Ionic modulation of QPX stability as a nano - switch regulating gene expression in neurons

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Ravari Soodeh Baghaee (Creator)
Institution
The University of North Carolina at Greensboro (UNCG )
Web Site: http://library.uncg.edu/
Advisor
Ethan Taylor

Abstract: G-quadruplexes (G-QPX) have been the subject of intense research due to their unique structural configuration and potential applications, particularly their functionality in biological process as a novel type of nano–switch. They have been found in critical regions of the human genome such as telomeres, promoter regions, and untranslated regions of RNA. About 50% of human DNA in promoters has G-rich regions with the potential to form G-QPX structures. A G-QPX might act mechanistically as an ON/OFF switch, regulating gene expression, meaning that the formation of G-QPX in a single strand of DNA disrupts double stranded DNA, prevents the binding of transcription factors (TF) to their recognition sites, resulting in gene down-regulation. Although there are numerous studies on biological roles of G-QPXs in oncogenes, their potential formation in neuronal cells, in particular upstream of transcription start sites, is poorly investigated. The main focus of this research is to identify stable G-QPXs in the 97bp active promoter region of the choline acetyltransferase (ChAT) gene, the terminal enzyme involved in synthesis of the neurotransmitter acetylcholine, and to clarify ionic modulation of G-QPX nanostructures through the mechanism of neural action potentials. Different bioinformatics analyses (in silico), including the QGRS, quadparser and G4-Calculator programs, have been used to predict stable G-QPX in the active promoter region of the human ChAT gene, located 1000bp upstream from the TATA box. The results of computational studies (using those three different algorithms) led to the identification of three consecutive intramolecular G-QPX structures in the negative strand (ChAT G17-2, ChAT G17, and ChAT G29) and one intramolecular G-QPX structure in the positive strand (ChAT G30). Also, the results suggest the possibility that nearby G-runs in opposed DNA strands with a short distance of each other may be able to form a stable intermolecular G-QPX involving two DNA complementary strands (ds ChAT G21). Formation of G-QPX structures, by blocking the availability of the transcription factor binding site (TFBS) on double stranded DNA, can interfere with transcriptional activation. This suggests that there is competition between TFBS binding to dsDNA and the conversion to high order non-B form secondary structures (G-QPXs) in the active promoter region. TFBS mapping analysis of the active promoter region of the human ChAT gene revealed that it contains multiple consensus AP-2a and Sp1 binding sites and consensus sites for other TF, including multiple sites for GR-alpha, Pax-5, p53 and GC box proteins. To get a better understanding of how modulation of G-QPX structures might affect the ChAT promoter activity, an artificial GFP reporter vector (modified GFP) was constructed, synthesized and used for reporter gene measurement. As known human ChAT promoter activators, nerve growth factors (HNGF and TGB) and cytokines (IL-ß and TNF-a) were used for activation of the artificial promoter driving GFP. Also, the G-QPX stabilizing drug TMPYP4 and aconitine, a Na+ channel opening drug, were used as G-QPX stability modulating factors. It was observed that aconitine potentiated the action of the transcriptional activator NGF, suggesting that the effect of sodium is contrary to that of TMPYP4, i.e., that an increase in promoter activity may be due to instability of G-QPX structures in a high Na+ environment, which results in melting these structures, enabling dsDNA formation required for the binding of TF to their recognition sites for initiation of transcription. The results were confirmed in several independent sets of experiments, using GFP reporter gene measurement by plate reader, by flow cytometry and using fluorescent microscopy. Moreover, quantitative RT-PCR was conducted to evaluate the effect of the same factors under similar conditions on the actual ChAT mRNA expression. It was observed that TMPY4 knocked down the ChAT mRNA expression by 87%, suggesting that G-QPX stabilization inhibits promoter activity as expected and that aconitine along with HNGF increases ChAT mRNA expression up to 2.8 fold. Aconitine-mediated influx of Na+ ions, possibly by inhibiting the formation of stable G-QPX structures, resulted in an Unique G-QPX structures can be stabilized with certain metal cations or small cationic molecule ligands such as TMPYP4, through occupying the space between the layers of G-tetrads. Although G-QPX are reported to have high stability in potassium solution, the diversity of G-QPX structures (due to diversity in sequence and size of G-runs, sequence and size of loops) will lead to diversity in physical behavior of G-QPX structures. Therefore, to get a clear image of folding topology and stability of identified G-QPX structures, physical studies including CD spectroscopy and AFM imaging were conducted. CD results showed that the identified ChAT G-QPX structures formed a hybrid, stable configuration in potassium environment (10mM) while being instable in sodium solution (100mM). AFM imaging demonstrated star-shaped structures (involving clusters of DNA strands) due to incubation with TMPYP4, where a greater number of these G-rich sequences have converted to G-QPX structures. The results of both an artificial engineered reporter gene system and actual ChAT mRNA expression (in vitro), plus physical characterization studies, strongly support the novel hypothesis that a neural action potential ionic mechanism regulates G-QPX formation/ deformation in the promoter region, due to movement of monovalent cations across the membrane, which is consistent with gene silencing and expression during neuronal resting and firing.

Additional Information

Publication
Dissertation
Language: English
Date: 2016
Keywords
Biochemistry, Gene Expression, Nanoscience, Nano-switch
Subjects
Quadruplex nucleic acids
Acetyltransferases
Neurons
Genetic regulation
Gene expression
Biochemistry
Nanoscience

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