Telomeres are crucial chromatin regions located at the ends of linear chromosomes that prevent genome instability and control the cellular replicative life span. Telomeres shorten with each cell division until they reach a critically short length, becoming unprotected and leading to cellular senescence. Unprotected or damaged telomeres constitute a major source of genome instability and massive genome rearrangements characteristic of tumorigenesis, while the activation of telomere maintenance mechanisms ensures cell immortalisation in cancer.
Telomeres are considered "difficult-to-replicate sites" due to their intrinsic characteristics, including the ability to be transcribed into long non-coding RNAs known as TERRAs. TERRAs can hybridise to the DNA template, forming DNA-RNA hybrids and displacing the non-template single-stranded DNA, constituting the so-called R-loops. Unscheduled R-loop formation is known to cause significant hindrances for DNA replication leading to transcription-replication conflicts; thus, contributing to genome instability when accumulated under pathological conditions. However, they also have important physiological regulatory roles, including at telomeres. Many efforts have been made to identify cellular factors that prevent R-loop-associated genome instability, but we have very little knowledge about the mechanisms that regulate TERRA DNA-RNA hybrid metabolism or whether these structures might differentially modulate telomere homeostasis in different cell types or conditions.
Understanding the molecular mechanisms that regulate telomeric R-loops in human cells may help us identify potential targets to combat some telomere-related diseases or certain types of cancers where telomeric dysregulation of these structures promotes DNA damage leading to disease development. A relevant case involves the subset of 10-15% of cancer types that use a telomerase-independent but recombination-dependent pathway, based on break-induced telomere synthesis, to elongate telomeres and achieve immortalisation, known as Alternative Lengthening of Telomeres (ALT). ALT cancers comprise a variety of cancers typically of mesenchymal or neuroendocrine origin, including some types of paediatric cancers, that are very aggressive with poor prognosis and without an established specific treatment. The molecular mechanism of ALT is not fully understood, but recent studies have identified TERRA R-loops as key intermediates that promote ALT recombination. Therefore, the idea of targeting telomeric R-loops has arisen as a promising therapy to specifically target ALT cancer cells without affecting telomerase-positive or primary human cells, where TERRA levels are generally much lower.
This project aims to gain insight into the molecular mechanisms that regulate telomeric DNA-RNA hybrids in normal and cancer cells that use different telomere maintenance mechanisms. By characterising telomeric DNA-RNA hybrids along the cell cycle and focusing on the consequences of the deregulation of new factors implicated in telomeric R-loop homeostasis, we seek to conduct a detailed analysis of R-loop control at telomeres. This will provide potential clues for targeting R-loop-associated malignancies.
