The nuclease hypersensitivity element III1 (NHE III1) upstream of the P1 and P2 promoters of c-MYC controls 80-90% of the transcriptional activity of this gene. The purine-rich strand in this region can form a G-quadruplex structure that is a critical part of the silencer element for this promoter. We have demonstrated that this G-quadruplex structure can form a mixture of four biologically relevant parallel-loop isomers, which upon interaction with the cationic porphyrin TMPyP4 are converted to mixed parallel/antiparallel G-quadruplex structures.

A polypurine (guanine)/polypyrimidine (cytosine)-rich sequence within the proximal promoter region of the human RET oncogene has been shown to be essential for RET basal transcription. Specifically, the G-rich strand within this region consists of five consecutive runs of guanines, which is consistent with the general motif capable of forming intramolecular G-quadruplexes. Here we demonstrate that, in the presence of 100 mM K+, this G-rich strand has the ability to adopt two intramolecular G-quadruplex structures in vitro. Moreover, comparative circular dichroism (CD) and DMS footprinting studies have revealed that the 3'-G-quadruplex structure is a parallel-type intramolecular structure containing three G-tetrads. The G-quadruplex-interactive agents TMPyP4 and telomestatin further stabilize this G-quadruplex structure. In addition, we demonstrate that the complementary C-rich strand forms an i-motif structure in vitro, as shown by CD spectroscopy and chemical footprinting. This 19-mer duplex sequence is predicted to form stable intramolecular G-quadruplex and i-motif species having minimum symmetrical loop sizes of 1:3:1 and 2:3:2, respectively. Together, our results indicate that stable G-quadruplex and i-motif structures can form within the proximal promoter region of the human RET oncogene, suggesting that these secondary structures play an important role in transcriptional regulation of this gene.

It is generally accepted that DNA predominantly exists in duplex form in cells. However, under torsional stress imposed by active transcription, DNA can assume nonduplex structures. The BCL2 promoter region forms two different secondary DNA structures on opposite strands called the G-quadruplex and the i-motif. The i-motif is a highly dynamic structure that exists in equilibrium with a flexible hairpin species. Here we identify a pregnanol derivative and a class of piperidine derivatives that differentially modulate gene expression by stabilizing either the i-motif or the flexible hairpin species. Stabilization of the i-motif structure results in significant upregulation of the BCL2 gene and associated protein expression; in contrast, stabilization of the flexible hairpin species lowers BCL2 levels. The BCL2 levels reduced by the hairpin-binding compound led to chemosensitization to etoposide in both in vitro and in vivo models. Furthermore, we show antagonism between the two classes of compounds in solution and in cells. For the first time, our results demonstrate the principle of small molecule targeting of i-motif structures in vitro and in vivo to modulate gene expression.

Most transcription of the MYC proto-oncogene initiates in the near upstream promoter, within which lies the nuclease hypersensitive element (NHE) III 1 region containing the CT-element. This dynamic stretch of DNA can form at least three different topologies: single-stranded DNA, double-stranded DNA, or higher order secondary structures that silence transcription. In the current report, we identify the ellipticine analog GQC-05 (NSC338258) as a high affinity, potent, and selective stabilizer of the MYC G-quadruplex (G4). In cells, GQC-05 induced cytotoxicity with corresponding decreased MYC mRNA and altered protein binding to the NHE III 1 region, in agreement with a G4 stabilizing compound. We further describe a unique feature of the Burkitt's lymphoma cell line CA46 that allowed us to clearly demonstrate the mechanism and location of action of GQC-05 within this region of DNA and through the G4. Most importantly, these data present, as far as we are aware, the most direct evidence of intracellular G4-mediated control of a particular promoter.The MYC proto-oncogene is a key component of normal cell growth and differentiation, with roles in a multitude of cellular processes. Normally, this gene is subject to tight transcriptional regulation; however, aberrant MYC expression is a common feature in an estimated 80% of all human malignancies (1-3); it is estimated that one-seventh of cancer deaths in the United States are associated with alterations in the MYC gene or its expression (4). Deregulation can arise through a variety of mechanisms (5-13), but most often MYC is activated through alterations in cell signaling that lead to increased transcription (14).Deregulated MYC can lead to transformation (15), often as an early step in the process of multistage cancer development, and one on which all other mutations are based (16,17). Cancer cells appear to be addicted to a deregulated MYC (18), which can be the "Achilles heel", offering the potential for a therapeutic window (19,20). The ability to selectively and potently down-regulate MYC would have considerable potential for both efficacy and safety in a variety of tumor types.There are several upstream elements within the MYC promoter that can potentially undergo strand separation to form either single-stranded or other non-B-DNA structures (21), which play a critical role in transcriptional control of MYC: the distant Far Upstream Element acts as a cruise control element, Z-DNA found both in the far upstream and the promoter regions, and a GC-rich region within the proximal promoter that acts as an on/off switch (22-28). This near upstream core promoter region, which is responsible for the initiation of 80 -90% of MYC transcription (29), contains the GC-rich nuclease hypersensitive element (NHE) 2 III 1 to which doublestranded (Sp1) and single-stranded (CNBP and hnRNP k) transcriptional factors bind. Within the MYC gene's NHE III 1 , a 31-base pair element consisting of five repeats of the sequence (C/T)C(C/T)TCCCCA serves as the "on/off switch" for MYC trans...