification of C0057684, an inducer of hTERT Expression and Telomerase Activity.

Beril Karakas, Chris Foster, Erica Gipson, Lancer K. Brown, Robert Blake, Rathnam Chaguturu, William H. Andrews, James Fleming, Jian Zhang, Dave Monda, Hamid Mohammadpour, Stephanie Fraser, Laura A. Briggs

Sierra Sciences, LLC, 250 S. Rock Blvd Suite 130, Reno, NV 89502

www.sierrasci.com

Abstract

The presence of telomerase activity in human cells is strongly related to the expression of the telomerase catalytic protein component, hTERT. Transient expression of hTERT in telomerase-negative normal cells reinstates telomerase activity and extends their life span (JW Shay, WE Wright, J. Pathol, 211, 114-123, 2007). The present study was designed to identify repressor sites within the hTERT minimal promoter and thus better our understanding of the regulation of the expression of the endogenous protein. We first made deletions of the minimal promoter to identify the minimum amount of DNA necessary for repression and activation of the hTERT minimal promoter. This region includes bases -107 to -37 relative to the "A" of the telomerase translation initiation codon (ATG). We called this region the "Minimized Minimal hTERT Promoter" (MMP). Fine mapping of this region by changing every base to every other base one base at a time provided us a map of regions within the MMP responsible for the promoter regulation. Analysis of this region in transcription factor databases has not exposed any likely transcription factor sites, except possibly a very weak resemblance to E2F or CDE/CHR consensus.

Figure 4. Transcriptional activity of single mutant hTERT minimal promoter. The x-axis represents the minimized minimal hTERT promoter sequence. The point mutations introduced to every base at a time are shown on the corner of each graph. SEAP activity from each plasmid was plotted and bars shown in red represent wild-type expression.

Methodology

SEAP reporter plasmids were constructed under the control of hTERT minimal promoter containing single or double site-specific mutations. The hTERT promoter was placed upstream of the SEAP reporter gene (Figure 2) and transiently transfected into human fetal lung fibroblast MRC5 cells using Lipofectamine 2000 (Invitrogen). Transfections with Basic (i.e no promoter) and SV40 (early promoter lacking enhancer) promoter were also carried out as negative and positive controls, respectively.

Figure 2.The map of the promoter assay plasmid.

In 96 well tissue culture plates, 1.5 x 104 MRC5 cells were seeded per well and exposed to lipid-mediated transfection media containing 0.25 mg of promoter assay plasmid. SEAP assays were performed 48 hr post transfection according to Clontech’s protocol. To normalize possible day to day variations and transfection efficiency, data were normalized relative to -65 C-->A mutation. Controls used in this experiment are shown in Figure 3.

Figure 3. Transfection efficiency with control assay plasmids.

Results and Conclusions

The transcriptional activity of the hTERT minimal promoter in human fibroblast MRC5 cells was studied by a transient expression assay. Expression of SEAP activity under the control of single mutant hTERT promoters were assayed to test the ability of each mutant to de-repress SEAP activity in transient transfections (Figure 4). This fine mapping revealed that mutations from -70 to -54 had the greatest effect on SEAP transcriptional activity. The significance of these sequences is presently unknown. However, if transient regulation of hTERT is similiar to endogenous promoter activity, then bases -70 to -54 could play a role in the regulation of the endogenous hTERT promoter. Analysis of the resulting data from Genomatix (www.Genomatix.de) very weakly supports E2F or CDE/CHR as a candidate repressor.

Introduction

Sierra Sciences has previously studied 118 deletions and hundreds of point mutations within the telomerase minimal promoter. Each deletion and mutation was constructed in triplicate and transiently transfected into MRC5 cells to test for reporter gene expression (Secreted Alkaline Phosphatase; SEAP). The deletion spanning -67 to -58 relative to the "A" of the telomerase translation initiation codon (ATG) has eliminated a significant repressor binding site resulting in de-repression of SEAP expression in MRC5 cells. We named this region "Site C". This analysis also exposed that one particular base change -65 C-->A promoted SEAP expression in MRC5 cells as much as the complete deletion of Site C. In our present study, we first made deletions of the telomerase minimal promoter from both ends to find a minimum amount of promoter necessary to give repression and activation of the telomerase promoter. This region called the "Minimized Minimal Promoter " extends from -107 to -37. This study represents a complete mutational analysis of this region in which every base of the minimized minimal promoter was converted one at a time to every other base. The region including -107 to -37 is shown in Figure 1. It is noted that this region does not include either E box/Myc sites that have been previously reported to affect hTERT promoter regulation (Takakura et al., Cancer Research, 59, 551-557, 1999 and numerous other publications).

Figure 1. A schematic representation of the hTERT minimal promoter.