MRE11 and TREX1 control senescence by coordinating replication stress and interferon signaling

Oncogene-induced senescence (OIS) arrests cell proliferation in response to replication stress (RS) induced by oncogenes. OIS depends on the DNA damage response (DDR), but also on the cGAS-STING pathway, which detects cytosolic DNA and induces type I interferons (IFNs). Whether and how RS and IFN responses cooperate to promote OIS remains unknown. Here, we show that the induction of OIS by the H-RASV12 oncogene in immortalized human fibroblasts depends on the MRE11 nuclease. Indeed, treatment with the MRE11 inhibitor Mirin prevented RS, micronuclei formation and IFN response induced by RASV12. Overexpression of the cytosolic nuclease TREX1 also prevented OIS. Conversely, overexpression of a dominant negative mutant of TREX1 or treatment with IFN-β was sufficient to induce RS and DNA damage, independent of RASV12 induction. These data suggest that the IFN response acts as a positive feedback loop to amplify DDR in OIS through a process regulated by MRE11 and TREX1.

� D A description of all covariates tested � D A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons D � A full description of the statistical parameters including central tendency {e.g.means) or other basic estimates {e.g.regression coefficient) AND variation {e.g.standard deviation) or associated estimates of uncertainty {e.g.confidence intervals) □ l'vl For null hypothesis testing, the test statistic {e.g.F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted � Give P values as exact values whenever suitable.
� D For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings � D For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes � D Estimates of effect sizes (e.g.Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Policy information about availability of computer code
Data collection RNA-seq used Spliced Transcripts Alignment to a Reference (STAR).

Data analysis
The DNA fibers were measured by MetaMorph Microscopy Automation and Image Analysis Software (Molecular Devices) and statistical analysis was performed with Graph Pad Prism 8 (GraphPad Software).The quality of sequencing data was assessed with FastQC (http:// www.bioinformatics.babraham.ac.uk/projects/fastqc). Flow cytometry data were analyzed using FlowJo 10 (LLC).
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Reporting on sex and gender Non applicable
Reporting on race, ethnicity, or Non applicable other socially relevant groupings

Recruitment
Ethics oversight

Non applicable
Non applicable

Non applicable
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Field-specific reporting
Please select the one below that is the best fit for your research.If you are not sure, read the appropriate sections before making your selection.
IZJ Life sciences For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
Sample size was determined according to field standards.For DNA fiber spreading, at least 150 fibers were measured.For comet assay, tail length was measured in 30-50 tailing cells.Mean fluorescence intensity (MFI) quantification was performed on more than 400 cells.
Data exclusions No data were excluded.

Blinding
Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.Randomization is not relevant to our study.Each sample has a specific treatment.It is not possible to randomize samples between different treatments.

Materials & experimental systems
For DNA fiber assays, sample treatments were given a number instead of naming an exact treatment.

Eukaryotic cell lines
Policy information about cell lines and Sex and Gender in Research Cell line source(s)

Mycoplasma contamination
Commonly misidentified lines (See ICLAC register)

Plants
Normal human fibroblasts BJ-hTERT were a gift of Dr. D. Peeper (The Netherlands Cancer Institute, Amsterdam).IMR90-ER/ RASV12 cells were a gift of Masashi Narita (Young et al. 2010).RPEl-hTERT cells were from ATCC (CRL-4000).
None of the cell lines have been authenticated by Standards for Cell Line Authentification (Almeida et al. 2016).However, RPEl-hTERT cells have been purchased from ATCC and authenticated by this organization with certificates.
Mycoplasma testing by qPCR was performed periodically to ensure that the cell lines are mycoplasma free.
No commonly misdefined cell lines were used in the study.

Seed stocks non applicable
Novel plant genotypes non applicable '-

Plots
Confirm that: k8J The axis labels state the marker and fluorochrome used (e.g.CD4-FITC).
k8J The axis scales are clearly visible.Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).k8J All plots are contour plots with outliers or pseudocolor plots.k8J A numerical value for number of cells or percentage (with statistics) is provided.

Software
Cell population abundance

Gating strategy
Cells were pulse-labeled with EdU and fixed in 1% PFA.EdU click chemistry was performed prior to flow cytometry analyses.

MACSQuant analyser
FlowJo (LLC) 10.000 cells were collected for the analyses.
Cellular integrity was first selected according to the SSC and FSC.Single cell population was then gated according to the DNA content.The fluorescence intensity increase was further gated as cells in S phase, using non-EdU-labeled cells as a negative control.
k8J Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.