Purification of Transcription Factors Using Promoter Trapping

With the growing number of cancer sufferers, it’s necessary to understand all the mechanisms that contribute to cell immortality. Human telomerase reverse transcriptase (hTERT) is an enzyme that adds telomeres to the ends of chromatids and high hTERT activity found in most cancer cells. Understanding the regulation of hTERT may give insight on how to control activity. A novel method is employed for the purification of hTERT transcriptional complexes.

 

Nuclear extract and duplexed DNA containing a single stranded (GT)₅ tail are incubated to allow hTERT complex assembly. The complex is bound to a (CA)₅-Sepharose column capturing the promoter complex by annealing the GT tail to the column. The column is then washed with a low salt buffer and the promoter complex eluted off the column with a high salt buffer. Eluate was subjected to two-dimensional gel electrophoresis.

Characterization and identification will be accomplished with the use of mass spectrometry, Southwestern blots, and Western blots. As of now, positive results have been achieved using Western blots with the identification of some general and specific transcription factors. To increase our confidence the spots will also be excised for de novo sequencing.

 

The c-Jun promoter has been successfully characterized using promoter trapping chromatography (Jiang, 2006). Transcription factors were identified by immunoblotting and have shown to have the appropriate elements to successfully transcribe the c-Jun transcript. These previous results give high confidence that hTERT will be fully characterized by a similar approach. This will allow in uncovering new regulatory factors.