Las hipótesis de la “koiné”

We next sought to investigate the molecular factors responsible for the mitosis- senescence decision and its associated CDK bifurcation. To determine which DDR pathway was responsible for the cell fate upon S-phase exit in S phase-damaged cells, we pre-treated with small molecule inhibitors for ATM, ATR, CDK2, or knockdown of p21, before NCS treatment, and monitored cell fate outcomes. Combining ATM inhibitor or p21 knockdown with NCS treatment led to drastic increase in the mitosis population (Figure 4.4A, Figure S4.3A-B). In contrast, ATR inhibitor did not significantly affect the cell fate upon NCS treatment (Figure 4.4A), indicating that NCS-related DNA damage during replication led to arrest primarily through the ATM pathway.

In response the DNA damage, cells receive signals from a continuous space (damage level) and decode the signal into a binary decision— either mitosis or

senescence. We asked where in the DDR pathway did the information translated into a binary response. We examined whether CDK activity bifurcation was continuous or binary. If the bifurcation was continuous, increasing the proportion of mitosis population by inhibiting ATM or p21 may lead to a delay in CDK activity drop. In contrast, if the bifurcation was binary, we would expect the CDK activity bifurcation to remain

unchanged, while only the proportion of high CDK versus low CDK cells changed. We found the medians of CDK activity under ATM, ATR, DNA-PK, and p21 inhibition

remained unchanged (Figure 4.4B), consistent with a binary CDK activity response upon S-phase exit. In contrast, inhibiting the CDK activity using a small molecule inhibitor for CDK2 sped up the CDK activity drop in the senescence population (Figure 4.4B), suggesting the dynamics of CDK activity drop in the bifurcation was robust to perturbations to molecules upstream of the CDK, but not robust to perturbations to CDK activity itself.

Notably, p21 knockdown completely abolished the arrest population (Figure 4.4A, 4C) without significantly affect the DSB repair rate (Figure 4.4D). In addition, p21 level in G1 has been shown to play a crucial role in the proliferation-quiescence

decision by synthesizing the endogenous DNA damage from the previous cell

cycle7,22,31,34_{, but the p21 accumulation upon S-phase exit in response to DNA damage} is largely unexplored. Therefore, we examined the p21 dynamics in relationship to the CDK activity bifurcation and asked whether the p21 response was continuous or binary. We used time-lapse microscopy to follow the CDK activity and treated the cells with NCS. At the end of the movie, we fixed the cell and quantified p21 level by

immunofluorescence (Figure 4.4E). We labeled the cells as either high or low CDK activities based on the CDK activity level and trend at the end point of the movie. We observed that p21 level gradually increased and started to diverge as the cells exit S phase (Figure 4.4F), indicating p21 level bifurcated concurrently with CDK bifurcation. In addition, the p21 level inversely correlated with CDK activity (Figure 4.4G),

confirming that the CDK activity was largely controlled by the p21 level.

p21 is one of the key cell cycle regulator targets of p53, and activation of both p53 and p21 is required for sustained G2 arrest and mitosis skip upon DNA

damage148,149_{. To determine whether the role of p21 in mediating the CDK bifurcation} was p53-dependent, we knockdown p53 and quantified the CDK activity. Surprisingly, the p53 knockdown altered the CDK bifurcation dynamics by delaying the CDK activity drop (Figure 4.4H, Figure S4.3C-D). Interestingly, we observed a significant proportion (17%) of cells that sustained high CDK activity in G2 to the end of the movie but failed to enter mitosis, which we did not observe in p21 knockdown or ATM inhibition. Taken together, our results suggested that p53 is required for the immediate CDK drop in the arrest cells upon S-phase exit, but p21 has a p53-independent role in suppressing the arrest cell fate choice.

We next asked whether the p21 response upon S-phase exit was binary or continuous. A binary response is consistent with a cell-fate decision upstream of p21, whereas a continuous response supports a model where the decision point is

somewhere between p21 and CDK activity. We treated cells with NCS and quantified CDK activity and p21 6 hours post damage in G2 cells using DAPI (Figure 4.4I). We found a strong inverse correlation between p21 level and CDK activity. In addition, both the p21 level and CDK activity exhibited a bimodal distribution, consistent with a binary p21 response in response to damage.

Figure 4.1. DNA damage in S phase lead to heterogeneous cell fates in the subsequent G2. A, Schematic of the simplified DNA damage response network in S phase. B, DNA damage during S phase leads to partial accumulation of G2 population. C, Representative images of SA- β-gal staining. Cells were synchronized in S phase by double thymidine block, and then released into fresh media for 2 hours before treated with or without 200 ng/mL NCS. 6 days-post treatment, cells were serially passaged at 1:20 dilution to assess proliferation and stained for SA-β-gal. D, Quantification of percentage of SA-β-gal positive cells treated with NCS during S phase from Figure 4.1C. Scoring was performed with blinding to the treatment conditions. n≥4. Within each replicate, cell number ≥ 25. Error bars represent standard deviation. p-value = 4e-81 based on Fisher-exact test.

Figure 4.2. The mitosis-senescence decision is controlled by a bifurcation in CDK activity at S-phase exit. A, Time-lapse images of PCNA-CFP and DHB-mCherry channels of single cells that are treated with 100 ng/mL NCS during S phase. Examples of a S-phase damaged cell that enters mitosis and a S-phase damaged cell that permanently arrests in the subsequent G2. B,

Quantification of CDK activity in cells damaged with 100 ng/mL NCS during S phase. CDK activity was quantified based on the DHB-mCherry reporter as the ratio of mean cytoplasmic fluorescence intensity over mean nuclear fluorescence intensity. Cells are aligned in silico to the S/G2 transition and colored based on the cell fate (permanent G2 arrest or mitosis). C, The difference in CDK activity between the senescence versus mitosis group as a function of time. The p-value was calculated based on two-sample t-test between the mitotic population’s and the senescent population’s CDK activities. D, Quantification of CDK activity in cells damaged with 300 ng/mL aphidicolin during S phase. Cells are aligned in silico to the S/G2 transition and colored based on the cell fate (permanent G2 arrest or mitosis).

Figure 4.3. The mitosis-senescence decision is contributed by the DNA damage repair process, but not the timing of damage or the damage level incurred. A, Schematic illustrating the quantification of time in S phase before damage and time in S phase post damage. B, The timing within S phase when the damage occurred does not correlate with the cell fate. p- value was calculated based on the Kolmogorov–Smirnov test. n=134. C, Erlang Time-lapse images of RPE cells with the 53BP1-EYFP reporter upon NCS addition. D, Quantification of 53BP1 foci number in single cells entering senescence or mitosis upon DNA damage in S phase. E, The statistical difference in 53BP1 foci number between mitotic and senescent populations. The p-value was calculated based on

two-sample t-test between the mitotic population’s and the senescent population’s 53BP1 foci number. F, The amount the DNA damage induced does not correlate with the cell fate. The 53BP1 foci number at damage was determined as the maximal foci number within the first hour post NCS treatment. p-value was calculated based on the Kolmogorov–Smirnov test. n=132. G, The repair rate of DSBs correlates with the cell fate. The 53BP1 foci half-life was obtained by fitting the 53BP1 foci number time course to an exponential distribution. p-value was calculated based on the Kolmogorov–Smirnov test. See Figure S2E for detailed description. n=107. H, 53BP1 foci repair rate upon NCS treatment in the population pretreated with 1µM DNA-PK inhibitor. 53BP1 foci time trajectories were fitted with an exponential distribution. Only data within the 1-12 hours post NCS treatment were fitted. I, 53BP1 foci repair rate in single cells treated with 100 ng/mL NCS during S phase. 53BP1 foci time trajectories for each cell were fitted with an

exponential distribution. p-value was calculated based on the Kolmogorov–Smirnov test. n=90. J, Single- cell traces of CDK activity treated with 100 ng/mL NCS pretreated pretreated with 1µM DNA-PKi. Only cells treated with NCS during S phase were quantified. The thick line indicates the median of the population.

Figure 4.4. The CDK activity bifurcation is mediated by p21 and is partially dependent on p53.A, Inhibition of the selected molecular factors in the DDR pathway and

quantification of the mitotic population. In the time-lapse imaging experiment, cells were treated with 100 ng/mL NCS. Cells treated within S phase were followed to determine the cell fate (entering mitosis or G2 arrest until the end of the movie). p-values were calculated based on Fisher-exact test. B, Median CDK activity of single cell traces treated with 100 ng/mL NCS in conjunction with indicated small molecular inhibitors. Only cells treated with NCS during S phase were quantified. (need to remake with more DNA- PKi) C, Single-cell traces of CDK activity treated with 100 ng/mL NCS pretreated with siRNA for p21. Only cells treated with NCS during S phase were quantified. The thick line indicates the median of the

population. n=47. D, 53BP1 foci repair rate in single cells treated with 100 ng/mL NCS during S phase. 53BP1 foci time trajectories for each cell were fitted with an exponential distribution. p-value was

calculated based on the Kolmogorov–Smirnov test. n=100. E, p21 level bifurcates concurrently with CDK activity upon S-phase exit. Asynchronously proliferating cells were treated with 50 ng/mL NCS. 10 or 13 hours post treatment, cells were fixed and quantified for p21 level. Only cells treated during S phase are quantified. The thick line indicates the median of the population. n=187. F, p21 level at the time of fixation as a function of time. Same data as in Figure 4.4E. G, p21 level and CDK activity at the time of fixation. Same data as in Figure 4.4E. H, Quantification of CDK activity in cells pretreated with siRNA for p53, damaged with 100 ng/mL NCS during S phase. Cells are aligned in silico to the S/G2 transition and colored based on the cell fate (permanent arrest or mitosis). The thick line indicates the median of the population. n=58. I, Bimodal distribution in p21 level leads to a bimodal distribution in the CDK activity upon S-phase exit. Cells were treated with 200 ng/mL NCS. 6 hours after treatment, cells were fixed, and p21 and CDK2 activity were quantified. DAPI was used to gate for G2 cells. n > 1700.

Figure S4.1. Characterization of cell cycle phase distribution. A, Example illustration of measuring the cell cycle phase of cells damaged in S phase. RPE cells were treated with a pulse of 10 μM EdU for 10 mins, followed by PBS washed and 200 ng/mL NCS. The cells were then incubated for 48 hours to allow for cell cycle progression before fixation, staining, and quantification for EdU incorporation and DAPI content. The colors represent the density of data points: yellow, high density; blue, low density. B, The reporters did not alter the cell cycle phase distribution. n>7600. C, Cells were imaged every 10 mins with the same imaging settings for acquiring phase duration data for 72 hours before 30 mins EdU pulse and quantification of cell cycle phase distributions. The control groups were taken from matched location of the replicated plate growing inside the incubator for 72 hours before the EdU pulse. n>750 for each condition.

Figure S4.2. Characterization and quantification of the 53BP1-EYFP reporter.A, Images of RPE cells with the 53BP1-YFP reporter treated with 100 ng/mL NCS treatment. 1 hour post treatment, cells were fixed and stained for γH2AX and YFP. B, Quantification of damage foci using the 53BP1-YFP reporter and the γH2AX stain 1 hour post 100 ng/mL NCS treatment. Data were fitted with linear regression. n=1305. C, Quantification of average γH2AX foci number binned based on the 53BP1- YFP reporter foci number. Same data as in Figure S4.2C. Data were fitted with linear regression. D, Histogram of 53BP1 foci number of cells growing in the incubator or cells being imaged under the

microscope every 10 minutes for 72 hours, with or without 75 ng/mL NCS treatment. p-value between the two groups were calculated based on 2-sided Kolmogorov–Smirnov test. E, Representative 53BP1 foci half-lives. The number of 53BP1 foci for each cell was fit to an exponential function to obtain the half-lives of 53BP1 foci.

Figure S4.3. Knockdown efficiency by siRNA for p21 and p53. A, Western blot for p21 and β-actin. Cells were treated with 100 ng/mL NCS and siRNA 24 hours before harvest. Luc represents luciferase. B, Quantification of nucleus p21 level by immunofluorescence. Cells were treated with 100 ng/mL NCS and siRNA 24 hours before fixation. p-value was calculated based on 2-sided Kolmogorov– Smirnov test. C, Western blot for p53 and β-actin. Cells were treated with 100 ng/mL NCS and siRNA 24 hours before harvest. Luc represents luciferase. D, Quantification of nucleus p53 level by

immunofluorescence with siRNA treatment. Cells were treated with 100 ng/mL NCS and siRNA 24 hours before fixation. p-value was calculated based on 2-sided Kolmogorov–Smirnov test.

4.5 Materials and Methods 4.5.1 Cell culture

hTERT retinal pigment epithelial cells (RPE) were obtained from the ATCC (ATCC® _CRL-4000™_{) and cultured in DMEM medium supplemented with 10% fetal calf} serum (FBS) and penicillin/streptomycin.Cells were passaged using Trypsin

(25300054, Gibco) as needed. When required, the medium was supplemented with selective antibiotics (2 μg/mL puromycin, A1113803, Gibco).

4.5.2 Chemical and genetic perturbation of the cell cycle phases

For NCS treatment, medium was replaced with fresh medium supplemented with neocarzinostatin (N9162, Sigma-Aldrich) during experiments. For aphidicolin treatment, medium was replaced with fresh medium supplemented with 300 nM aphidicolin

(A0781, Sigma-Aldrich) for 8 hours during experiments, washed off once with PBS, and then replenished with imaging media. For CDK2 inhibition, cells were treated with 2.5 μM CVT-313 (221445, Santa Cruz) prior to starting the imaging.

4.5.3 Cell line construction

The pLenti-PGK-hygro-TK-NLS-mTurq-PCNA plasmid was subcloned from eGFP-PCNA (Gift from S. Angus) using the Gateway system (Life Technologies) following manufacturers protocols. pLenti PGK hygro DEST (w530-1) was a gift from Eric Campeau (Addgene plasmid # 19066). The DHB-mCherry reporter was a gift from S. Spencer. The plasmid was stably expressed into RPE cells by first transfecting the plasmid into 293T cells to generate replication-defective viral particles using standard protocols (TR-1003 EMD Millipore), which were used to stably infect the RPE cell lines. The cells were maintained in selective media and hand-picked to generate a clonal population.

4.5.4 Time-lapse microscopy

Prior to microscopy, cells were plated in poly-D-lysine coated glass-bottom plates (Cellvis) with FluoroBrite™ DMEM (Invitrogen) supplemented with FBS, 4 mM L-

glutamine, and penicillin/streptomycin. Fluorescence images were acquired using a Nikon Ti Eclipse inverted microscope with a Nikon Plan Apochromat Lambda 40X objective with a numerical aperture of 0.95 using an Andor Zyla 4.2 sCMOS detector. In addition, we employed the Nikon Perfect Focus System (PFS) in order to maintain focus of live cells throughout the entire acquisition period. The microscope was surrounded by a custom enclosure (Okolabs) in order to maintain constant temperature (37o_{C) and} atmosphere (5% CO2). The filter set used for mCherry was: 560/40 nm; 585 nm; 630/75 nm (excitation; beam splitter; emission filter) (Chroma). Images were acquired every 20 mins for U2OS cells and every 10 minutes for RPE cells in the mCherry channel. We acquired 2-by-2 stitched large image for RPE cell. NIS-Elements AR software was used for image acquisition and analysis.

4.5.5 Image analysis

Image and data analysis was performed in Fiji97 _{(version 1.51n, ImageJ NIH) and} MATLAB (R2017b, MathWorks). Images from time lapse experiments (16 bit) were processed with rolling ball background subtraction algorithm prior to analysis. PCNA channel was selected for segmentation of nuclear regions of interest (ROIs) and tracking of individual cells by in-house developed ImageJ scripts with a user-assisted approach. Briefly, user-defined tracks were used for local automated segmentation of ROIs based on intensity thresholding followed by morphological operations to define an oval shape and a watershed algorithm to separate adjacent nuclei. Defined ROIs were used to analyze all fluorescent channels.

PCNA pattern (PCNA variance) was defined within nuclear ROIs from which nucleoli (dark regions) were eliminated using Remove Outliers algorithm (ImageJ). Images were smoothed with a Gaussian filter (sigma=1x) and then processed with a variance filter (sigma=2x) to enhance PCNA pattern. Intensity and standard deviation of variance images was measured within 70% central region of defined ROIs to avoid edge artefacts. Beginning and end of S phase was defined as a transition from low to high and high to low variance respectively and detected automatically from a one

dimensional signal.

4.5.6 Immunofluorescence

Cells were fixed with 4°C 4% paraformaldehyde for 10 minutes, permeabilized with 0.5% Triton-X 100 for 10 minutes, and incubated with primary antibodies (anti- phospho-H2AX Ser139 (JBW301, EMD Millipore 05-636), anti-GFP (abcam ab6556)) overnight at 4°C. Primary antibodies were visualized using a secondary antibody conjugated to Alexa Fluor-488/-647 and imaged with appropriate filters. EdU incorporation and staining was performed using the Click-iT™ EdU kit (Invitrogen C10337).