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Deconvoluting the Structural and Drug-Recognition Complexity of the G-Quadruplex-Forming Region Upstream of the bcl-2 P1 Promoter
文摘
The human bcl-2 gene contains a GC-rich region upstream of the P1 promoter that has beenshown to be critically involved in the regulation of bcl-2 gene expression. We have demonstrated that theguanine-rich strand of the DNA in this region can form any one of three distinct intramolecular G-quadruplexstructures. Mutation and deletion analysis permitted isolation and identification of three overlapping DNAsequences within this element that formed the three individual G-quadruplexes. Each of these wascharacterized using nondenaturing gel analysis, DMS footprinting, and circular dichroism. The centralG-quadruplex, which is the most stable, forms a mixed parallel/antiparallel structure consisting of threetetrads connected by loops of one, seven, and three bases. Three different G-quadruplex-interactive agentswere found to further stabilize these structures, with individual selectivity toward one or more of theseG-quadruplexes. Collectively, these results suggest that the multiple G-quadruplexes identified in thepromoter region of the bcl-2 gene are likely to play a similar role to the G-quadruplexes in the c-myc promoterin that their formation could serve to modulate gene transcription. Last, we demonstrate that the complexityof the G-quadruplexes in the bcl-2 promoter extends beyond the ability to form any one of three separateG-quadruplexes to each having the capacity to form either three or six different loop isomers. These resultsare discussed in relation to the biological significance of this G-quadruplex-forming element in modulationof bcl-2 gene expression and the inherent complexity of the system where different G-quadruplexes andloop isomers are possible.
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