G-quadruplex formation at human DAT1 gene promoter: Effect of cytosine methylation

The dopamine transporter gene (DAT1), a recognized genetic risk factor for attention deficit hyperactivity disorder (ADHD) is principally responsible for the regulation of dopamine synaptic levels and serves as a key target in many psychostimulants drugs. DAT1 gene methylation has been considered an epigenetic marker in ADHD. The identification of G-rich sequence motifs potential to form G-quadruplexes is correlated with functionally important genomic regions. Herein, biophysical and biochemical techniques are employed to investigate the structural polymorphism along with the effect of cytosine methylation on a 26-nt G-rich sequence present in the promoter region of the DAT1 gene. The gel electrophoresis, circular dichroism spectroscopy, and UV-thermal melting data are well correlated and conclude the formation of a parallel (bimolecular), as well as antiparallel (tetramolecular) G-quadruplex in Na+ solution. Interestingly, the existence of uni-, bi-, tri-, and tetramolecular quadruplex structures in K+ solution exhibited only the parallel type G-quadruplex. The results demonstrate that in presence of either cation (Na+ or K+) the cytosine methylation reserved the structural topologies unaltered. However, methylation lowers the thermal stability of G-quadruplexes and the duplex structures, as well. These findings provide insights to understand the regulatory mechanisms underlying the formation of the G-quadruplex structure induced by DNA methylation.