Defining the Role of DNA Secondary Structures and Transcriptional Factors in the Control of c-myc and bcl-2 Expression

Abstract

In this dissertation, we explore the transcriptional regulatory roles of Gquadruplex- forming motifs and the involvement of specific transcriptional factors, which interact with the same elements, in the control of human c-myc and bcl-2 gene expression. The G-quadruplex structures within the NHE III1 region of the c-myc promoter and their ability to repress transcription has been well established. However, a longstanding unanswered question is how these stable DNA secondary structures are transformed to activate c-myc transcription. NDPK-B has been recognized as an activator of c-myc transcription via interactions with NHE III1 region of the c-myc gene promoter. Through the use of RNAi, we confirmed the transcriptional regulatory role of NDPK-B. We demonstrate that NDPK-B has DNA binding activity and the nuclease activity results from a contaminating protein. NDPK-B preferentially binds to the singlestranded guanine-rich strand of the c-myc NHE III₁. Potassium ions and G-quadruplexinteractive agents, which stabilize G-quadruplex structures, had an inhibitory effect on NDPK-B DNA binding activity. Based on our studies, we have proposed a stepwise trapping-out of the NHE III1 region in a single-stranded form, thus allowing singlestranded transcription factors to bind and activate c-myc transcription. This model provides a rationale for how the stabilization of G-quadruplexes within the c-myc gene promoter region can inhibit NDPK-B from activating c-myc transcription. Similarly, the human bcl-2 gene contains a GC-rich region within its promoter region, which is critical in the regulation of bcl-2 expression. We demonstrate that the guanine rich strand within this region can form three intramolecular G-quadruplex structures. Based on NMR studies, the central G-quadruplex forms a mixed parallel/antiparallel structure with three tetrads connected by loops of one, seven, and three bases. The Gquadruplex structures in the bcl-2 promoter extends beyond the ability to form any one of three separate G-quadruplexes to each having the capacity to form either three or six different loop isomers. This suggests that targeting these individual structures could lead to different biological outcomes. We also found that Telomestatin upregulates bcl-2 gene expression, which we propose is a result of inhibiting the binding of the WT1 repressor protein by the formation of a drug-stabilized G-quadruplex structure.