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Basal P21 Controls Population Heterogeneity in Cycling and Quiescent Cell Cycle States

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Basal P21 Controls Population Heterogeneity in Cycling and Quiescent Cell Cycle States Basal p21 controls population heterogeneity in cycling and quiescent cell cycle states K. Wesley Overtona, Sabrina L. Spencerb, William L. Noderera, Tobias Meyerb, and Clifford L. Wanga,1 Departments of aChemical Engineering and bChemical and Systems Biology, Stanford University, Stanford, CA 94305 Edited by Charles S. Peskin, New York University, Manhattan, NY, and approved August 27, 2014 (received for review May 27, 2014) Phenotypic heterogeneity within a population of genetically identical SCF/Skp2 then ubiquitinates p21, targeting it for proteasomal cells is emerging as a common theme in multiple biological systems, degradation (13). Thus, p21 both regulates and, through the ac- including human cell biology and cancer. Using live-cell imaging, flow tion of E3 ubiquitin ligase complexes that target p21, is regulated cytometry, and kinetic modeling, we showed that two states—quies- by active CDK2 bound to Cyclin E. cence and cell cycling—can coexist within an isogenic population of The p21–CDK2 control scheme is an example of a double- human cells and resulted from low basal expression levels of p21, a negative feedback loop. When stochastic gene expression leads Cyclin-dependent kinase (CDK) inhibitor (CKI). We attribute the p21- to fluctuations in factors involved in positive or double-negative dependent heterogeneity in cell cycle activity to double-negative feedback regulation, distinct cellular states within a population feedback regulation involving CDK2, p21, and E3 ubiquitin ligases. can arise (1, 14–18). Because of the role of p21 in the double- In support of this mechanism, analysis of cells at a point before cell negative feedback regulation of cell cycle activity, we hypothesized cycle entry (i.e., before the G1/S transition) revealed a p21–CDK2 axis that p21 controlled population heterogeneity in quiescent and that determines quiescent and cycling cell states. Our findings suggest cycling cell states. In mammalian systems, there are few examples a mechanistic role for p21 in generating heterogeneity in both normal where inactivation of a single gene leads to loss of heterogeneity in tissues and tumors. cell cycle activity. Here, in comparing wild type (WT), deficient, and ectopically restored p21 genetic backgrounds, we reveal a di- tumor heterogeneity | cell dormancy | synthetic uORF | rect role for low basal levels of p21 in controlling population nongenetic cell heterogeneity | positive feedback loop heterogeneity in cell cycle activity. population of genetically identical cells can exhibit phe- Results A notypic heterogeneity (1, 2). That is, even when cells with Live-Cell Imaging Revealed p21-Dependent Population Heterogeneity. the same DNA sequence and epigenetic markings experience the To gauge cell cycle activity, we tracked the activity of CDK2 same environment, there can be cell to cell variability. Researchers in single cells by using a fluorescent reporter consisting of the have reported that cells within a clonal population can vary by size C-terminal CDK2 phosphorylation domain of DNA helicase and morphology (3), cell cycle activity (4, 5), lifespan (6), and B (DHB) fused to YFP (19). In G0 or G1 phase cells, the un- receptor sensitivity to cytokines (7, 8). Furthermore, population phosphorylated reporter is located primarily in the nucleus (Fig. heterogeneity can determine biological outcome: instead of a 1A and Fig. S1A). During the G1/S transition, S, and G2 phases, homogenous stem cell population where cells renew equally, a CDK2 phosphorylates the reporter, causing it to translocate from subpopulation can remain dormant until needed (5), a bacterial the nucleus to the cytoplasm (Movie S1). As a result, the cyto- colony can harbor a subpopulation resistant to antibiotics (3), plasmic-to-nuclear ratio of DHB-YFP can be used to monitor and a clonal tumor can harbor a subpopulation that survives CDK2 activity and cell cycle progression (12). radiation or chemotherapy (9). Although many instances of nongenetic population heterogeneity have been documented, Significance fewer underlying regulatory mechanisms governing this hetero- geneity have been reported. When cells exhibit heterogeneity in cell cycle activity, one Population heterogeneity can make the treatment of tumors subpopulation actively cycles, whereas another subpopulation more challenging. Whereas a therapeutic agent may be effec- tive against one fraction of a population, it may be less effec- remains quiescent. The main activators of the cell cycle are tive against another fraction. Although heterogeneity can be Cyclin-dependent kinases (CDKs) bound to Cyclin proteins, and genetic and attributed to mutations, there can also be non- a major class of cell cycle inhibitors is CDK inhibitors (CKIs). genetic heterogeneity, where a clonal population can harbor One CKI, p21, was first discovered in the mid-1990s as an in- distinct subpopulations. Here, we identified a single gene, p21, hibitor of CDKs in G1 phase (10) and a factor that was tran- that was responsible for population heterogeneity in cell cycle scriptionally activated by p53 (11). Since its discovery, much of activity and explain that this heterogeneity can arise from the research involving p21 has focused on its role in arresting the regulatory relationships of p21 with Cyclin-dependent kinase 2 cell cycle in response to activation by p53 after DNA damage. (CDK2) and E3 ubiquitin ligases. We suggest that, instead of However, cells without exogenous DNA damage still express using CDK inhibitors (CKIs) in cancer therapy, CKIs themselves p21. For example, single-cell analysis of p21 and CDK2 activity should be targeted. Given concurrently with chemotherapy led Spencer et al. (12) to propose that p21 is involved in the agents, CKI inhibitors would reduce tumor heterogeneity and cellular decision to enter the cell cycle under normal conditions. thus increase chemotherapy efficacy. Because p21 is a potent inhibitor of Cyclin-CDK complexes, the cell must actively regulate p21 levels under normal conditions Author contributions: K.W.O. and C.L.W. designed research; K.W.O. performed research; to promote the transition from G1 to S phase. At this stage of the S.L.S., W.L.N., and T.M. contributed new reagents/analytic tools; K.W.O. and C.L.W. ana- cell cycle, CDK2 activity begins to increase and causes inactivation lyzed data; and K.W.O. and C.L.W. wrote the paper. of the anaphase promoting complex (APC). The inactivation of The authors declare no conflict of interest. the APC allows Skp2 levels to increase, because Skp2 is a target of This article is a PNAS Direct Submission. the APC-Cdh1 complex. The E3 ubiquitin ligase complex of Skp1/ 1To whom correspondence should be addressed. Email: [email protected]. Cullin/F box (SCF) and Skp2 recognizes p21 that has bound This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Cyclin E-CDK2 complexes and been phosphorylated by CDK2. 1073/pnas.1409797111/-/DCSupplemental. E4386–E4393 | PNAS | Published online September 29, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1409797111 Downloaded by guest on October 2, 2021 A PNAS PLUS G1 S G2 M G1 Time after Mitosis (hrs) 1.4 3.6 8.8 19 20.6 21 22.4 G1 G2 G1 hhrsrs hrsrsr hrshrss hrshhrrsrs hrs hrshrh s hrsrsr 2 S M H2B- mTurq 1 CDK2 Activity YFP DHB- (DHB-YFP cyt/nuc) 0 0 12 24 DHB-YFP 0.62 0.78 1.20 1.32 0.64* 0.64* 0.67 Time after (cyt/nuc) Mitosis (hrs) B C p21-/- 0.2 ng/mL EGF 5 ng/mL EGF 20 ng/mL EGF GFP-p21-ER 0.05% Serum 1.25% Serum 5% Serum 2 2 2 WT 1 1 1 0 0 0 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 /- YFP cyt/nuc) 2 2 2 - - CDK2 Activity CDK2 Activity ACC/ACC/ACC ACC/ACC ACC/ACC/ACC/ACC CGA GGG/ACC UAA WT UCU/ACC WT+IR p21 p21-/- 1 1 1 GCCACC GFP- (DHB p21-ER 0 0 0 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 Time (hrs) p21 Beta- actin D p21-/- RFP-p21-ER ACC ACC ACC FP-p21-ER 0.2 ng/mL EGF 5 ng/mL EGF 20 ng/mL EGF ytivitc 0.05% Serum 1.25% Serum 5% Serum ) 2 2 2 -YFP nuc A 1 1 1 2KDC cyt/ (DHB 0 0 0 SYSTEMS BIOLOGY 3 10 103 103 -ER 1 1 1 p21 10 10 10 -1 -1 -1 RFP- 10 10 0 10 20 30 40 10 0 10 20 30 40 0 10 20 30 40 Time (hrs) Fig. 1. p21 causes population heterogeneity in cell cycle activity. (A) Single-cell tracking of cell cycle progression using a DHB-YFP reporter of CDK2 activity. (Left) CDK2-dependent translocation of DHB-YFP from the nucleus to the cytoplasm occurs during G1/S, S, and G2 phases. DHB-YFP returns to the nucleus after mitosis (M) and remains during early G1. H2B-mTurquoise (H2B-mTurq) is the nuclear marker. *During mitosis, after nuclear membrane collapse, the cytoplasmic-to-nuclear ratio (cyt/nuc) is not well-defined. (Right) CDK2 activity (cyt/nuc DHB-YFP) vs. time for cell in Left; arrows indicate depicted time points. − − (B) Single-cell traces of CDK2 activity in MCF10A WT and p21-deficient ( / ) cycling (blue) and quiescent (red) cells. Traces for cycling cells were aligned to the second mitosis. B and D show results for populations grown under varying growth factor concentrations (EGF and serum). (C) Immunoblot of p21. The right three immunoblot lanes show WT and p21−/− cells without ionizing irradiation (IR) and WT with 10 Gy IR. The left eight immunoblot lanes show expression of GFP-p21-ER in p21−/− cells. Expression levels were tuned by varying translation initiation site bases preceding the GFP-p21-ER gene and/or synthetic uORFs.
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