Purification and identification of a transcription factor, USF‐2, binding to E‐box element in the promoter of human telomerase reverse transcriptase (hTERT)

Controversy remains about the identity of the transcription factor(s) (TFs), which bind to the two E‐box elements (CACGTG, proximal and distal) of the human telomerase (hTERT) gene promoter, the essential elements in the regulation of telomerase. Here, systematic oligonucleotide trapping supplemented with 2‐DE and proteomic methods was used to identify E‐box binding TFs. Although insufficient purity was obtained from the proximal E‐box element trapping, further fractionation provided by 2‐DE and specific identification from Southwestern blotting analysis allow us to clearly identify an E‐box binding TF. The protein spot was cut from 2‐DE and in‐gel digested with trypsin for LC‐nanospray ESI‐MS/MS analysis. This identified upstream stimulatory factor 2 (USF2). Western blotting analysis with specific antibodies clearly shows USF2 present in the purified fraction and USF2 antibody supershifts the specific DNA‐binding complex on non‐denaturing gels. Furthermore, a novel method was developed in which the specific DNA‐TF complex was separated on a non‐denaturing gel, the band was cut and applied to SDS‐PAGE for a second dimension. Western blots of this second gel also confirmed the presence of USF2.