Analysis of the Effects of BCL-2 Promoter G-Quadruplex Formation on Protein Expression

Abstract

Bcl-2 is an anti-apoptotic protein that has been implicated in a number of human diseases, including some cancers. Within the P1 promoter region, which is involved in 80-90% of bcl-2 transcriptional control, a G-rich region (Pu39) can form various monomeric G-quadruplex folding structures, each with four of the six runs of multiple guanines present. The midG4 and 5'5G4 structures have been found to be the most stable and are the potential targets of comparative studies on transcriptional effects. Plasmids containing a luciferase reporter gene under the control of this bcl-2 P1 promoter region, along with mutants to alter or eliminate G-quadruplex formation, were constructed in order to study the ability of Pu39 to control transcription. Initial luciferase assay results in HeLa and HEK293 cells suggest that mutants designed to isolate certain G-quadruplex structures caused an overall decrease in protein expression while mutants designed to knock out major folding structures caused an increase in protein expression. DNA mutant constructs in conjunction with quadruplex-specific, quadruplex-interacting drugs appear to consistently decrease protein activity, supporting the idea that the G-quadruplex has an inhibitory effect on transcription.