Abstract
Replication stress (RS), a term that collectively refers to slowing down or stalling of the replication forks, is a recognized causative event in tumorigenesis that has been exploited as a cancer chemotherapeutic strategy to limit the uncontrolled proliferation of cancer cells. A battery of cellular mechanisms has evolved to cope with RS in order to ensure the fidelity of DNA replication and genome stability. Among them, replication fork reversal (RFR) -i.e. the transient remodeling of a replication fork from a 3-way to a 4-way junction-, is followed by helicase RECQ1-mediated replication fork restart and acts as a protective mechanism in response to RS. Interestingly, RFR is not limited to forks directly encountering a hindrance, but also extends to forks that are not directly damaged. This implies that signaling pathways and 3-D organization are able to coordinate with each other and orchestrate the conversion of local responses into global modulation of the replication program. Lamin A/C is a crucial nuclear organization factor, mostly known as a structural component of the nucleus providing mechanical support. However, recent studies reveal the involvement of Lamin A/C in different nuclear processes, ranging from regulation of transcription, nuclear trafficking and positioning of nuclear pore complexes, to DNA replication and repair. Mutations in LMNA genes have been associated with numerous organ-specific or multisystem diseases, collectively known as laminopathies. On the other hand, upregulation or downregulation of Lamin A/C levels can be prominent in different types of cancer. Interestingly, recent studies using prolonged depletion of Lamin A/C have demonstrated strong defects in DNA repair pathways or RS upon prolonged fork stalling. However, the role of Lamin A/C in the early response to mild genotoxic treatments -the most relevant in the clinic- is not yet elucidated. Importantly, it is yet unknown which fraction of Lamin A/C is involved in RS response. In the present PhD thesis, I am shedding light on the fascinating role of Lamin A/C in the early response to mild RS, as well as show that the nucleoplasmic pool of Lamin A/C is responsible for controlling replication fork dynamics. In particular, I show that depletion of Lamin A/C during RS impairs replication fork slowing and leads to unrestrained fork progression mediated by the RECQ1 helicase. Interestingly, Lamin A/C depletion reduces ADP-ribosylation at replication forks, which is known to regulate RECQ1 activity via its interaction with PARP1. Moreover, restoring ADP-ribosylation levels rescues fork slowing in the absence of Lamin A/C. I also show that Lamin A/C physically interacts with PARP1, suggesting that physical association of these proteins underlie the control of fork progression. In addition, as it was already established that ADP-ribosylation can impact chromatin compaction upon DNA damage, I aimed to elucidate whether Lamin A/C affects chromatin compaction at forks. Importantly, a heterochromatic mark recently reported at stalled forks (H3K9me3) is also accumulating upon mild RS and requires Lamin A/C function, likely by limiting its removal from nascent DNA. Overall, Lamin A/C modulates RS response via control of chromatin compaction and ADP-ribosylation at replication forks, potentially regulating the accessibility of RECQ1 and KDM3A. Strikingly, I show that depletion of the LAP2α – a specific Lamin A/C interactor within the nucleoplasm – phenocopies acute Lamin A/C inactivation, suggesting that the role of Lamin A/C upon RS is largely uncoupled from its structural function at the nuclear periphery. Overall, the results from my PhD unveil a novel function of Lamin A/C in the RS response regulating replication dynamics in 3-D. These findings are ultimately expected to broaden our knowledge on the mechanisms and molecular causes of different human diseases and may open novel therapeutic avenues for treatment of tumors that rely prominently on Lamin A/C or laminopathies that bear mutations in the LMNA gene.
Apart from the aforementioned results, that have been included in the manuscript submitted, I am hereby describing other interesting and related findings, as well as others that were difficult to reproduce due to technical challenges. Moreover, during the first period of my PhD studies I also established a super-resolution microscopy-based strategy to monitor replication fork reversal, the preliminary results of which I report later on. Last but not least, I am including fascinating results that show that replication fork slowing takes place as a response to extremely low levels of RS, even down to femptomolar concentrations -1 million times lower doses than the ones typically used in the clinical setting-, suggesting that fork slowing is part of an immediate and local to global response, regulated in 3-D context of the nucleus.
Cherdyntseva, Veronica