It has been reported that binding of STAT3 protein to the 5′-flanking region of the relaxin gene may result in downregulation of the relaxin expression. There is a Guanine(G)-rich segment located in about 3.8 Kb upstream of the relaxin gene and very close to the STAT3's binding site. In our study, NMR spectroscopy revealed the formation of G-quadruplex by this G-rich strand, and the result was confirmed by ESI mass spectrometry and CD spectroscopy. The theoretical structure of RLX G-quadruplex was constructed and refined by molecular modeling. When this relaxin G-quadruplex was stabilized by berberine(ΔTm = 10°C), a natural alkaloid from a Chinese herb, the gene expression could be up-regulated in a dose-dependent manner which was proved by luciferase assay. This result is different from the general G-quadruplex function that inhibiting the telomere replication or down-regulating many oncogenes expression. Therefore, our study reported a novel G-quadruplex in the relaxin gene and complemented the regulation mechanism about gene expression by G-quadruplexes.
References
[1]
Sherwood OD, editor. (1994) The physiology of reproduction. New York: Raven Press. pp. 861–1009.
[2]
Bryantgreenwood GD, Schwabe C (1994) Human Relaxins - Chemistry and Biology. Endocrine Reviews 15: 5–26.
[3]
Negri AL (2004) Prevention of progressive fibrosis in chronic renal diseases: Antifibrotic agents. Journal of Nephrology 17: 496–503.
[4]
Du XJ, Samuel CS, Gao XM, Zhao L, Parry LJ, et al. (2003) Increased myocardial collagen and ventricular diastolic dysfunction in relaxin deficient mice: a gender-specific phenotype. Cardiovascular Research 57: 395–404.
[5]
Dschietzig T, Richter C, Bartsch C, Laule M, Armbruster FP, et al. (2001) The pregnancy hormone relaxin is a player in human heart failure. Faseb Journal 15: 2187–2195.
[6]
Soloff MS, Gal S, Hoare S, Peters CA, Hunzicker-Dunn M, et al. (2003) Cloning, characterization, and expression of the rat relaxin gene. Gene 323: 149–155.
[7]
Grand CL, Bearss DJ, Von Hoff DD, Hurley LH (2002) Quadruplex formation in the c-MYC promoter inhibits protein binding and correlates with in vivo promoter activity. European Journal of Cancer 38: S106–S107.
[8]
Han HY, Hurley LH (2000) G-quadruplex DNA: a potential target for anti-cancer drug design. Trends in Pharmacological Sciences 21: 136–142.
[9]
Muller S, Kumari S, Rodriguez R, Balasubramanian S (2011) Small-molecule-mediated G-quadruplex isolation from human cells. Nature Chemistry 2: 1095–1098.
[10]
Smith FW, Feigon J (1992) Quadruplex Structure of Oxytricha Telomeric DNA Oligonucleotides. Nature 356: 164–168.
[11]
Mergny JL, Helene C (1998) G-quadruplex DNA: A target for drug design. Nature Medicine 4: 1366–1367.
[12]
Neidle S, Parkinson G (2002) Telomere maintenance as a target for anticancer drug discovery. Nature Reviews Drug Discovery 1: 383–393.
[13]
Rizzo A, Salvati E, Porru M, D'Angelo C, Stevens MF, et al. (2009) Stabilization of quadruplex DNA perturbs telomere replication leading to the activation of an ATR-dependent ATM signaling pathway. Nucleic Acids Research 37: 5353–5364.
[14]
Ambrus A, Chen D, Dai JX, Jones RA, Yang DZ (2005) Solution structure of the biologically relevant g-quadruplex element in the human c-MYC promoter. implications for g-quadruplex stabilization. Biochemistry 44: 2048–2058.
[15]
Cheatham TE, Young MA (2001) Molecular dynamics simulation of nucleic acids: Successes, limitations, and promise. Biopolymers 56: 232–256.
[16]
Case DA, Cheatham TE, Darden T, Gohlke H, Luo R, et al. (2005) The Amber biomolecular simulation programs. Journal of Computational Chemistry 26: 1668–1688.
[17]
Goodsell DS, Olson AJ (1990) Automated Docking of Substrates to Proteins by Simulated Annealing. Proteins-Structure Function and Genetics 8: 195–202.
[18]
Morris GM, Goodsell DS, Huey R, Olson AJ (1996) Distributed automated docking of flexible ligands to proteins: Parallel applications of AutoDock 2.4. Journal of Computer-Aided Molecular Design 10: 293–304.
[19]
Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, et al. (1998) Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. Journal of Computational Chemistry 19: 1639–1662.
[20]
Xu M, Liu XH, Gai YL, Liu F, Gao W, et al. (2010) Trimetazidine inhibits pressure overload-induced cardiac fibrosis through NADPH oxidase-ROS-CTGF pathway. Cardiovascular Research 88: 150–158.
[21]
Zhang YY, Liao WQ, Wang SY, Han CD (2005) 14-3-3 Proteins regulate glycogen synthase 3 beta phosphorylation and inhibit cardiomyocyte hypertrophy. Febs Journal 272: 1845–1854.
[22]
Cosconati S, Marinelli L, Trotta R, Virno A, Mayol L, et al. (2009) Tandem Application of Virtual Screening and NMR Experiments in the Discovery of Brand New DNA Quadruplex Groove Binders. Journal of the American Chemical Society 131: 16336-+.
[23]
da Silva MW (2007) NMR methods for studying quadruplex nucleic acids. Methods 43: 264–277.
[24]
Yang DZ, Hurley LH (2006) Structure of the biologically relevant G-quadruplex in the c-MYC promoter. Nucleosides Nucleotides & Nucleic Acids 25: 951–968.
[25]
Hounsou C, Guittat L, Monchaud D, Jourdan M, Saettel N, et al. (2007) G-quadruplex recognition by quinacridines: a SAR, NMR, and biological study. Chemmedchem 2: 655–666.
[26]
Dai JX, Punchihewa C, Ambrus A, Chen D, Jones RA, et al. (2007) Structure of the intramolecular human telomeric G-quadruplex in potassium solution: a novel adenine triple formation. Nucleic Acids Research 35: 2440–2450.
[27]
Phan AT, Kuryavyi V, Gaw HY, Patel DJ (2005) Small-molecule interaction with a five-guanine-tract G-quadruplex structure from the human MYC promoter. Nature Chemical Biology 1: 167–173.
[28]
Kypr J, Kejnovska I, Renciuk D, Vorlickova M (2009) Circular dichroism and conformational polymorphism of DNA. Nucleic Acids Research 37: 1713–1725.
[29]
Paramasivan S, Rujan I, Bolton PH (2007) Circular dichroism of quadruplex DNAs: Applications to structure, cation effects and ligand binding. Methods 43: 324–331.
[30]
Zhou J, Yuan G, Liu JJ, Zhan CG (2007) Formation and stability of G-quadruplexes self-assembled from guanine-rich strands. Chemistry-a European Journal 13: 945–949.
[31]
Li HH, Liu YQ, Lin S, Yuan G (2009) Spectroscopy Probing of the Formation, Recognition, and Conversion of a G-Quadruplex in the Promoter Region of the bcl-2 Oncogene. Chemistry-a European Journal 15: 2445–2452.
[32]
Ma Y, Ou TM, Tan JH, Hou JQ, Huang SL, et al. (2009) Synthesis and evaluation of 9-O-substituted berberine derivatives containing aza-aromatic terminal group as highly selective telomeric G-quadruplex stabilizing ligands. Bioorganic & Medicinal Chemistry Letters 19: 3414–3417.
[33]
Arora A, Balasubramanian C, Kumar N, Agrawal S, Ojha RP, et al. (2008) Binding of berberine to human telomeric quadruplex - spectroscopic, calorimetric and molecular modeling studies. Febs Journal 275: 3971–3983.
[34]
Zhang WJ, Ou TM, Lu YJ, Huang YY, Wu WB, et al. (2007) 9-substituted berberine derivatives as G-quadruplex stabilizing ligands in telomeric DNA. Bioorganic & Medicinal Chemistry 15: 5493–5501.
[35]
Franceschin M, Rossetti L, D'Ambrosio A, Schirripa S, Bianco A, et al. (2006) Natural and synthetic G-quadruplex interactive berberine derivatives. Bioorganic & Medicinal Chemistry Letters 16: 1707–1711.
[36]
Ranjan N, Andreasen KF, Kumar S, Hyde-Volpe D, Arya DP (2010) Aminoglycoside Binding to Oxytricha nova Telomeric DNA. Biochemistry 49: 9891–9903.
[37]
Mergny JL, Lacroix L (2003) Analysis of thermal melting curves. Oligonucleotides 13: 515–537.
[38]
Bishop GR, Ren JS, Polander BC, Jeanfreau BD, Trent JO, et al. (2007) Energetic basis of molecular recognition in a DNA aptamer. Biophysical Chemistry 126: 165–175.
[39]
Phan AT (2010) Human telomeric G-quadruplex: structures of DNA and RNA sequences. Febs Journal 277: 1107–1117.
[40]
Keniry MA (2000) Quadruplex structures in nucleic acids. Biopolymers 56: 123–146.
[41]
Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, et al. (2004) UCSF chimera - A visualization system for exploratory research and analysis. Journal of Computational Chemistry 25: 1605–1612.
[42]
Sanner MF (1999) Python: A programming language for software integration and development. Journal of Molecular Graphics & Modelling 17: 57–61.
[43]
Guo K, Gokhale V, Hurley LH, Sun D (2008) Intramolecularly folded G-quadruplex and i-motif structures in the proximal promoter of the vascular endothelial growth factor gene. Nucleic Acids Research 36: 4598–4608.
[44]
Kumari S, Bugaut A, Huppert JL, Balasubramanian S (2007) An RNA G-quadruplex in the 5′ UTR of the NRAS proto-oncogene modulates translation. Nature Chemical Biology 3: 218–221.
[45]
Beaudoin JD, Perreault JP (2010) 5′-UTR G-quadruplex structures acting as translational repressors. Nucleic Acids Research 38: 7022–7036.
