G-quadruplex-binding proteins and their functional relevance in human cancer cells

Background: DNA can adopt a variety of different conformations based on particular sequence motifs and interactions with various proteins. Guanine-rich motifs can form non-canonical structures known as G-quadruplexes. The existence of such structures in vivo has been definitively proved by their direct visualization in human cells. G-quadruplex-forming sequences are located in strategic functional regions of the genome, such as telomeres and oncogene promoters, and play important roles in cancer biology. Hypothesis: There is compelling evidence that G-quadruplex DNA structures are strongly involved in tumorigenic processes, with regulatory functions in telomere maintenance and oncogene expression. Experimental evidences imply that an ensemble of interacting proteins modulates the formation of telomeric G-quadruplex DNA to achieve biological effects, as well as proteins are involved in the recognition of such structures in oncogene promoters to control their expression. However, most of G-quadruplex- interacting proteins, as well as their functional relevance remain elusive. The identification of those proteins is crucial to clarify the mechanisms in which G-quadruplexes are involved. These new knowledge may greatly push forward the research in this field. Aims: The biological functions of G-quadruplex DNA at the telomere, including its role in tumorigenic processes, are modulated by cellular factors such as proteins. Human telomeric G-quadruplexes display a large structural heterogeneity, that may influence the binding partners and, thus, have biological relevance. Therefore, the first principal aim of this project is the identification of proteins that recognize the telomeric G-quadruplex DNA in its different conformations, along with the assessment of the biological relevance of validated interactions. The second main aim is to discover the proteins that specifically bind the G- quadruplexes in selected oncogene promoters and to evaluate the functional relevance of the binding. Experimental Design: We have already demonstrated that the use of a chemoproteomic-driven approach represents a successful strategy for the identification of G-quadruplex-binding proteins. We also showed the effectiveness of the combined use of biophysical and biological experiments to validate the selected targets. This promising strategy will be applied to all the G-quadruplex folding topologies formed by human telomeric DNA and to selected oncogene promoter G-quadruplexes. Expected Results: The identification and characterization of the proteins that recognize the different G-quadruplex structures in human telomere will allow us to shed light, for the first time, on the biological relevance of the structural heterogeneity of telomeric DNA. The discovery of the binding partners of the investigated oncogene promoter G-quadruplexes, and of their role, will contribute to expand the knowledge on these DNA structures and to understand the mechanisms underlying regulation of oncogene expression. Impact On Cancer: The proposed project is likely to have significant impact on cancer research. Our studies will help to elucidate the role of G- quadruplexes in the control of telomere functions and oncogene expression in cancer cells. In addition to clarify the elusive biological mechanisms in which such non-canonical DNA structures are implicated, our findings will have considerable implications in the field of drug discovery. Indeed, we could exploit these protein-DNA interactions for therapeutic intervention aimed at the development of a new generation of anticancer drugs targeted at specific molecular events