Downloaded by guest on September 26, 2021 oto fteRsrcinPitb badp21 and Rb by Moser Point Justin Restriction the of Control www.pnas.org/cgi/doi/10.1073/pnas.1722446115 restim- serum starvation, serum Based from fore. emerging the cells :cyclin- in to and work coming on complexes (Rb) (CDK) kinase retinoblastoma dependent the Restriction the with for Point, basis systems-level and molecular the covered of absence the in even division, of round . one whereas complete Point, mitogens will on Restriction dependent and the longer at no are arrest Point can post-Restriction cells and uncommitted cycle are cell the Point to pre-Restriction cells late-G1 Cells to and mid- cells Point. a cycling to Restriction subject both were Based after that starvation serum (5). emerged h from model interval emerging 3–4 a this first data, in the these Point cells on only Restriction cycling for the withdrawal putting that , serum suggested to cells sensitive asyn- 3T3 are of round Swiss microscopy a cycling to time-lapse cells Similarly, chronously shown suffi- commit 4). and (3, was later Point proliferation exposure Restriction was of the It cross constant, to (2). than cient again rather pulsed, once between become that proliferation conditions point mitogen single to and a serum favorable until at phase arrest S cells and mitosis that suggested synchro- cells serum Early nized in withdrawn. are experiments mitogens current or withdrawal–restimulation–withdrawal the serum cross complete if must will even cells cycle, they proliferate, cell which and after cycle Point, cell Restriction preventing the the to while commit To maintenance (1). and development tissue T replication sensi- DNA cycle is cell inherited also Point but Restriction Point mitogens, pre-Restriction the . only via a where stress not to and to returns in mitosis tive cells model of a after subset support state a data these only Together, which in Point. CDK2 G2 Restriction the in the from escape rising with begins fur- cides p21 pre-Restriction We CDK2 the inhibitor into state. Point, mitosis CDK exit daughters Point whose the cells mito- pre-Restriction mother that exits uncommitted show that an ther subpopulation into and another Point sis and one Restriction cycling delineating the , “uncrosses” continues after never minutes Rb. that hypophosphorylated within subpopulation of present and hypophosphorylation are hyper- into vs. Rb vs. with bifurcate hyper- increasing techniques subpopulations cells and by Notably, single-cell activity cancer pre-Restriction marked of CDK2 and anaphase, a low variety noncancer after into a subpopulations both mitosis two use that Point exit show we Restriction cells to Here, the that state. of notion Point location G1. the the late and in about paradigm, current occurs questions the challenged then raising have Rb; Point studies cyclin- single-cell hypophosphorylated Restriction low Recent the and mitosis have through activity exit they passage cells (CDK) where that state, kinase to posit Point dependent models suggested pre-Restriction cycle later a moment cell pro- was into retinoblastoma Current and the the (Rb). cycle as of tein cell hyperphosphorylation defined the with 2017) to 27, originally coincide December commit review was for cells (received 2018 Point that 10, July approved Restriction and Kingdom, The United London, UK, Research Cancer Hunt, Tim by Edited 80309 CO a eateto iceity nvriyo ooaoBudr ole,C 00;and 80303; CO Boulder, Colorado–Boulder, of University Biochemistry, of Department oeua ilgcladbohmclivsiain ae un- later investigations biochemical and biological Molecular setsae sciia o raimlhat,a hsenables this as health, organismal for qui- and critical proliferative is between states transition escent to cells of ability he | etito point restriction low a,b anMiller Iain , tt.Frhroe erdto fp1coin- p21 of degradation Furthermore, state. | quiescence a,b ya Carter Dylan , low | G0 tt n asg through passage and state | CDK2 a,b n arn .Spencer L. Sabrina and , 1073/pnas.1722446115/-/DCSupplemental ulse nieAgs 5 2018. 15, August online Published at online information under supporting contains article This distributed is article 1 BY-NC-ND). (CC 4.0 access License NonCommercial-NoDerivatives open This Submission. Direct PNAS a is article This interest. wrote of S.L.S. conflict and no J.M. declare and authors The project; the research; of performed conceived D.C. S.L.S. paper. data; and the analyzed I.M., S.L.S. J.M., and research; J.M. designed J.M. contributions: Author once are cells and activity dephosphorylated, CDK2 becomes mitosis, the Rb after follow in off, cells wherein Some mitosis turns sensor paradigm, (14). after activity expected MCF10A behavior line the CDK2 cellular cell a epithelial of mammary of classes challenged development two have revealed The cells cycling paradigm. asynchronously this in activity studies CDK recent low of state to a rise giving into Rb. cycle, born hypophosphorylated cell and again the cells of daughter round one two to Rb, of committed Point hyperphosphorylation are the Restriction cells uncom- with the hypophos- mitosis an crossing after and On into hours (13). activity several born 1A) CDK are (Fig. low cells Rb a by phorylated which to characterized in led state cycle observations mitted cell these the the CDK2 of of through of synthesis model passage activation The indicate Thus, Point. Rb 12). Restriction the of 11, underlies hyperphosphorylation (7, and system and Point this hyper- in Restriction of to CDKs, the hypo- bistability of from at activity switch Rb the The phosphorylated triggers (10). then G1 anaphase CDK2, late early particular in through begin continue to shown and was Rb hypophosphorylated hyperphosphorylated to from switch the released, subsequently the and to entry. leads S-phase for and critical genes releases other of cell fully production marking This (8), (9). Rb commitment hyperphosphorylated the cycle with triggers to complex that hypo- loop in from feedback switch positive These the A. generate initiates help and which CDK2 E E2F, (7). some cyclins phosphorylation liberates of complex Rb this transcription in model, of which canonical process (6), the the D In cyclin initiates of CDK4/6, buildup with the in results ulation owo orsodnesol eadesd mi:[email protected] Email: addressed. be should correspondence whom To rgniyi hscl aedcso,wihmyb exploitable be het- may gain. which significant therapeutic decision, for experience fate cell can this cells in erogeneity cancer This even cycle. while that cell state, another a shows Point to only pre-Restriction commits that a immediately into remainder such the that, born split is populations shows cells Restriction cell of and the subset the cells tested, examines cases noncancerous six work and in cancerous This in model. Point this of challenged are have aspects studies nutrients single-cell sufficient cell recent if However, the later available. to hours the several cross uncommitted and Point are 2 Restriction kinase they cyclin-dependent which activate will are in but cells cycle, state that a suggests into model born Point Restriction canonical The Significance lhuhti oe ftecl yl swdl accepted, widely is cycle cell the of model this Although nocodazole by mitosis in synchronized cells in work on Based b iFotesIsiue nvriyo ooaoBudr Boulder, Colorado–Boulder, of University Institute, BioFrontiers a,b,1 PNAS . | o.115 vol. www.pnas.org/lookup/suppl/doi:10. raieCmosAttribution- Commons Creative | o 35 no. | E8219–E8227

CELL BIOLOGY A B Recent papers suggest that entry into quiescence is facilitated Canonical Model Alternative Model during the maternal G2 (14, 19) and that spontaneous entry into the CDK2low state is caused in part by low-level endoge- window of signal integration nous DNA replication stress that is passed from mother cells to Rb dephosphorylation daughter cells (20–23). Furthermore, the CDK2 inhibitor, p21, Rb dephosphorylation was found to be up-regulated in these stressed cells and was R G0 required for entry into the CDK2low state after both endogenous Rb hyper- and exogenous replication stress (20, 21). Thus, p21 seems to be a M M phosphorylation G1 critical component of the Restriction Point decision, but its exact connection to the Restriction Point is unclear. G2 G2 G1 R Rb hyper- Based on these recent findings, we propose here that healthy phosphorylation cells in optimal growth conditions cycle continuously without ever G1 “uncrossing” the Restriction Point or losing Rb phosphorylation (Fig. 1B), whereas the classic Restriction Point model predicts that S S cells are born into a pre-Restriction Point state with hypophos- phorylated Rb and low CDK2 activity (Fig. 1A). Distinguishing between these two models and establishing their generalizability are necessary for improving our understanding of how cells behave both in cell culture and in vivo. The canonical model of the ignores the behavioral heterogeneity as well as its underlying CDK2 activity CDK2 activity 0481216 0481216 causes that have been unearthed in recent years. It thus incom- Time relative to anaphase (hr) Time relative to anaphase (hr) pletely acknowledges the various mechanisms and timing thereof that regulate cell cycle commitment, exit, and reentry. In this manuscript, we show the applicability of the alternative model across five widely used transformed and nontransformed cell lines and one type of primary cell, all of which have func- phospho-Rb phospho-Rb tional Rb and , establishing its validity beyond MCF10A 04812 1604812 16 cells. We show that all of these cell lines display a bifurcation Time since anaphase (hr) Time since anaphase (hr) in CDK2 activity and Rb phosphorylation at mitotic exit. This result contrasts with the canonical cell cycle model, in which Fig. 1. Two models of the Restriction Point depicted using simulated data. cells have hypophosphorylated Rb after anaphase. Furthermore, (A) The canonical model of the cell cycle predicts that cells are born into by tracking p21 under endogenous control in single cells over a pre-Restriction Point state characterized by low CDK2 activity, hypophos- multiple cell cycles, we detect the up-regulation of p21 in G2 phorylated Rb, mitogen sensitivity, and lack of cell cycle commitment. This phase of mother cells whose daughters enter the CDK2low state state has been referred to variously as “early G1” and “G1pm” (5, 7). Then, low 3–4 h after mitosis, cells reach the Restriction Point (R). Given optimal con- after mitosis. We further show that these CDK2 cells reen- ditions, cells cross the Restriction Point and become committed to the cell ter the cell cycle by degrading p21 at the Restriction Point. cycle. This state has been referred to as “late G1” and “G1ps” (5, 7). (B) An Together, these data reveal that only a fraction of cells in the alternative cell-cycle model that takes into account recent studies, in which cell types examined here display features of the canonical model cells integrate mitogen and stress signaling during a maternal window of and exit mitosis into a pre-Restriction Point state of hypophos- signal integration that informs the G0–G1 decision that occurs after mitosis. phorylated Rb. Our results instead support an alternative One subpopulation is born with moderate CDK2 activity and hyperphospho- rylated Rb, is insensitive to mitogen withdrawal, and is committed to the model in which, under saturating mitogens, the proliferation– cell cycle, whereas the other subpopulation is born with low CDK2 activity quiescence decision is influenced strongly by the G2/M levels and hypophosphorylated Rb and remains sensitive to mitogen withdrawal, of p21, which if high enough, result in the dephosphorylation of similar to the cells diagrammed in A. The proliferative subpopulation thus Rb in , and the reset of those cells to a pre-Restriction never uncrosses the Restriction Point, whereas the CDK2low subpopulation is Point state. born into a pre-Restriction Point state that can be considered a transient G0. These cells emerge from the CDK2low state to reengage with the cell cycle Results by building up CDK2 activity and hyperphosphorylating Rb. A Bifurcation in CDK2 Activity and Rb Phosphorylation After Mito- sis Is Present in both Transformed and Nontransformed Cell Types. The observation of a bifurcation in CDK2 activity after mito- low low again mitogen sensitive (“CDK2 cells”). CDK2 cells are not sis in unperturbed cells was initially made in nontransformed senescent, and cells can remain in this state anywhere from a MCF10A mammary epithelial cells (14). To determine the gen- few hours to multiple days before reentering the cell cycle (15). eralizability of this finding, we examined several noncancerous While the CDK2low state could be conceptualized as “early G1,” (MCF10A and retinal RPE-hTERT) and cancerous (mammary it also has many features consistent with a transient G0 or qui- MCF7, osteosarcoma U2OS, and colorectal HCT116) cells as escence, including declining Ki67 protein levels (16), little to no well as primary human lung fibroblasts (HLFs). HLFs were CDK2 activity, hypophosphorylated Rb, and elevated p21 (14). recently reported to follow the canonical model depicted in Fig. In contrast, other cells show increasing CDK2 activity after mito- 1A rather than the alternative model in Fig. 1B (17). We trans- sis, hyperphosphorylated Rb, and mitogen insensitivity, and they duced all six cell types with fluorescent histone 2B (H2B) as a immediately commit to another cell cycle (“CDK2inc cells”) (14). nuclear marker as well as the CDK2 activity sensor and mon- Moreover, the CDK2 activity sensor can be used to visualize pas- itored CDK2 activity by time-lapse imaging and cell tracking sage through the Restriction Point: cells in the CDK2low state in asynchronously cycling cells. Despite the wide variety of cell are mitogen sensitive and become locked in the CDK2low state types examined, each showed a bifurcation in CDK2 activity if mitogens are withdrawn, but after cells build up CDK2 activ- at mitotic exit (Fig. 2A and Movies S1–S6). Notably, the can- low ity above a threshold level, they become mitogen insensitive and cer lines did not always show a lower fraction of CDK2 cells continue progression through the cell cycle, even if mitogens are than MCF10A. Only HCT116 cells, with 1% CDK2low, were withdrawn or the MAPK pathway is inhibited (14, 17, 18). significantly more proliferative than MCF10A cells, with 26%

E8220 | www.pnas.org/cgi/doi/10.1073/pnas.1722446115 Moser et al. Downloaded by guest on September 26, 2021 Downloaded by guest on September 26, 2021 oe tal. et Moser n el nlzd el eeiae o 4h xd n tie o hsh-b n h muoursec mg a eitrdt h nlfaeo the of frame all final for the time-since-anaphase to in of registered as was function time-since-anaphase image a of immunofluorescence function as the a and (S807/811) as phospho-Rb, phospho-Rb p21 for of of stained measurement measurement and Single-cell fixed, Single-cell h, (B) 24 MCF10A, 1%. for movie. HCT116, imaged were and Cells 17%; analyzed. U2OS, cells 18%; MCF7, 23%; HLF, CDK2 of CDK2 for 2. Fig. ,2 el;U2OS, cells; 1,929 = HCT116 U2OS MCF7 HLF RPE-hTERT MCF10A ABC igecl rcso D2activity CDK2 of traces Single-cell (A) expression. p21 and phosphorylation, Rb activity, CDK2 in bifurcation the of generalizability the for Evidence inc inc n CDK2 and 0.2 0.2 0.2 0.2 0.2 0.2

el bu)adCDK2 and (blue) cells CDK2 activity CDK2 activity CDK2 activity CDK2 activity CDK2 activity CDK2 activity 2 20 -20 2 20 20 -20 20 -20 -20 20 20 -20 -20 1 2 1 2 1 2 1 2 1 2 1 2 n -15 -15 -15 -15 -15 -15 546cls RPE-hTERT, cells; 15,446 = Time relativeto anaphase(hr) Time relativetoanaphase(hr) Time relativetoanaphase(hr) Time relativeto anaphase(hr) Time relativetoanaphase(hr) Time relativetoanaphase(hr) low n -10 -10 -10 -10 -10 -10 ,5 el;adHCT116, and cells; 4,958 = rcspotdhr r rprinlt h rcin fec ttepplto ee.CDK2 level. population the at each of fractions the to proportional are here plotted traces -5 -5 -5 -5 -5 -5 515 05 low 515 05 15 05 15 05 15 05 15 05 el rd.Frcaiy 5 aulyvrfidtae r lte o ahcl ieo huad nlzd h numbers the analyzed; thousands of line cell each for plotted are traces verified manually 250 clarity, For (red). cells 10 10 10 10 10 10 n 196cls HLF, cells; 11,936 = n 2 cells. 825 =

log2(phospho-Rb S807/811) log2(phospho-Rb S807/811) log2(phospho-Rb S807/811) log2(phospho-Rb S807/811) log2(phospho-Rb S807/811) log2(phospho-Rb S807/811) 10 11 10 12 13 10 12 13 10 12 13 10 12 13 14 10 12 11 11 11 11 11 8 9 8 9 8 9 7 8 9 9 8 9 015 10 5 0 051015 051015 01 025 20 15 10 5 0 01 025 20 15 10 5 0 015 10 5 0 n 4 el;MCF7, cells; 547 = Time sinceanaphase (hr) Time sinceanaphase(hr) Time sinceanaphase(hr) Time sinceanaphase(hr) Time sinceanaphase (hr) Time sinceanaphase(hr) MCF10A, B. n n 902cls RPE-hTERT, cells; 19,052 = ,5 el;U2OS, cells; 7,659 = 025 20 025 20 025 20 025 20

log (p21) log (p21) log (p21) log (p21) log (p21) log (p21) n 2 2 2 2 2 2 10 12 13 14 10 12 13 14 15 11 11 10 12 13 14 10 12 13 10 12 13 10 12 13 11 11 11 11 ,4 el;adHCT116, and cells; 5,348 = 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 015 10 5 0 015 10 5 0 015 10 5 0 015 10 5 0 015 10 5 0 015 10 5 0 n ,0 el;HLF, cells; 2,809 = low PNAS Time sinceanaphase (hr) Time sinceanaphase(hr) Time sinceanaphase(hr) Time sinceanaphase(hr) Time sinceanaphase (hr) Time sinceanaphase(hr) C1A 6;REhET 31%; RPE-hTERT, 26%; MCF10A, : | o.115 vol. n ,1 el;MCF7, cells; 1,019 = n | ,5 el.(C cells. 8,157 = o 35 no. 025 20 025 20 025 20 025 20 025 20 025 20 | E8221 )

CELL BIOLOGY CDK2low, whereas MCF7 and U2OS cells were intermediate with AB 18 and 17% CDK2low, respectively. HLF cells were compara- ble with these, with 23% of cells entering the CDK2low state, whereas RPE-hTERT cells were the least likely to immediately enter another cell cycle, with 31% entering the CDK2low state after mitosis under optimal growth conditions. We next examined the phosphorylation state of Rb in these six cell types. Not only is Rb a canonical CDK2 substrate, but the phosphorylation status of this protein is considered an indi- C cator of a cell’s position relative to the Restriction Point (18, 24, DNA 25). After 24 h of time-lapse imaging, we fixed and stained cells for phospho-Rb at Serine 807/811. Phospho-Rb is bimodally dis- pHH3

tributed (SI Appendix, Fig. S1), and we consider the high mode to pRb represent hyperphosphorylated Rb as was done previously (14, 23, 26). Through image registration, we linked the immunoflu- orescence data to the time-lapse data to determine how long DNA ago each cell underwent anaphase (Fig. 2B and SI Appendix, Fig. S1). Phospho-Rb staining already shows clear bimodality pHH3 one frame after anaphase (12 or 20 min depending on cell type) pRb in all six cell types examined, indicating that the proliferative Early Anaphase subpopulation maintains hyperphosphorylation of Rb from one cycle to the next as has previously been shown for MCF10A (26). DNA Strikingly, the fraction of cells with hyperphosphorylated Rb pHH3 immediately after anaphase is similar to the fraction of the pop- inc ulation born into the CDK2 state (SI Appendix, Fig. S1). We pRb then repeated this experiment, staining for p21 after 24 h of time- Late Anaphase *** lapse imaging. A weakly bimodal or long-tailed distribution of D p21 abundance is apparent within the first frame after anaphase DNA (Fig. 2C and SI Appendix, Fig. S1). Both the hypophosphory- lated Rb state and the high p21 population decrease as time- pRb since-anaphase increases, highlighting the transient nature of 12 Minutes Post-Anaphase ** the CDK2low state. Previous work by this group and others has shown that in DNA MCF10A, RPE-hTERT, and Hs68 primary human fibroblasts, pRb

high p21 levels in G0/G1 cells are associated with 53BP1 nuclear 24 Minutes

Post-Anaphase ** bodies, a marker of DNA lesions (20, 21). We therefore asked if this relationship was conserved in the cancer cell lines used Fig. 3. A subset of cells matching the CDK2low population up-regulates p21 here. As was previously shown with MCF10A and RPE-hTERT, before mitosis but does not dephosphorylate Rb until chromatin decon- we find that G0/G1 cells with 53BP1 nuclear bodies have higher densation begins. (A and B) p21 signal (A) or phospho-Rb (pRb; S807/811) p21 levels than G0/G1 cells without 53BP1 nuclear bodies (SI signal (B) as a function of pHH3 signal in asynchronously cycling MCF10A Appendix, Fig. S2). Thus, the three cancer cell lines tested here cells as measured by flow cytometry. Thirty-three percent of mitotic (pHH3+) seem to face the CDK2inc vs. CDK2low decision in the same cells have high p21 levels; 12% of mitotic cells have falling pRb, but there way as reported previously for nontransformed cells, although is no discrete hypophosphorylated Rb population (n = 10,000 cells). Gates the fraction of cells born committed to each fate varies across were set according to the saddle point for p21 and pRb. (C) Asynchronously growing MCF10A cells were stained for pHH3, pRb (S807/811), and Hoechst. cell lines. Mitotic cells were identified based on Hoechst staining and displayed as mock film strips. Anaphase cells are ordered based on distance between p21 Up-Regulation and Rb Dephosphorylation Are Temporally chromosomes. Histone H3 is dephosphorylated starting in late anaphase Separated. Given that asynchronously cycling cells already show (green stars), whereas Rb is not dephosphorylated until after anaphase differential Rb phosphorylation and p21 levels immediately after is complete and chromatin decondensation begins. (D) pRb (S807/811) in mitosis, we next asked when exactly this bifurcation in Rb sta- MCF10A cells from Fig. 2B. Shown are newly born cells fixed 12 min (Upper) tus and p21 expression occurs. To this end, we fixed and stained or 24 min (Lower) after anaphase was detected. Cells are ordered based on asynchronously cycling, wild-type MCF10A cells for the mitotic chromatin decondensation and distance between sister cells. The numbers marker phospho-histone H3 (pHH3) and either phospho-Rb or of cells with hyper- and hypophosphorylated Rb presented here are pro- portional to the total population. Green stars mark cells that are pHH3−; p21 and measured these three signals by flow cytometry. p21 was magenta stars mark cells with hypophosphorylated Rb. up-regulated in 30% of pHH3+ cells, mirroring the fraction of cells born into the CDK2low state (Fig. 3A, upper right quad- rant). In contrast, there was no discrete hypophosphorylated Rb previous literature (27–30), whereas Rb is not dephosphorylated population among pHH3+ cells. We therefore asked whether until after late anaphase (Fig. 3 C and D), explaining why no dis- this unexpected result could be explained by the facts that phos- crete pHH3+/hypophosphorylated Rb population is detectable phorylation of histone H3 is lost before anaphase and that by flow cytometry. Synthesizing our flow cytometry, film strip, phosphorylation of Rb is lost in CDK2low cells after anaphase. and time-lapse microscopy data (where we have immunofluores- To more carefully examine the timing of dephosphorylation cence data for phospho-Rb beginning 12 min after anaphase), of Rb, we repeated the experiment using immunofluorescence we conclude that Rb dephosphorylation in CDK2low cells occurs microscopy, computationally isolated cells at each phase of mito- in telophase after chromosome decondensation has started. The sis based on Hoechst staining, and then evaluated the levels of differential timing of p21 up-regulation in G2 and Rb dephos- pHH3 and phospho-Rb in the resulting mock film strips. His- phorylation in telophase suggests that p21 up-regulation (by tone H3 is dephosphorylated in late anaphase, in agreement with inhibiting CDK activity) causes entry into the CDK2low state,

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CDK2 heterogeneity of in levels the rise monitored the and of time CDK2 from the traces to and activity aligned CDK2 we Point, single-cell Restriction averaged the cross and CDK2 reactivate Point. Restriction CDK2 the when at Initiated Is Degradation p21 reentry. cycle cell of time the around levels p21 in the sustaining CDK2 anaphase, the after levels p21 CDK2 CDK2 increase These to 4B). (Fig. continue line depend- cell anaphase, the CDK2 before on the h ing 5–10 enter p21 G2 that up-regulating previous CDK2 cells start the mitosis general daughter in the p21 while a enter of M, not levels that and low is cells maintain up-regulation mitosis daughter after p21 Instead, state that G2. find of we feature 37), (36, sion aligned 4 and (Fig. groups anaphase of four time these the within to averaged then were traces after each CDK2 from activity Cells CDK2 CDK2 S2–S6). to (Movies sensor according fluo- CDK2 h CDK2 anaphase: a classified 24 the with were for and transduced population imaged marker was then nuclear lev- line and the H2B endogenous cell at tagged at Each promoter rescently protein (35)]. inducible [U2OS fluorescent an els a from with expressed (34)] HCT116 or and terminus MCF7, (21), C [RPE-hTERT locus endogenous the at tagged cell S4). alter Fig. dramatically Appendix, not (SI does dynamics N and cycle CDK2, the p21, with at wild-type interact tagged still to p21 can similarly degrades 31). been normally, N (14, have localizes p21 the terminus constructs functional targeted tagged maintain we similarly to 33), shown because (32, p21 p21 of ubiquitylation for high- terminus mechanism or have regulatory acylation studies a N-terminal some as for Although potential S4). the and lighted S3 Figs. Appendix, otayt al oeso elccedpnetp1expres- p21 cycle-dependent cell of models early to Contrary is p21 which in lines cell other three examined also We low h emerge7–10 low tt – r71 fe npae(CDK2 anaphase after h 7–10 or 4–7 state tt.I otat CDK2 contrast, In state. low low emerge tt n hnretrtecl yl hwadecline a show cycle cell the reenter then and state el ni h etito on,a hc point which at Point, Restriction the until cells epciey.Tesnl-elCK n p21 and CDK2 single-cell The respectively). , inc el onrglt 2 eaiet hnthey when to relative p21 down-regulate cells low CDK2 , tt fe Mitosis. After State mre– h emerge4–7 low n el hteeg rmthe from emerge that cells and , A and el htrcie h rg3– drug the received that cells emerge and B). el htiiilyenter initially that cells h iiaiybetween similarity The i.S5). Fig. Appendix, SI low CDKN1A agtrclsthen cells daughter odetermine To low mre– h emerge4–7 low tt after state emerge el exit cells ou ( locus cells and SI inc tedgnu eesofa nuil rmtr(2S rfrom or (RPE- (U2OS) promoter expressed terminus endogenous inducible is the an C p21 off fluorescent the levels the at endogenous if or at protein or fluorescent U2OS) HCT116) a MCF7, (MCF10A, hTERT, to terminus fused not N is is pattern the p21 this whether Notably, on anaphase. after dependent and before both CDK2 mother CDK2 p21 in of the G2 mothers enter during whereas daughters increase into whose to cells begins all cells send p21 to endogenous sufficient that is CDK2 G2 the in p21 of overexpression p21 that CDK2 Point pre-Restriction the of immunofluorescence cell fixed simple phospho-Rb. from estimated be can fraction the for and even CDK2 Rb true fraction hyperphosphorylated with be the population between can the similarity this of state. the find Point Additionally, we pre-Restriction HLFs. (17), a primary work into recent mitosis to exits Contrary cells only that of argue the subset therefore, cross results, a they Our as 17). and (14, rises long Point activity Restriction as CDK2 after inhibition perturbations CDK2 these Mek the and in are withdrawal they serum (14). CDK2 G1 to contrast, early sensitive In in state. perturbed Point so post-Restriction are CDK2 anaphase, they after serum if immediately Thus, further even to cycle, is subjected cell proliferation when CDK2 rent inhibition, to Mek that, acute committed or observation a withdrawal born the that conclusion by is The supported cells p21. of with has high mitosis remainder subset and the exits while Rb cells p21, of hypophosphorylated low phos- subset and Rb a Rb hetero- of where hyperphosphorylated staining this p21, immunofluorescence and sensor, pop- by phorylation a CDK2 visible in the is cells geneity of CDK2 of independent subset uncommitted a Furthermore, the only enters that transformed, ulation types, not cell but cancerous immortalized show, primary, and to of analysis number fixed-cell a and across imaging time-lapse single-cell of (14, cycle. signaling cell MAPK previous of the during blockade 41) or 26, 23, 40) (20–23, stress rienced etn nta ht natvl yln el,teuncommitted the sug- cells, model, cycling actively this of CDK2 in of rise that, The aspects instead behavior. challenged cell gesting has in analysis heterogeneity cycle mask single-cell the and cell perturb cycle on which cell analysis, predominately population relied bulk and model cycle, uncom- synchronization studies this an cell groundbreaking into established the The born that of state. cross again Point round are they pre-Restriction cells one After mitted daughter to Point. resulting committed the Restriction deter- are and the to they cross signals point, can environmental this anaphase, they integrate after if hours to mine few thought first are are uncom- the cells are cells For they that proliferation. which suggests in to state cycle mitted Point cell pre-Restriction a the into of born model long-standing A Conclusions and Discussion CDK2 in Point. rise Restriction the the with at coincident activity begins p21 of degradation that tra- CDK2 that increasing conclude their We from deviate CDK2 not in p21. do jectory reaccumulate levels rapidly p21 MLN4924 contrast, In receiving Restric- cells the Point, crossing after tion p21 down-regulate to continue vehicle eea ie feiec ugs htacueo nr into entry of cause a that suggest evidence of lines Several combination a uses study this trend, recent this with line In inc low −/ ugssta h ieo h rlfrtv subpopulation proliferative the of size the that suggests tt ssmldol yasbe fcls(4 htexpe- that (14) cells of subset a by only sampled is state low el aeyetrteCDK2 the enter rarely cells tt fe ioi 1,2,3) ee eshow we Here, 31). 20, (14, mitosis after state CDKN1A low el ntetetwt L42 Fg 4E). 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CELL BIOLOGY CDK2inc CDK2emerge 4-7hr AB1 C CDK2low CDK2emerge 7-10hr H 0 1.6 2.3 2 1.4 1.4 2.2 1.9 mean mCit-p21 2.1 1.2 1.2 2 1.8 1 1 1.9 1.7 0.8 1.8 0.8 1.7 1.6

MCF10A 0.6 0.6 1.6 1.5 0.4 mean mCit-p21 1.5 0.4

mean CDK2 activity 1.4 0.2 1.4 mean CDK2 activity 0.2 -15 -10 -5 0 5 10 15 -15 -10 -5 0 5 10 15 -30 -20 -10 0 10 20 Time relative to anaphase (hr) Time relative to anaphase (hr) Time relative to R-point (hr) 1 H 0 1.8 2.6 1.8 2.3

1.6 2.5 1.6 mean p21-GFP 1.4 1.4 2.4 2.2 1.2 1.2 1 2.3 1 2.1 0.8 2.2 0.8 0.6 0.6 RPE-hTERT 2.1

mean p21-GFP 2 0.4 2 0.4 mean CDK2 activity 0.2 mean CDK2 activity 0.2 -15 -10 -5 0 5 10 15 -15 -10 -5 0 5 10 15 -30 -20 -10 0 10 20 Time relative to anaphase (hr) Time relative to anaphase (hr) Time relative to R-point (hr)

1 H 0 1.6 1.9 1.4 1.6 mean p21-mVenus 1.4 1.8 1.2 1.7 1.5 1.2 1.6 1 1.4 1 1.5 0.8 0.8 1.4 1.3

MCF7 0.6 0.6 1.3 1.2 1.2 0.4 0.4 1.1 1.1 mean CDK2 activity mean p21-mVenus 0.2 1 mean CDK2 activity 0.2 -15 -10 -5 0 5 10 15 -15 -10 -5 0 5 10 15 -20 -10 0 10 Time relative to anaphase (hr) Time relative to anaphase (hr) Time relative to R-point (hr)

1 H 0 2 2.4 2.8 mean mVenus-p21 1.8 1.6 2.6 1.6 1.4 2.2 2.4 1.2 1.4 2 1.2 2.2 1 1 2

U2OS 0.8 1.8 0.8 1.8 0.6 0.6 1.6 1.6 0.4 1.4 0.4 mean mVenus-p21 mean CDK2 activity 0.2 1.2 mean CDK2 activity 0.2 1.4 -15 -10 -5 0 5 10 15 -15 -10 -5 0 5 10 15 -20 -10 0 10 Time relative to anaphase (hr) Time relative to anaphase (hr) Time relative to R-point (hr) 1 H 0 1.6 1.7 1.4 1.6 1.2 1 1.5 0.8 1.4 1.3

HCT116 0.6 0.4 1.2 mean CDK2 activity 0.2 mean p21-mVenus 1.1 -15 -10 -5 0 5 10 15 -15 -10 -5 0 5 10 15 Time relative to anaphase (hr) Time relative to anaphase (hr) D E 1.8 2.1 1.8 2.1 1.8 2.1 1.8 2.1 mean mCit-p21 mean mCit-p21 mean mCit-p21 mean mCit-p21 1.9 1.9 1.4 1.4 1.4 1.9 1.4 1.9 +DMSO +MLN4924 +MLN4924 1.7 1.7 +DMSO 1.7 1.7 1 1 1 1 1.5 1.5 1.5 1.5 0.6 0.6 0.6 1.3 0.6 1.3 1.3 1.3

mean CDK2 activity 0.2 1.1 mean CDK2 activity 0.2 1.1 mean CDK2 activity 1.1 mean CDK2 activity 0.2 1.1 -8 -4 0 4 8 -8 -4 0 4 8 0.2 -8 -4 0 4 8 12 -8 -4 0 4 8 12 Time relative to R-point (hr) Time relative to R-point (hr) Time relative to anaphase (hr) Time relative to anaphase (hr)

Fig. 4. p21 up-regulation precedes mitosis in mother cells whose daughters enter the CDK2low state after mitosis, and p21 is degraded at the Restriction Point. (A–C) Cells expressing the CDK2 sensor and fluorescent p21 from the endogenous locus (with the exception of U2OS, which were induced to endoge- nous levels with doxycycline) were imaged for 24 h, tracked, classified according to CDK2 activity after anaphase, averaged, and aligned to either the time of anaphase (A and B) or the Restriction Point (C), which was defined as the time that CDK2 activity begins to rise. (A) CDK2 activity traces were classified as CDK2low if the CDK2 activity dropped below the cutoff indicated in Materials and Methods and remained below this cutoff for the remainder of the imaging period. Traces were classified as CDK2emerge 4–7 h or CDK2emerge 7–10 h if the CDK2 activity dropped below the cutoff indicated in Materials and Methods and rose above this cutoff 4–7 or 7–10 h after anaphase, respectively; otherwise, traces were classified as CDK2inc. A CDK2emerge population does not exist for HCT116 cells because few cells enter the CDK2low state. (B) Averaged p21 traces for the categories described in A. The purple line above each plot indicates the hypothesis testing result of a two-sample t test with P value < 0.05, comparing p21 in CDK2inc with CDK2low cells at each time point. (C) Averaged p21 and CDK2 activity traces for CDK2emerge7–10 h cells aligned to the Restriction Point. (D) Averaged p21 (orange) and CDK2 activity (black) traces from MCF10A CDK2emerge4–7 h cells aligned to the Restriction Point. Only those cells receiving DMSO (Left) or 1.4 µM MLN4924 (Right) 3–4 h (gray shading) after CDK2 had begun to rise are included. (E) Averaged p21 (orange) and CDK2 activity (black) traces from MCF10A CDK2low cells that received DMSO (Left) or 1.4 µM MLN4924 (Right) 3–5 h (gray shading) after anaphase. Error bars represent SEM. Data presented here are representative of two biological replicates. Cell counts for each group can be found in SI Appendix. mCit-p21, mCitrine-tagged p21 from the endogenous CDKN1A locus. p21 axis units are in log(10).

E8224 | www.pnas.org/cgi/doi/10.1073/pnas.1722446115 Moser et al. 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CELL BIOLOGY data support the alternative model in Fig. 1B, where the decision GCGCCATGTCAGAACCGGCTGGG. Cells were transfected in antibiotic-free to proliferate is informed by events occurring at the end of medium and supplemented with 1 µM SCR7, an inhibitor of nonhomol- the previous cell cycle and only a subset of cells resets to a ogous end joining (SML1546; Sigma). Cells were allowed to recover and pre-Restriction Point state after mitosis. expand before the YFP-positive cells were isolated via FACS. Clonal lines were then expanded and validated by PCR, genomic DNA sequencing, Materials and Methods immunofluorescence, and Western blotting (SI Appendix, Figs. S3 and S4). Cells used for time-lapse live-cell microscopy were transduced with a Cell Culture and Maintenance. MCF10A (ATCC CRL-10317) were maintained nuclear marker [H2B-mTurquoise or H2B-miFP (53)] and the CDK2 sensor DHB in DMEM/F12 supplemented with 5% horse serum, 100 ng/mL cholera toxin, (14) using established lentivirus protocols, and double-positive cells were 20 ng/mL EGF, 10 µg/mL insulin, 0.5 µg/mL hydrocortisone, and 100 µg/mL sorted by FACS. CDK2 activity was read out as the cytoplasmic to nuclear ratio each penicillin and streptomycin. RPE-hTERT [ATCC CRL-4000 and those of the DHB sensor. Primary HLFs were transduced with H2B-mTurquoise and tagged with p21-GFP (21)] were maintained in DMEM/F12 supplemented DHB-mCherry at passage 2 and were imaged at passage 5. with 10% FBS, 1× Glutamax, and 100 µg/mL each penicillin and strepto- mycin. MCF7 were maintained in RPMI supplemented with 10% FBS, 1× Glu- tamax, and 100 µg/mL each penicillin and streptomycin. U2OS and HCT116 Flow Cytometry. MCF10A were harvested through trypsinization and resus- were maintained in McCoy’s 5A supplemented with 10% FBS and 100 µg/mL pended in DMEM/F12 supplemented with 20% horse serum. The cell pellet was washed twice with PBS, and the cells were fixed and permeabilized each penicillin and streptomycin. Primary fetal HLFs (507-75f; Cell Applica- ◦ tions) were maintained in Human Lung Fibroblast Growth Medium (516-500; with ice-cold methanol at −20 C. The population was then split, and cells Cell Applications), in contrast with the growth conditions used in ref. 17. were stained for pHH3 (CST 9706) and either p21 (CST 2947S) or phospho-Rb For live-cell imaging, each cell line was maintained in a phenol red-free ver- (S807/811; CST 8516S) followed by secondary antibody staining. Fluores- sion of their growth media; HLFs were imaged in Human Lung Fibroblast cence intensities for each signal were read on a MoFlo Cytomation and Growth Medium. U2OS were seeded with 10 ng/µL doxycycline to induce analyzed using custom MATLAB scripts. endogenous-level expression of fluorescent p21 (35) for at least 24 h before imaging. All cell lines were grown in a humidified incubator (5% CO2, Imaging and Image Processing. Time-lapse imaging, immunofluorescence, 37 ◦C). MCF7 and HCT116 cells with p21 tagged at the endogenous locus image processing, and classification of populations were conducted as previ- were provided by the laboratory of Galit Lahav, Department of Systems Biol- ously described (15, 20). Both fixed and time-lapse microscopy images were ogy, Harvard Medical School, Boston (34). RPE-hTERT with p21 tagged at the processed as previously described (26). The tracking code is available for endogenous locus was provided by the laboratory of Chris Bakal, Division of download here: https://github.com/scappell/Cell tracking. Additional detail Cancer Biology, Institute of Cancer Research, London (21). U2OS cells with is available in SI Appendix. inducible p21 were provided by the laboratory of Jeanette Cook, Depart- ment of Biochemistry and Biophysics, University of North Carolina at Chapel ACKNOWLEDGMENTS. We thank Galit Lahav, Jean Cook, and Chris Bakal Hill, Chapel Hill, NC (35). for cell lines with fluorescently tagged p21 and the members of the labora- tory of S.L.S. for general help, especially Chen Yang for H2B-mIFP lentivirus. This work was supported by NIH Training Grant T32 GM 8759-16 (to J.M.), Cell Line Generation. Low-passage wild-type MCF10A were transfected with 0 NIH Instrumentation Grant S10OD021601, NIH K22 Early-Career Investigator a plasmid encoding CRISPR-Cas9, single-guide (sg)RNA targeting the 5 Award 1K22CA188144-01, a Boettcher Webb-Waring Early-Career Investi- end of CDKN1A, and a repair template encoding the mCitrine gene; gator Award, a Kimmel Scholar Award SKF16-126, a Pew-Stewart Scholar the left and right homology arms extended for 1,000 bp upstream and for Cancer Research Award, a Searle Scholar Award SSP-2016-1533, and a downstream of the CDKN1A start codon. The sgRNA sequence used was Beckman Young Investigator Award (to S.L.S.).

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E8226 | www.pnas.org/cgi/doi/10.1073/pnas.1722446115 Moser et al. Downloaded by guest on September 26, 2021 Downloaded by guest on September 26, 2021 4 tuiL rn H(08 elccehtrgniydrcstetmn fnua stem neural of timing the directs heterogeneity cycle generated Cell lesions (2018) AH DNA Brand around L, Otsuki form 44. bodies nuclear 53bp1 (2011) during al. et required C, in are Lukas domains OPT D1 43. 53bp1-containing induces stress cyclin Replication (2011) al. and et JA, Ras Harrigan 42. Cellular (1999) DW Stacey M, a Hitomi via 41. stability p21 Nov of C, Bakal inhibition S, Cdk2-dependent Cooper AR, (2005) Barr FS, CG Heldt Maki 40. L, cyclin- Nie G1 a H, p53 p27kip1, Zhu and of 39. degradation expression Cell-free (1999) cycle A Cell Koff DM, (1994) Gitig Y H, Nguyen Xiong 38. MA, Nichols CW, Jenkins Y, Li 37. oe tal. et Moser elatvto rmquiescence. from activation cell stress. replication under chromosomes 243–253. of transmission mitotic by cells. 3t3 NIH cells. G1 cycling in periods cycle 4632. cell different human in damage DNA cells. endogenous to response in decision proliferation–quiescence motif. cyclin-binding C-terminal proteasome. the and activity CDK2 Biol on Cell dependent p21. is inhibitor, kinase inhibitor dependent kinase cyclin-dependent the 2268. of regulation rcNt cdSiUSA Sci Acad Natl Proc elBiol Cell J 19:1190–1201. 193:97–108. 115:2532–2537. Science ilChem Biol J 360:99–102. kB(08 opeesv oe o the for model comprehensive A (2018) B ak ´ 280:29282–29288. o elBiol Cell Mol Oncogene a elBiol Cell Nat 19:4623– 9:2261– Mol 13: 3 uD ta.(05 aual ooei nrrdfloecn rti o protein for protein fluorescent infrared monomeric naturally A (2015) al. et D, Yu induce 53. synergistically inhibitors autophagy and critical CDK4/6 (2017) is al. USP11 et S, by Vijayaraghavan p21 52. of and stabilization regulators and cycle Deubiquitylation (2018) Cell al. inhibitors: et CDK T, (2008) Deng JM 51. Roberts SF, Cdc25 Dowdy through A, checkpoints Besson mammalian Regulating 50. (2003) a GF in Ras Draetta on M, depends cells Donzelli cycling in 49. production D1 Cyclin (1999) suppression. DW tumor Stacey p53 M, Hitomi of mediator 48. potential a WAF1, (1993) al. et vary WS, damage El-Deiry DNA to 47. response and in dynamics ubiquitylation p53 (2017) p21 G Lahav of J, regulation Stewart-Ornstein 46. PCNA-dependent (2008) al. et T, Abbas 45. aeigi vivo. in labeling . negative E cyclin cytoplasmic positive Rb in senescence responses. damage DNA 4678–4683. and progression cell-cycle for beyond. inactivation. manner. cell-cycle-specific kinase 75:817–825. the of activity and repair DNA of efficiency by shaped ATM. are and lines complex. cell across ligase ubiquitin CRL4cdt2 the 2506. via degradation c Signal Sci e Cell Dev MORep EMBO 10:eaah6671. a Methods Nat 14:159–169. 4:671–677. urBiol Curr 12:763–765. 9:1075–1784. PNAS | o.115 vol. rcNt cdSiUSA Sci Acad Natl Proc a Commun Nat ee Dev Genes | o 35 no. | 22:2496– 8:15916. E8227 115: Cell

CELL BIOLOGY