Purification and identification of a transcription factor, USF-2, binding to E-box element in the promoter of human...

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.