Promoter trapping method: transcription factor purification using human telomerase reverse transcriptase promoter.

BACKGROUND

Transcription factors bind to response elements on the promoter regions of genes to regulate transcriptional activity. One of the major problems with identifying transcription factors is their low abundance relative to other proteins in the cell. Developing a purification technique specific for transcription factors is crucial to the understanding of gene regulation. Promoter trapping is a method developed that uses the promoter regions as bait to trap proteins of interest and then purified using column chromatography. Here we utilize this technique to study the telomerase promoter, which has increased transcriptional activity in cancer cells. Gaining insight on how to control the enzyme at the promoter level may give new routes towards cancer treatments.

RESULTS

Our findings show that the telomerase promoter (-170 - +91) and Promoter Trapping isolate a transcriptionally active and reproducible complex, when analyzed by liquid chromatography tandem mass spectrometry. We were also able to identify transcription factors, including AP-2 and SP1 known to bind this promoter, as well as show that these two proteins can bind to each other's response element.

CONCLUSION

Here we focus on verifying the ability and versatility of Promoter Trapping coupled with additional well-characterized methods to identify already known factors responsible for telomerase transcriptional regulation.