Promoter G-quadruplexes and their Interactions with Ligands and Proteins

G-quadruplex secondary structures are four-stranded globular nucleic acid structures that form in specific DNA and RNA G-rich sequences with biological significance, such as those found in human telomeres, oncogene promoter regions, replication initiation sites, and 5’- and 3’-untranslated (UTR) regions, which have been identified as novel drug targets. The non-canonical G-quadruplex secondary structures readily form under physiologically relevant ionic conditions, and exhibit great diversity in their topologies and loop conformations depending on the DNA or RNA sequences at hand. The structural diversity of these unique secondary structures is essential to their specific recognition by different regulatory proteins or small molecule compounds. A significant amount of research has been done in this field that provides compelling evidence for the existence, biological significance, and potential druggability of G-quadruplexes. In this dissertation, I explore G-quadruplex formation in the promoters of BCL2, PDGFR-β and c-Myc oncogenes and their interactions with small molecule compounds or proteins. Firstly, I investigated a newly-identified G-quadruplex (P1G4) forming immediately upstream of the human BCL2 gene, which has been found to be overexpressed in several human tumors. In this research, I have found that P1G4 acts as a transcription repressor, and that its inhibitory effect can be enriched by the G-quadruplex-interactive compound, TMPyP4. Both P1G4 and the previously reported Pu39 G-quadruplexes form independently in adjacent regions within the BCL2 P1 promoter, but P1G4 appears to play a more dominant role in repressing transcriptional activity. NMR and CD studies have shown that the P1G4 G-quadruplex appears to comprise a novel dynamic equilibrium of two parallel structures, one regular, with two 1-nt loops and a 12-nt middle loop, and another broken-stranded, with three 1-nt loops and an 11-nt middle loop; both structures adopt a novel hairpin (stem-loop duplex) conformation in the long central loop. This dynamic equilibrium of two closely-related G-quadruplex structures with a unique hairpin loop conformation may provide a specific target for small molecules to modulate BCL2 gene transcription. I also explored the 3’ end G-quadruplex that forms within the core promoter of PDGFR-β, which has also been observed to be present at abnormal levels in a variety of clinical pathologies, including malignancies. The 3′-end G-quadruplex formed in the PDGFR-β promoter NHE appears to be selectively stabilized by an ellipticine analog, GSA1129, which can shift the dynamic equilibrium in the full-length sequence to favor the 3′-end G-quadruplex, and can repress PDGFR-β activity in cancer cell lines. NMR studies in combination with biophysical experiments have shown that in the wild-type extended 3ʼ-end NHE sequences, two novel intramolecular G-quadruplexes can be formed in a potassium solution, one with a 3’-flanking distant guanine inserted into the 3’-external tetrad (3’-insertion G-quadruplex), and another with a 5’-flanking distant guanine inserted into the 5’-external tetrad (5’-insertion G-quadruplex). Further investigation of the elongated PDGFR-β 3′-end sequence containing both the 5’- and 3’- flanking guanine sequences showed the formation of a combination of the two G-quadruplexes existing in equilibrium. Importantly, it was observed that GSA1129 can bind to and increase the stability of each of the end-insertion G-quadruplexes, raising their Tₘ by 25 degrees. This study highlights the dynamic nature of the 3′-end NHE sequence and the importance of identifying the proper sequence for the formation of biologically relevant G-quadruplex structures. Significantly, the dynamic nature of the 3′-end G-quadruplex suggests that it may be an attractive target for drug regulation. I then analyzed two proteins, Nucleolin and NM23-H2, which interact with the c-Myc G-quadruplex structure that forms in the proximal promoter region of the c-Myc gene; this is one of the most commonly deregulated genes in the human neoplasm. Nucleolin is known to be a transcriptional repressor for c-Myc, binding to and stabilizing the c-Myc G-quadruplex, whereas NM23-H2 is known to be a transcriptional activator that unwinds and destabilizes the c-Myc G-quadruplex. An investigation of the molecular mechanisms of the interaction between the c-Myc G-quadruplex and nucleolin showed that the minimal binding domains required for a tight binding of the protein to the c-Myc G-quadruplex are the four RNA binding domains (RBDs) of nucleolin, referred to as Nuc1234, and that the RGG domain is unnecessary for c-Myc G-quadruplex binding. The stable G-quadruplex formed within Pu27 using G-tract runs I, II, IV and V was determined to be the best substrate (Myc1245T) for nucleolin binding, showing the highest affinity. 3D NMR experiments performed on the free protein Nuc1234 and its complex with the Myc1245T G-quadruplex have shown that upon complex formation, only the disordered linker regions of the protein display significant chemical shift changes, whereas most other residues show chemical shift values similar to those of the free protein. The c-Myc G-quadruplex has three loops that flip outward in a solvent containing K⁺, according to its structure. The hypothesis for this association is that nucleolin wraps around the G-quadruplex and interacts specifically with the flipped-outward loop regions of the c-Myc G-quadruplex via its own inter-RBD linker regions, with little structural change in the RBDs themselves. A definitive determination of the 3D molecular structure of nucleolin and its complex with Myc1245T is currently in development. Biophysical and structural studies were then conducted to investigate the interactions of the protein NM23-H2/NDP kinase B with the c-Myc G-quadruplex. NM23-H2 binds to single-stranded guanine- and cytosine-rich sequences, but not to double-stranded DNA in the NHE III₁ region; the binding therefore appears structure-specific, rather than sequence-specific. Moreover, increasing concentrations of the strong G-quadruplex-interactive compound TMPyP4, a porphyrin-based drug, inhibits the binding of NM23-H2 to the NHE III₁ region; this suggests that the stabilization of the G-quadruplex hinders the recognition and remodeling function of the NM23-H2. By conducting Forster Resonance Energy Transfer (FRET) assays in combination with Circular Dichroism (CD) studies, I demonstrated that NM23-H2 can actively resolve the c-Myc G-quadruplex. Taken together, these results show that the use of small molecules to prevent NM23-H2 from binding to and resolving the NHE III₁ region G-quadruplex may have the potential to inhibit c-Myc transcription for cancer therapeutic purposes. This underlines the importance of understanding the mechanism of function operating between NM23-H2 and the c-Myc G-quadruplex. Understanding molecular mechanism between NM23-H2 and c-Myc is under investigation.Release after 26-Sep-201

Solution Structure of a MYC Promoter G‑Quadruplex with 1:6:1 Loop Length

The important MYC oncogene is deregulated in many cancer cells and comprises one of the most prominent G-quadruplex (G4) forming sequences in its promoter regions, the NHE III1 motif. Formation of G4s suppresses MYC transcription and can be modulated by drug binding, establishing these DNA structures as promising targets in cancer therapy. The NHE III1 motif can fold into more than one parallel G4s, including 1:2:1 and 1:6:1 loop length conformers, with the 1:2:1 conformer shown as the major species under physiological conditions in solution. However, additional factors such as protein interactions may affect the cellular folding equilibrium. Nucleolin, a protein shown to bind MYC G4 and repress MYC transcription, is reported herein to preferably bind to the 1:6:1 loop length conformer suggesting a physiological significance of this species. The high-resolution NMR solution structure of the 1:6:1 conformer is determined, which reveals a 5'-capping structure distinctive from the 1:2:1 form, with the 6 nt central loop playing an essential role for this specific capping structure. This suggests that each parallel G-quadruplex likely adopts unique capping and loop structures determined by the specific central loop and flanking sequences. The resulting structural information at the molecular level will help to understand protein recognition of different G4s, contribution of G4 polymorphism to gene regulation, and to rationally design small molecules selectively targeting the 1:6:1 MYC G4.National Institutes of Health [R01CA177585, P30CA023168]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

A New G‑Quadruplex with Hairpin Loop Immediately Upstream of the Human BCL2 P1 Promoter Modulates Transcription

The abnormal overexpression of the BCL2 gene is associated with many human tumors. We found a new 28-mer G-quadruplex-forming sequence, P1G4, immediately upstream of the human BCL2 gene P1 promoter. The P1G4 is shown to be a transcription repressor using a promoter-driven luciferase assay; its inhibitory effect can be markedly enhanced by the G-quadruplex-interactive compound TMPyP4. G-quadruplex can readily form in the P1G4 sequence under physiological salt condition as shown by DMS footprinting. P1G4 and previously identified Pu39 G-quadruplexes appear to form independently in adjacent regions in the BCL2 P1 promoter. In the extended BCL2 P1 promoter region containing both Pu39 and P1G4, P1G4 appears to play a more dominant role in repressing the transcriptional activity. Using NMR spectroscopy, the P1G4 G-quadruplex appears to be a novel dynamic equilibrium of two parallel structures, one regular with two 1-nt loops and a 12-nt middle loop and another broken-strand with three 1-nt loops and a 11-nt middle loop; both structures adopt a novel hairpin (stem–loop duplex) conformation in the long loop. The dynamic equilibrium of two closely related structures and the unique hairpin loop conformation are specific to the P1G4 sequence and distinguish the P1G4 quadruplex from other parallel structures. The presence of P1G4 and Pu39 in adjacent regions of the BCL2 P1 promoter suggests a mechanism for precise regulation of BCL2 gene transcription. The unique P1G4 G-quadruplex may provide a specific target for small molecules to modulate BCL2 gene transcription

The Consequences of Overlapping G‑Quadruplexes and i‑Motifs in the Platelet-Derived Growth Factor Receptor β Core Promoter Nuclease Hypersensitive Element Can Explain the Unexpected Effects of Mutations and Provide Opportunities for Selective Targeting of Both Structures by Small Molecules To Downregulate Gene Expression

The platelet-derived growth factor receptor β (PDGFR-β) signaling pathway is a validated and important target for the treatment of certain malignant and nonmalignant pathologies. We previously identified a G-quadruplex-forming nuclease hypersensitive element (NHE) in the human PDGFR-β promoter that putatively forms four overlapping G-quadruplexes. Therefore, we further investigated the structures and biological roles of the G-quadruplexes and i-motifs in the PDGFR-β NHE with the ultimate goal of demonstrating an alternate and effective strategy for molecularly targeting the PDGFR-β pathway. Significantly, we show that the primary G-quadruplex receptor for repression of PDGFR-β is the 3′-end G-quadruplex, which has a GGA sequence at the 3′-end. Mutation studies using luciferase reporter plasmids highlight a novel set of G-quadruplex point mutations, some of which seem to provide conflicting results on effects on gene expression, prompting further investigation into the effect of these mutations on the i-motif-forming strand. Herein we characterize the formation of an equilibrium between at least two different i-motifs from the cytosine-rich (C-rich) sequence of the PDGFR-β NHE. The apparently conflicting mutation results can be rationalized if we take into account the single base point mutation made in a critical cytosine run in the PDGFR-β NHE that dramatically affects the equilibrium of i-motifs formed from this sequence. We identified a group of ellipticines that targets the G-quadruplexes in the PDGFR-β promoter, and from this series of compounds, we selected the ellipticine analog GSA1129, which selectively targets the 3′-end G-quadruplex, to shift the dynamic equilibrium in the full-length sequence to favor this structure. We also identified a benzothiophene-2-carboxamide (NSC309874) as a PDGFR-β i-motif-interactive compound. In vitro, GSA1129 and NSC309874 downregulate PDGFR-β promoter activity and transcript in the neuroblastoma cell line SK-N-SH at subcytotoxic cell concentrations. GSA1129 also inhibits PDGFR-β-driven cell proliferation and migration. With an established preclinical murine model of acute lung injury, we demonstrate that GSA1129 attenuates endotoxin-mediated acute lung inflammation. Our studies underscore the importance of considering the effects of point mutations on structure formation from the G- and C-rich sequences and provide further evidence for the involvement of both strands and associated structures in the control of gene expression