The Formation and Stabilization of a Novel G-Quadruplex in the 5′-Flanking Region of the Relaxin Gene

et al. (2012) The Formation and Stabilization of a Novel G-Quadruplex in the 59-Flanking Region of the Relaxin Gene. PLoS ONE 7(2): e31201. doi:10.1371/journal.pone.0031201 The Formation and Stabilization of a Novel G-Quadruplex in the 59-Flanking Region of the Relaxin Gene Sen Lin 0 1 Huiping Gu 0 1 Ming Xu 0 1 Xiaojie Cui 0 1 Youyi Zhang 0 1 Wei Gao 0 1 Gu Yuan 0 1 Mark Isalan, Center for Genomic Regulation, Spain 0 Ethics Statement The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The experi- ments were approved by the Committee on Ethics of Animal Experiments and were conducted in accordance with the Guidelines for Animal Experiments, Peking University Health Science Center 1 1 Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University , Beijing , China , 2 Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Ministry of Health, Key Laboratory of Molecular Cardiovascular Sciences of Ministry of Education, Institute of Vascular Medicine, Third Hospital, Peking University , Beijing , China It has been reported that binding of STAT3 protein to the 59-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 Gquadruplex 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 Gquadruplex was stabilized by berberine(DTm = 10uC), 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 Gquadruplex 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. - Funding: This work was supported by the National Natural Science Foundation of China (no. 90913004 and 81070196) and Program for New Century Excellent Talents in University and Beijing Talents Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Relaxin (RLX), a polypeptide hormone, was first discovered in 1926 by Frederick Hisaw [1]. The main functions of relaxin are associated with female reproductive physiology and in particular connective tissue remodeling in the interpubic symphysis and uterine cervix to facilitate birth [1,2]. In addition to its reproductive associated functions, recent studies revealed diverse actions of relaxin on non-reproductive tissues. Relaxin has been reported to reduce fibrosis in the kidney, heart, lung, and liver [3]. Relaxin could regulate fibroblast proliferation, differentiation, and collagen deposition. Relaxin deficient mice had significantly high left ventricular end-diastolic pressures and age-dependent increasing left ventricular collagen content [4]. Clinical studies also showed increased RLX concentrations positively correlated with severity of congestive heart failure [5]. Relaxin is subjected to many transcriptional factors regulation. Among these factors, STAT3 was considered as the most important one. When STAT3 binds in the 59UTR of the relaxin gene, it negatively regulates the relaxin expression [6]. Gquadruplex nucleic acids have been reported to be involved in gene expression [7,8], telomere maintenance [9,10,11,12] and replication stalling [13]. There is a G-rich segment adjacent to the binding site in the 59-flanking region of the RLX gene (In Figure 1). This G-rich segment has four consecutive G-tracts which have the potential to form a G-quadruplex structure. Our research found that berberine could stabilize the G-quadruplex resulting in upregulating the expression of the relaxin gene. The elevation of promoter activity might be attributed to the formation and stabilization of the RLX G-quadruplex near STAT3s binding site which could interfere this suppressors binding to the relaxin gene. The report of this novel discovery will fertilize our knowledge about G-quadruplex functions in cells and provide new clues to develop the G-quadruplex as a target for the therapeutic intervention of relaxin-related diseases. Materials and Methods Oligonucleotides and Natural Product The oligonucleotides were purchased from Sangon (Shanghai, China). Berberine was purchased from National Institute for the Control of Pharmaceutical and Biological Products (China). Mass Spectrometry ESI mass spectra were obtained in the negative-ion mode with a Finnigan LCQ Deca XP Plus ion-trap mass spectrometer (San Jose, CA). The direct-infusion flow rate was 2.0 mL/min. The electrospray-source conditions were 2.7 kV spray voltage and 120uC capillary temperature. Circular Dichroism The CD spectra of DNA were measured by using a J-810 spectropolarimeter (JASCO Co., Ltd., Japan) with a 0.1 cm pathlength quartz cell at 25uC. All the samples were annealed at 90uC for 10 min and then cooled to room temperature overnight. Thermal Denaturation Melting curves were obtained by monitoring the changing at a wavelength 264 nm which has the highest CD value. The CD spectra were measured by using a J-810 spectropolarimeter (JASCO Co., Ltd., Japan) with a 0.1 cm path-length quartz cell. The procedures to analyze the thermodynamic parameters were included in Text S1. NMR Titrations The strand concentration of NMR samples was typically 0.81 1.31 mM; the solutions contained potassium phosphate (pH 7.0) as buffer, as well as 10% D2O. 5 mM Berberine solution was titrated into the sample solution to achieve different molar ratios. All experiments were performed on 600 MHz Bruker spectrometers at 298 K. Molecular Modeling The solution structure of quadruplex in human c-MYC promoter (PDB code 1xav) [14] was used as an initial model and then treated by some necessary replacement and deletion to construct the RLX G-quadruplex structure. Each system was neutralized by Na+ and solvated with a truncated octahedral box containing an 8 angstrom buffer of TIP3P water. Energy minimization was performed in these explicit solvents using the sander module of AMBER 10 [15,16]. First, the system of Gquadruplex soaked in water was minimized for 1000 steps with holding the DNA (...truncated)