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Research activity

Our Research Mission

Regulation of genomic functions is a complex process that involves several protein factors and nucleic acid structures. Particularly in eukaryotes, our understanding of functional roles of protein factors is much more advanced than the role of genomic DNA itself and RNA molecules. However, in the last decade, thanks mainly to the development of parallel sequencing technologies (known as Next Generation Sequencing, NGS), it has become possible to investigate how particular secondary structures of nucleic acids can affect fundamental genomic functions such as gene expression, DNA replication, genome recombination and stability. Non-B DNA structures have been established to play major roles in cell transformation and neuron degeneration as they markedly influence expression profiles and genome stability.

In this context, our research focuses on how non-B DNA structures affect chromatin organization, genome functions and stability, on one side, and how they are regulated by DNA topoisomerases, on the other. We are particularly interested in R loops and G-quadruplex (G4). R-loops are formed during transcription upon re-annealing of the nascent RNA to the DNA template strand, forming an RNA:DNA hybrid and forcing the non-template strand to loop out in a single-stranded status (see Figure 1). G4s are instead generated by single-stranded guanine-rich DNA sequences that fold into stable four-stranded structures wherein three sets of four guanines are arranged in three stacking planar quartets (see Figure 2).

Figure 1. The general structure of an R loop (Wikimedia Commons).

Figure 2. The general structure of G-quadruplex (G4). Note that G4 structures are very polymorphic (Wikimedia Commons).

NGS techniques have allowed the mapping of non-B structures along the genome. The mapping data show that R loops and G4s are prevalent in mammalian genomes, where they form dynamically over conserved regions. In particular, R-loops have been linked to genomic instability by causing interference between the replication and transcription machineries.  R loops and G4s are DNA structures particularly sensitive to DNA supercoilings. Negative DNA supercoiling generated behind an elongating RNA polymerase is thought to facilitate R-loop formation by inducing an underwound DNA state favorable to the re-annealing of the nascent transcript. Interestingly, mapping data in human cells showed that active promoters are free of nucleosomes, have more negative supercoils than other genomic sites and are regions where R loops and G4s can form more easily.

Figure 3. DNA topoisomerase family (to read more, Capranico et al., Type I DNA topoisomerase, J Med Chem. 2017 Mar 23;60(6):2169-2192).

Among several DNA topoisomerases present in a mammalian cell (see Figure 3), DNA Topoisomerase I is a main cellular factor controlling topological homeostasis of the genome. Thus, we have recently studied the role of Topoisomerase I in R loop formation in human cells by siRNA gene silencing and NGS technology. The findings were somewhat surprising as they demonstrate that Topoisomerase I can indeed regulate an R-loop, but it can either favor or dis-favor its formation depending on the precise chromatin context. This paper has recently been published in Genome Biology (first authors Stefano G. Manzo and Stella Hartono) in a close collaboration with Fred Chedin at UCS, Davis.

Other projects are aimed at establishing the role of G4s in gene functions and genome stability by using specific and selective G4 binders developed by some chemist colleagues in our Department. The findings show that G4 binder analogs can bind specifically to a particular set of G4 conformations, and that this action can be relevant for their biological activity.

In summary, our research goal is to establish molecular and epigenetic mechanisms dependent on non-B DNA structures of transcription regulation and genome instability. As several enzymes and other factors recognize these structures and regulate their stability and formation, mutations of these enzymes can affect diseases progression and therapeutic intervention. Thus, the discovery of regulation mechanisms involving R loops and G4s can reveal unexpected opportunities to develop strategies for personalized medicine in oncology and neurodegenerative diseases.

Research activity in the lab is supported by funds from "Associazione Italiana per la Ricerca sul Cancro" (AIRC).

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