Structural characterization of a G-rich region within EGFR promoter

R. Rigo1, A. Kotar2, J. Plavec2, C. Sissi1

1Pharmaceutical and Pharmacological Sciences Dept., Università degli studi di Padova,Via Marzolo 5, 35131 Padova, Italy

2Slovenian NMR centre, National Institute of Chemistry, Hajdrihova 19, 1001 Ljubljana, Slovenia

riccardo.rigo@ceitec.muni.cz

G-quadruplexes (G4) are nucleic acid secondary structures formed by planar arrays of four guanines (G-quartets), held together through Hoogsteen hydrogen bonds, stacking one on each other to generate a non-canonical tetra-helix. Many studies reported their involvement in several cellular processes, such as gene expression, DNA replication and telomere maintenance. G-rich sequences are enriched in promoter regions of oncogenes and at these sites G-quadruplex formation has been implicated in modulation of gene transcription, suggesting potential for G4-based anticancer therapies [1].

Whereas it is quite easy to map the distribution of putative G-quadruplex forming sequences (PQS) by in silico analytical tools, the real folding properties of these sequences are currently poorly predictable due to the highly polymorphic behaviour of the G4 arrangements. Indeed, both the nucleic acid sequence and the environment greatly affect the structure, stability and folding kinetics of these elements. Thus, in order to assess any potential biological role for these non-canonical DNA structures, a detailed evaluation of the ability of these PQS to fold into stable G4 is required [2].

In this context, we considered EGFR (Epidermal Growth Factor Receptor) oncogene as a case-study. It encodes a receptor tyrosine kinase, which overexpression or mutations, that make it constitutively active, induce uncontrolled cell proliferation, survival, adhesion, migration and differentiation [3]. A bioinformatic search identified some G-rich regions within the EGFR promoter gene. Here we explore the conformational equilibria of one of them, a 30 residue long sequence located at 272 bp upstream the transcription start site (EGFR-272). By merging spectroscopic and electrophoretic analysis performed on the wild-type sequence as well as on a wide panel of related mutants, we were able to prove that in the presence of K+ ions this sequence folds into two main G-quadruplex structures, presenting  parallel and hybrid topology. They show comparable thermal stabilities and affinities for the metal ion and, indeed, they are always co-existing in solution. The folding process is driven by a hairpin occurring in the domain corresponding to the terminal loop which is an important stabilizing element for both the identified G-quadruplexes.

 

1. D. Rhodes, H. J. Lipps, Nucleic Acids Res., 43, (2015), 8627–8637.

2. R. Rigo, M. Palumbo, C. Sissi, Biochim. Biophys. Acta, 1816, (2017), 1399-1413.

3. J. R. Goffin, K. Zbuk, Clin. Ther., 35, (2013), 1282-1303.