# Multigenerational silencing dynamics control cell aging

1. aSection of Molecular Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093;
2. bBioCircuits Institute, University of California, San Diego, La Jolla, CA 92093;
3. cThe San Diego Center for Systems Biology, La Jolla, CA 92093;
4. dDepartment of Bioengineering, University of California, San Diego, La Jolla, CA 92093;
5. eMoores Cancer Center, University of California, San Diego, La Jolla, CA 92093
1. Edited by Jasper Rine, University of California, Berkeley, Berkeley, CA, and approved September 5, 2017 (received for review February 27, 2017)

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Fig. 2.

Dynamic patterns of silencing loss during aging. (A) Dynamics of silencing loss in cells aging with elongated daughters. (Top) Representative images of cell aging and death with elongated daughters. Blue arrows point to daughter cells, white arrows point to the living mother cell, and the red arrow points to the dead mother cell. (Bottom) Single-cell color map trajectories of reporter fluorescence. Each row represents the time trace of a single cell throughout its life span. Color represents the normalized fluorescence intensity as indicated in the color bar, which is used throughout to allow comparisons between different conditions. Cells are sorted based on their RLSs. (B) Dynamics of silencing loss in cells aging with rounded daughters. (C) Daughter cell morphology is correlated with the silencing state of mother cells. Color map trajectories of representative mothers are aligned with the morphology (round vs. elongated) of daughters produced throughout their life spans. A cross-correlation analysis of daughter morphology and mother silencing dynamics is shown in SI Appendix, Fig. S2.

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Fig. 3.

A quantitative analysis of silencing loss dynamics in single cells. (A) Dynamics of silencing loss in representative single cells with different life spans. For each cell, (Top) time-lapse images for the cell have been shown with (Bottom) the fluorescence time trace throughout its life span. Vertical dashed line represents each division time of the cell, in which the distance between two adjacent dashed lines indicates the cell cycle length. Reporter fluorescence is normalized to the baseline level. (B) A schematic illustrates the dissection of two phases based on the silencing loss dynamics. The rise times of early sporadic and final sustained silencing loss are defined as t1 and t2, respectively. (C) Scatter plots showing the relationships between the length of (Left) Intermittent Phase or (Right) Sustained Phase and life span at the single-cell level. Single-cell data are from Fig. 2A. Each circle represents a single cell. Correlation coefficient (R) is calculated and shown. (D) Bar graph showing the average durations of (blue) early sporadic and (red) final sustained silencing loss.

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Fig. 4.

A phenomenological model of cell aging. (A) Schematic diagram of the model. The circles indicate the cellular states, and the arrows depict transitions between the states. (B) The statistics of silencing state transitions as a function of age. The fraction of all cells at state 0 of a given generation that switch to state 1 at the next cell cycle (red) and the fraction of the cells at state 1 that return to state 0 at the next cell cycle (blue) have been computed as a function of age. (C) The transition rates from state 1 to death as a function of the number of consecutive generations in state 1. Blue squares are experimentally measured fractions of cells that died exactly after N consecutive generation in state 1 over the total number of cells that lived for at least N generations. Yellow straight line is a linear fit of these data (0 < N < 10). The red line and the error bars indicate the mean and SD of the fraction <mml:math><mml:mrow><mml:msub><mml:mi>f</mml:mi><mml:mrow><mml:mn>1</mml:mn><mml:mo>→</mml:mo><mml:mi>D</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:math>f1→D from simulations. (D) The average number of cells alive as a function of age and the SD in simulations (red line and error bars) and experimental data (blue line). (E) The average number of cells in state 1 as a function of the age and the SD in simulations (red line and error bars) and experimental data (blue line). (F) The distribution of the number of consecutive generations in state 1 until death in simulations (red line and error bars) and experimental data (blue line). All simulation results in CF were obtained from 200 stochastic simulations of 79 cells. (G) Single-cell state trajectories from a single stochastic simulation of the model with 79 cells at time 0. Each row represents the time trace of a single cell throughout its life span. Blue corresponds to state 0, and red to state 1. Cells are sorted based on their RLSs. (H) Single-cell state trajectories simulated using modified transition rates to reflect sustained silencing loss. (I) Single-cell state trajectories simulated using modified transition rates for four generations to reflect transient silencing loss. Details of the model are included in SI Appendix.

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Fig. 5.

Perturbations of silencing dynamics shorten life span. (A) Dynamics of silencing loss in sir2Δ cells. (Left) Single-cell color map trajectories of reporter fluorescence. Each row represents the time trace of a single cell throughout its life span. Color bar is identical to Fig. 2. Cells are sorted based on their RLS. The red dashed line represents the average length of Sustained Phase in WT cells, defined in Fig. 3B. (Right) Representative single-cell time traces with different life spans. Dynamics of silencing loss in WT cells are shown in response to chemical perturbations. (B) A 1,000-min 5 mM NAM pulse. (Inset) Expression of RNR3-mCherry in response to a 1,000-min 5 mM NAM pulse at indicated times. (C) A 240-min 5 mM NAM pulse. Inset shows the expression of RNR3-mCherry in response to a 240-min 5 mM NAM pulse at indicated times. (D) A constant treatment of 2.5 mM NA. For each condition, (Top) a schematic illustrating the time-based drug treatment has been shown with (Bottom) the corresponding single-cell color map trajectories of reporter fluorescence.

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