Universitą degli studi di Pavia
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Montecucco research activity
My research activity is focused on the cellular response to DNA damage. The integrity of the genome is continuously challenged by endogenous and exogenous DNA damaging agents, and to counteract the biological consequences of DNA injuries, cells have evolved an intricate network of genome surveillance mechanisms, collectively known as DNA damage response (DDR) pathways. In addition to specific repair processes, DNA damage triggers checkpoint pathways whose function in proliferating cells is to delay or arrest cell-cycle progression thus providing more time to repair mechanisms before replication or cell division resume. DNA repair is tightly connected with replication and transcription, therefore functional alterations in any of these processes and in their crosstalk may induce uncontrolled cell proliferation as well as programmed cell death, two opposite events that are harmful both at the cellular and organismal level
Cellular response to DNA damage during cell cycle in human cells
We are currently studying the cell response to chronic replication-dependent DNA damage using as a model system 46BR.1G1 cells established from a patient with a replicative LigI haplo-insufficiency. We have extensively characterized these cells and found that they show a delayed maturation of Okazaki fragments, which results in the accumulation of DNA breaks. Notably, the replication-dependent DNA damage in LigI-deficient cells fails to halt cell cycle progression and to induce apoptosis. Actually, 46BR.1G1 cells display only a moderate delay in cell cycle progression and do not activate the S-phase specific ATR/Chk1 checkpoint pathway while the ATM/Chk2 pathway is constitutively activated at basal level. Thus, 46BR.1G1 are a suitable tool to investigate the strategies used by the cells to cope with low levels of chronic DNA damage, a condition frequently encountered in tumors. Using this model system we have recently shown that i) a number of pre-mRNA processing factors are regulated during the DNA damage response, shifting the alternative splicing pattern of target genes to control cell survival; ii) the DNA damage-initiated ATM signaling directly impacts cell morphology, adhesion and migration and affects the expression profile of cell-cell receptors encoded by the cadherins family genes.
These observations extend the influence of the DDR checkpoint pathways and unveil a role for ATM kinase activity in modulating cell biology parameters relevant to cancer progression.
Cellular response to DNA damage in non-replicating cell
Although defective repair of DNA damage can lead to neurodegenerative diseases, the molecular processes of its production and signaling in non-replicating cells are largely unknown. We have set up a protocol for neuronal differentiation and we are interested in investigating the sensitivity to DNA damage and the signaling in response to endogenous (reactive oxygen species) and exogenous genotoxic agents in postmitotic cells.
Neurons might be particularly prone to produce DNA breaks as a result of high rates of oxygen consumption, which produces ROS that can be harmful for nuclear genome. Our recent observations on cell hybrids from Parkinson’s patients raise the possibility that accumulation of DNA damage due to a chronic oxidative stress might contribute to the neurodegenerative phenotype.
Epigenetic signature of damaged chromatin
In eukaryotic cells, DNA is densely packaged into chromatin that is highly regulated by epigenetic marks bound to DNA as well as to components of the nucleosome. Mechanisms that control the dynamics of chromatin structure are: i) reversible histone post-translational modifications, which include methylation, ubiquitylation, sumoylation, phosphorylation and acetylation, ii) histone variants, and iii) ATP-dependent chromatin remodeling enzymes that displace histones or slide whole nucleosomes along DNA. Changes in chromatin architecture induced by epigenetic mechanisms are essential for vital cellular processes such as gene expression, DNA repair, replication and cellular division. My laboratory participates in a project funded by EPIGEN, aimed at elucidating by genome-wide analysis, the epigenetic modifications linked to DNA repair.
Cellular response to DNA damage during cell cycle in human cells
We are currently studying the cell response to chronic replication-dependent DNA damage using as a model system 46BR.1G1 cells established from a patient with a replicative LigI haplo-insufficiency. We have extensively characterized these cells and found that they show a delayed maturation of Okazaki fragments, which results in the accumulation of DNA breaks. Notably, the replication-dependent DNA damage in LigI-deficient cells fails to halt cell cycle progression and to induce apoptosis. Actually, 46BR.1G1 cells display only a moderate delay in cell cycle progression and do not activate the S-phase specific ATR/Chk1 checkpoint pathway while the ATM/Chk2 pathway is constitutively activated at basal level. Thus, 46BR.1G1 are a suitable tool to investigate the strategies used by the cells to cope with low levels of chronic DNA damage, a condition frequently encountered in tumors. Using this model system we have recently shown that i) a number of pre-mRNA processing factors are regulated during the DNA damage response, shifting the alternative splicing pattern of target genes to control cell survival; ii) the DNA damage-initiated ATM signaling directly impacts cell morphology, adhesion and migration and affects the expression profile of cell-cell receptors encoded by the cadherins family genes.
These observations extend the influence of the DDR checkpoint pathways and unveil a role for ATM kinase activity in modulating cell biology parameters relevant to cancer progression.
Cellular response to DNA damage in non-replicating cell
Although defective repair of DNA damage can lead to neurodegenerative diseases, the molecular processes of its production and signaling in non-replicating cells are largely unknown. We have set up a protocol for neuronal differentiation and we are interested in investigating the sensitivity to DNA damage and the signaling in response to endogenous (reactive oxygen species) and exogenous genotoxic agents in postmitotic cells.
Neurons might be particularly prone to produce DNA breaks as a result of high rates of oxygen consumption, which produces ROS that can be harmful for nuclear genome. Our recent observations on cell hybrids from Parkinson’s patients raise the possibility that accumulation of DNA damage due to a chronic oxidative stress might contribute to the neurodegenerative phenotype.
Epigenetic signature of damaged chromatin
In eukaryotic cells, DNA is densely packaged into chromatin that is highly regulated by epigenetic marks bound to DNA as well as to components of the nucleosome. Mechanisms that control the dynamics of chromatin structure are: i) reversible histone post-translational modifications, which include methylation, ubiquitylation, sumoylation, phosphorylation and acetylation, ii) histone variants, and iii) ATP-dependent chromatin remodeling enzymes that displace histones or slide whole nucleosomes along DNA. Changes in chromatin architecture induced by epigenetic mechanisms are essential for vital cellular processes such as gene expression, DNA repair, replication and cellular division. My laboratory participates in a project funded by EPIGEN, aimed at elucidating by genome-wide analysis, the epigenetic modifications linked to DNA repair.
