Nucleoplasmic Lamin A/C controls replication fork restart upon stress by modulating local H3K9me3 and ADP-ribosylation levels

Background

The ability of cells to survive and proliferate depends on the faithful duplication of their DNA [1]. Inaccurate or incomplete DNA replication can result in genomic instability, cell death and cancer. Damage to our DNA by exposure to several internal (e.g. reactive oxygen species as a byproduct of cell metabolism) and external sources (e.g. chemotherapeutic drugs) can perturb or stall DNA replication and trigger replication stress (RS) [2]. At the same time, inducing RS has proven effective in cancer treatment, but the exact underlying mechanism remains unclear [3]. It became clear that mild RS conditions such as low doses of chemotherapeutics induce replication fork reversal, a mechanism that allows for a transient break of DNA synthesis until the damage is resolved [4].

Adding another layer of complexity, DNA replication, and thus RS, occur in chromatin. Chromatin packages DNA together with histone and non-histone proteins into a condensed structure. Through this packaging chromatin not only contributes to organizing DNA in the nucleus, but also regulates DNA repair and gene expression [5,6]. A major question in the field is how chromatin dynamics and the 3D nuclear architecture participate in the cellular response to RS.

Recently, the team of Dr. Nitika Taneja discovered that RS-induced heterochromatin acts as a fork protective mechanism [7]. Given that RS remodels forks which are not directly challenged, several other components preserving nuclear architecture have been implicated in the RS response such as nuclear F actin [8,9]. In this preprint the authors describe additional components of nuclear architecture involved in the RS response and they found nucleoplasmic lamin A/C to be involved in replication fork restart upon mild RS.

Key Findings

• Lamin A/C dynamically interacts with replication factories and promotes active fork slowing upon mild RS

With the aim of understanding whether Lamin A/C is present at nuclear replication factors, the authors performed a proximity ligation assay (PLA) between Lamin A/C and EdU which was incorporated into nascent DNA at replication forks. As expected, Lamin A/C was mostly associated with the nuclear lamina, however confocal imaging also showed Lamin A/C-EdU PLA foci in the nucleus suggesting that a subset of Lamin A/C interacts with replication factories via its nucleoplasmic interaction partner LAP2α (Figure 1A) [10]. Interestingly, induction of mild RS by treatment with low doses of genotoxic treatments such as the topoisomerase I inhibitor camptothecin or the topoisomerase II inhibitor etoposide decreased Lamin A/C-EdU PLA signal. Etoposide was shown to induce fork slowing, however, in Lamin A/C depleted cells this effect was abolished.

Taken together, the authors demonstrate that Lamin A/C and its interaction partner LAP2α are required for active fork slowing in response to mild RS and thereby preserve genome stability.

• Lamin A/C maintains fork slowing by promoting PARylation at stressed replication forks

In order to understand how Lamin A/C prevents unrestrained fork progression upon mild RS, the authors set out to perform DNA fiber assays by co-depleting RECQ1 and Lamin A/C. Interestingly, they found that RECQ1 depletion fully rescued unrestrained fork progression and maintained high levels of reversed forks in Lamin A/C and LAP2α depleted cells.

It was previously shown that RECQ1 is negatively regulated by its transient interaction with auto-modified PARylated PARP1, thereby preventing the restart of reversed forks until RS has been resolved [11]. Moreover, Lamin A/C was shown to interact with PARP [12]. Thus, the authors hypothesized that Lamin A/C may globally promote PARylation levels, thereby controlling RECQ1 activity and fork progression upon RS. To test this hypothesis, they performed experiments in the presence and absence of Lamin A/C including: PLA between PAR and EdU, and DNA fiber assays in the presence of PARGi which counteracts PAR chains removal. Indeed, they found that Lamin A/C controls a subset of PAR levels at stressed replication forks which is important to counteract RECQ1 activity and mediate active fork slowing upon RS.

• Lamin A/C promotes heterochromatin maintenance at replication forks

Given that heterochromatin is transiently formed upon replication stress by the histone methyltransferase G9A, which deposits H3K9me2/3 on stressed replication forks [7], and that PARP1 supposedly binds to heterochromatin marks, the authors asked two main questions: 1) whether transient heterochromatin could also form under mild RS and 2) whether this could lead to Lamin A/C dependent fork restart.

Indeed, the authors found the accumulation of heterochromatin marks on newly replicated DNA undergoing fork slowing upon mild RS conditions. Inhibiting these marks resulted in unrestrained fork progression, as well as reduction of PAR levels. Similarly to Lamin A/C loss, unrestrained fork progression in G9Ai cells depends on RECQ1. To gain insight into the second question, the authors performed PLA for H3K9me3 and EdU, as well as a single molecule approach called chromstretch to observe H3K9me3 at single replication tracts under mild RS conditions in the presence and absence of Lamin A/C. They found Lamin A/C loss to reduce H3K9me3 levels at forks challenged with mild RS.

What I like about this preprint

The preprint by Cherdyntseva and colleagues is of great interest to me since this study resonates with my own research which focuses on understanding the RS response in the context of chromatin. Their auxin inducible degron cell line to efficiently and transiently degrade lamin A/C, without compromising genome organization functions of Lamin A/C is a very elegant approach. Such a tool is essential since long term depletion or inactivation of nuclear architecture components could lead to unwanted side effects e.g. gene activity changes. I am also fascinated by how their single molecule molecule microscopy approach, allowed them to observe changes in histone marks at individual replication tracts as a consequence of mild RS. This unique tool provides valuable insight into how replication stress impacts chromatin at a detailed, molecular level.

Questions for the authors

Q1: Could you speculate on how RS-induced heterochromatin marks affect PAR levels? Is this directly through H3K9me3? Or through chromatin compaction?

Q2: You mention that ‘a subset of PARyation events’ to be controlled by Lamin A/C. How is the remaining fraction of PARylation under mild RS conditions controlled?

References

1. Zeman, Michelle K., and Karlene A. Cimprich. “Causes and Consequences of Replication Stress.” Nature Cell Biology 16, no. 1 (January 2014)
2. Gaillard, Hélène, Tatiana García-Muse, and Andrés Aguilera. “Replication Stress and Cancer.” Nature Reviews Cancer 15, no. 5 (May 2015): 276–89.
3. Forment, Josep V., and Mark J. O’Connor. “Targeting the Replication Stress Response in Cancer.” Pharmacology & Therapeutics 188 (August 2018): 155–67.
4. Neelsen, Kai J., and Massimo Lopes. “Replication Fork Reversal in Eukaryotes: From Dead End to Dynamic Response.” Nature Reviews Molecular Cell Biology 16, no. 4 (April 2015): 207–20.
5. Stewart-Morgan, Kathleen R., Nataliya Petryk, and Anja Groth. “Chromatin Replication and Epigenetic Cell Memory.” Nature Cell Biology 22, no. 4 (April 2020): 361–71.
6. Hsu, Chia-Ling, Shin Yen Chong, Chia-Yeh Lin, and Cheng-Fu Kao. “Histone Dynamics during DNA Replication Stress.” Journal of Biomedical Science 28, no. 1 (December 2021): 48.
7. Gaggioli, Vincent, Calvin S. Y. Lo, Nazaret Reverón-Gómez, Zuzana Jasencakova, Heura Domenech, Hong Nguyen, Simone Sidoli, et al. “Dynamic de Novo Heterochromatin Assembly and Disassembly at Replication Forks Ensures Fork Stability.” Nature Cell Biology 25, no. 7 (July 2023): 1017–32.
8. Lamm, Noa, Mark N. Read, Max Nobis, David Van Ly, Scott G. Page, V. Pragathi Masamsetti, Paul Timpson, Maté Biro, and Anthony J. Cesare. “Nuclear F-Actin Counteracts Nuclear Deformation and Promotes Fork Repair during Replication Stress.” Nature Cell Biology 22, no. 12 (December 2020): 1460–70.
9. Schie, Janne J. M. van, and Job de Lange. “The Interplay of Cohesin and the Replisome at Processive and Stressed DNA Replication Forks.” Cells 10, no. 12 (December 8, 2021): 3455.
10. Naetar, Nana, Konstantina Georgiou, Christian Knapp, Irena Bronshtein, Elisabeth Zier, Petra Fichtinger, Thomas Dechat, Yuval Garini, and Roland Foisner. “LAP2alpha Maintains a Mobile and Low Assembly State of A-Type Lamins in the Nuclear Interior.” eLife 10 (February 19, 2021): e63476.
11. Berti, Matteo, Arnab Ray Chaudhuri, Saravanabhavan Thangavel, Shivasankari Gomathinayagam, Sasa Kenig, Marko Vujanovic, Federico Odreman, et al. “Human RECQ1 Promotes Restart of Replication Forks Reversed by DNA Topoisomerase I Inhibition.” Nature Structural & Molecular Biology 20, no. 3 (March 2013): 347–54.
12. Mosler, Thorsten, H Irem Baymaz, Justus F Gräf, Ivan Mikicic, Georges Blattner, Edward Bartlett, Matthias Ostermaier, et al. “PARP1 Proximity Proteomics Reveals Interaction Partners at Stressed Replication Forks.” Nucleic Acids Research50, no. 20 (November 11, 2022): 11600–618.

Tags: chemotherapy, genomic stability, nuclear organization

doi: https://doi.org/10.1242/prelights.40687

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