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• W2012258557 abstract "•Increased Dyrk1a dosage steeply increases the duration and variability of G1 phase•A p21-cyclin D1 signaling map specifies cell cycle entry and exit decisions•Dosage of Dyrk1a controls the relative cyclin D1 and p21 protein levels•Extra-dosage of Dyrk1a explains the cell cycle changes in Down syndrome cells Mammalian cells have a remarkable capacity to compensate for heterozygous gene loss or extra gene copies. One exception is Down syndrome (DS), where a third copy of chromosome 21 mediates neurogenesis defects and lowers the frequency of solid tumors. Here we combine live-cell imaging and single-cell analysis to show that increased dosage of chromosome 21-localized Dyrk1a steeply increases G1 cell cycle duration through direct phosphorylation and degradation of cyclin D1 (CycD1). DS-derived fibroblasts showed analogous cell cycle changes that were reversed by Dyrk1a inhibition. Furthermore, reducing Dyrk1a activity increased CycD1 expression to force a bifurcation, with one subpopulation of cells accelerating proliferation and the other arresting proliferation by costabilizing CycD1 and the CDK inhibitor p21. Thus, dosage of Dyrk1a repositions cells within a p21-CycD1 signaling map, directing each cell to either proliferate or to follow two distinct cell cycle exit pathways characterized by high or low CycD1 and p21 levels. Mammalian cells have a remarkable capacity to compensate for heterozygous gene loss or extra gene copies. One exception is Down syndrome (DS), where a third copy of chromosome 21 mediates neurogenesis defects and lowers the frequency of solid tumors. Here we combine live-cell imaging and single-cell analysis to show that increased dosage of chromosome 21-localized Dyrk1a steeply increases G1 cell cycle duration through direct phosphorylation and degradation of cyclin D1 (CycD1). DS-derived fibroblasts showed analogous cell cycle changes that were reversed by Dyrk1a inhibition. Furthermore, reducing Dyrk1a activity increased CycD1 expression to force a bifurcation, with one subpopulation of cells accelerating proliferation and the other arresting proliferation by costabilizing CycD1 and the CDK inhibitor p21. Thus, dosage of Dyrk1a repositions cells within a p21-CycD1 signaling map, directing each cell to either proliferate or to follow two distinct cell cycle exit pathways characterized by high or low CycD1 and p21 levels. The term “dosage effect” is often used to describe a heterozygous gene loss or the presence of an extra gene copy that causes a profound change in phenotype. Reported heterozygous phenotypes are relatively rare in mammals, with ∼75% of known loss-of-function mutations in human diseases being recessive (Jimenez-Sanchez et al., 2001Jimenez-Sanchez G. Childs B. Valle D. Human disease genes.Nature. 2001; 409: 853-855Crossref PubMed Scopus (316) Google Scholar). This suggests that compensation mechanisms exist for many genes to accommodate 2-fold protein level changes. A model case of a mammalian dosage effect is Down syndrome (DS), where a third copy of chromosome 21 (trisomy 21) is associated with mental retardation, early onset of Alzheimer’s diseases, and a number of additional phenotypic changes (Coyle et al., 1988Coyle J.T. Oster-Granite M.L. Reeves R.H. Gearhart J.D. Down syndrome, Alzheimer’s disease and the trisomy 16 mouse.Trends Neurosci. 1988; 11: 390-394Abstract Full Text PDF PubMed Scopus (78) Google Scholar). To determine how dosage effects create phenotypes and to understand how dosage mechanisms might be used for cell regulation, we focused on the protein Dyrk1a, dual-specificity tyrosine (Y)-phosphorylation-regulated protein kinase 1A, whose gene is localized within the DS-critical region on chromosome 21. We selected Dyrk1a since it is one of the relevant contributors to the neurological abnormalities associated with DS (Park et al., 2009Park J. Song W.J. Chung K.C. Function and regulation of Dyrk1A: towards understanding Down syndrome.Cell. Mol. Life Sci. 2009; 66: 3235-3240Crossref PubMed Scopus (140) Google Scholar) and since it clearly shows dosage effects on neurogenesis and brain development on its own. For instance, Dyrk1a heterozygous knockout mice show reduced brain size whereas Dyrk1a overexpression was sufficient to induce learning defects and delay neuromotor development in mice (Altafaj et al., 2001Altafaj X. Dierssen M. Baamonde C. Martí E. Visa J. Guimerà J. Oset M. González J.R. Flórez J. Fillat C. Estivill X. Neurodevelopmental delay, motor abnormalities and cognitive deficits in transgenic mice overexpressing Dyrk1A (minibrain), a murine model of Down’s syndrome.Hum. Mol. Genet. 2001; 10: 1915-1923Crossref PubMed Scopus (324) Google Scholar, Fotaki et al., 2002Fotaki V. Dierssen M. Alcántara S. Martínez S. Martí E. Casas C. Visa J. Soriano E. Estivill X. Arbonés M.L. Dyrk1A haploinsufficiency affects viability and causes developmental delay and abnormal brain morphology in mice.Mol. Cell. Biol. 2002; 22: 6636-6647Crossref PubMed Scopus (251) Google Scholar). While most studies on Dyrk1a focused on neuronal defects, studies of Dyrk1a orthologs in yeast, C. elegans, Dictyostelium, and mammals have shown that Dyrk family proteins also have conserved roles in regulating proliferation (Becker, 2012Becker W. Emerging role of DYRK family protein kinases as regulators of protein stability in cell cycle control.Cell Cycle. 2012; 11: 3389-3394Crossref PubMed Scopus (72) Google Scholar, Litovchick et al., 2011Litovchick L. Florens L.A. Swanson S.K. Washburn M.P. DeCaprio J.A. DYRK1A protein kinase promotes quiescence and senescence through DREAM complex assembly.Genes Dev. 2011; 25: 801-813Crossref PubMed Scopus (175) Google Scholar). Since neurogenesis encompasses transitions from proliferating precursor cells to terminally differentiated cells, it is conceivable that Dyrk1a contributes to the neurogenesis defect indirectly through regulating the cell cycle exit decision during neurogenesis. Supporting such a hypothesis, Litovchick et al., 2011 identified a potential link from Dyrk1a to Lin52 and DREAM complex assembly, which in turn regulates quiescence and senescence. We recently identified Dyrk1a in an siRNA screen of nerve growth factor (NGF)-triggered differentiation using a PC12 model system (Chen et al., 2012Chen J.Y. Lin J.R. Cimprich K.A. Meyer T. A two-dimensional ERK-AKT signaling code for an NGF-triggered cell-fate decision.Mol. Cell. 2012; 45: 196-209Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Among a set of regulatory proteins that we targeted by siRNA, Dyrk1a was the strongest hit when we separated a group of genes that controlled the proliferative state under NGF stimulation. Other genes in this group were cell cycle regulatory proteins such as cyclin D1 (CycD1). This suggested a potential role for Dyrk1a in regulating the G1 phase, where CycD1 acts and where the cell cycle entry versus exit decision is made (Massagué, 2004Massagué J. G1 cell-cycle control and cancer.Nature. 2004; 432: 298-306Crossref PubMed Scopus (969) Google Scholar). However, it remained unclear whether Dyrk1a plays a role in cell cycle regulation under normal growth condition and, if so, which cell cycle steps are controlled by Dyrk1a. Moreover, it was unknown how Dyrk1a’s dosage might contribute to cell cycle regulation in normal versus DS-derived cells. Here, we investigated the role of Dyrk1a in controlling cell cycle entry and exit using long-term time-lapse microscopy, single-cell image analysis, and biochemical validation. We show that Dyrk1a mediates a dose-dependent increase in the duration and variability of the G1 phase via direct phosphorylation and subsequent degradation of CycD1. In contrast, knockdown of Dyrk1a greatly increased CycD1 protein levels and split cells into two fates, with one subpopulation accelerating the cell cycle with a significantly shortened G1 duration and the other entering an arrested state. We show that this arrested state results from the costabilization between CycD1 and p21, which is distinct from a quiescent state reached during serum starvation where p21 and CycD1 are both low. Finally, in DS-derived fibroblasts, we observed a prolongation of G1, a reduced percentage of cycling cells, and significantly lower CycD1 levels. These phenotypes could be rescued by knockdown or inhibition of Dyrk1a. Together, our study introduces a dosage effect for cell fate whereby Dyrk1a controls the relative CycD1 to p21 levels to guide cells either toward cell cycle entry or toward one of two distinct cell cycle exit pathways. We monitored the fraction of cells in S phase to test whether Dyrk1a has a role in regulating cell cycle progression under normal growth condition in nontransformed neonatal foreskin fibroblasts (BJ-5ta cells). Using two independent siRNAs, knockdown of Dyrk1a significantly enhanced the percentage of cells in S phase, consistent with prior observations (Figures 1A and 1B ) (Litovchick et al., 2011Litovchick L. Florens L.A. Swanson S.K. Washburn M.P. DeCaprio J.A. DYRK1A protein kinase promotes quiescence and senescence through DREAM complex assembly.Genes Dev. 2011; 25: 801-813Crossref PubMed Scopus (175) Google Scholar). Metazoans typically regulate proliferation by controlling exit and re-entry into the cell cycle in the G1 phase. We therefore examined whether Dyrk1a regulates the duration of G1 by combining fluorescent reporters with long-term time-lapse microscopy. We expressed two fluorescent protein conjugates: Histone 2B-mTurquoise (H2B-mTurq) to track cell nuclei over time and a cell cycle reporter, mCherry-Geminin (mChy-Geminin), to measure the time between anaphase and the G1/S transition in each cell (Figures S1A and S1B). Geminin is a substrate of the APC (anaphase-promoting complex) E3 ubiquitin ligase and is degraded at anaphase (tM) during mitosis and starts to gradually accumulate at the G1/S transition (ts) when APC is inactivated (Figures 1C and 1D) (Sakaue-Sawano et al., 2008Sakaue-Sawano A. Kurokawa H. Morimura T. Hanyu A. Hama H. Osawa H. Kashiwagi S. Fukami K. Miyata T. Miyoshi H. et al.Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.Cell. 2008; 132: 487-498Abstract Full Text Full Text PDF PubMed Scopus (1478) Google Scholar). The time spent between M and S phase (ts − tM1), defined in this work as “G1,” can be measured computationally by automated image analysis based on the rapid drop and subsequent rise in mChy-Geminin levels (Figure 1D). When we aligned individual traces to the time of mitosis, G1 phase was highly variable under normal growth conditions (Figure 1E). Strikingly, either knockdown or inhibition of Dyrk1a using Harmine (a Dyrk1a kinase inhibitor) greatly shortened G1 duration (median 12.4 hr versus 7–8 hr) and at the same time markedly reduced its variability (Figure 1F). Thus, Dyrk1a increases the median duration between mitosis and DNA replication and renders this time interval more variable. We next asked whether the variability of G1 duration under normal growth condition can be explained by a dosage effect whereby small changes in the expression of Dyrk1a may cause a nonlinear increase in the duration of the cell cycle. To gain control over the single-cell expression level of Dyrk1a, we knocked down endogenous Dyrk1a and replaced it with a tetracycline-inducible, mCitrine-tagged Dyrk1a (tet-mCit-Dyrk1a, designed to be Dyrk1a siRNA resistant) (Figure 2A) using BJ-5ta cells (stably expressing H2B-mTurq and mChy-Geminin). The tet-mCit-Dyrk1a rescued the Dyrk1a knockdown effect on G1 duration (Figure 2B), arguing that the conjugated form of Dyrk1a maintains the function of the endogenous kinase. Moreover, cells overexpressing wild-type Dyrk1a (WT) but not catalytically inactive Dyrk1a (K188R, hereafter “KR”) exhibited prolonged G1 (Figure 2B) as well as an increased fraction of noncycling cells (entirely blue traces, Figure 2C), suggesting that Dyrk1a’s kinase activity is required for its cell cycle effect. We then asked if small increases in the single-cell expression level of mCit-Dyrk1a alter G1 duration by quantifying the fluorescence intensity of mCit-Dyrk1a in each of the analyzed cells at the end of time-lapse imaging (Figure 2D). The absolute expression level was calibrated to endogenous expression of Dyrk1a (Figure S2 and Supplemental Experimental Procedures). This analysis revealed a sensitive Dyrk1a dose- and kinase activity-dependent increase in G1 duration. The sensitive region where mCit-Dyrk1a WT expression steeply increased G1 duration was in a window 100%–200% above the endogenous Dyrk1a levels (Figure 2E), exactly in the region where the dosage effect of Dyrk1a would be expected to occur in DS patients. Thus, given that the typical noise in single-cell protein expression level is ∼30% (Niepel et al., 2009Niepel M. Spencer S.L. Sorger P.K. Non-genetic cell-to-cell variability and the consequences for pharmacology.Curr. Opin. Chem. Biol. 2009; 13: 556-561Crossref PubMed Scopus (160) Google Scholar), the high variability of the G1 duration in the presence of Dyrk1a (Figure 1) can at least in part be explained by a combination of a dosage effect and a natural variation in endogenous Dyrk1a levels. We then focused on the mechanism by which Dyrk1a mediates its dosage effect on the G1 phase by examining whether Dyrk1a may regulate CycD1, a key driver of cell cycle entry. Immunostaining and single-cell analysis showed that increased levels of Dyrk1a significantly decreased the levels of CycD1 within the same range where a steep increase in G1 duration occurs (Figure 2F). This decrease in CycD1 level upon Dyrk1a induction was confirmed by western blot analysis (Figure 2G, lanes 2 and 4). We also tested whether reduced Dyrk1a activity could increase CycD1 levels. Indeed, a loss of function of Dyrk1a using either si-Dyrk1a or Harmine resulted in a marked increase in CycD1 expression (Figure 2G, lanes 1 and 2, and Figure 3A). We further showed that the proliferation increase upon Dyrk1a knockdown or inhibition can be suppressed by co-knockdown of CycD1 (Figure 3B). This argues that CycD1 acts downstream of Dyrk1a and that the regulation of CycD1 levels by Dyrk1a is an important mechanism through which Dyrk1a regulates the G1 phase of the cell cycle. We next tested whether CycD1 protein levels are directly regulated by Dyrk1a by examining the kinetics of CycD1 changes after inhibiting Dyrk1a. Harmine addition triggered a rapid increase in CycD1 with a half-time of 50 min (Figure 3C). In the same experiment, we observed an even more rapid decrease (t1/2 = 25 min) in CycD1 Thr286 phosphorylation following Harmine addition (Figure 3D). Since phosphorylation of CycD1 at Thr286 by other kinases has previously been associated with a decrease in CycD1 stability (Diehl et al., 1997Diehl J.A. Zindy F. Sherr C.J. Inhibition of cyclin D1 phosphorylation on threonine-286 prevents its rapid degradation via the ubiquitin-proteasome pathway.Genes Dev. 1997; 11: 957-972Crossref PubMed Scopus (647) Google Scholar), we considered that the regulation of CycD1 levels by Dyrk1a might result from phosphorylation-mediated CycD1 degradation. Indeed, using the proteasome inhibitor MG132, we found that the effect of Dyrk1a on the CycD1 concentration is lost (Figure 3E). Thus, the Dyrk1a-mediated CycD1 downregulation is controlled by proteasome-driven degradation. The rapid dephosphorylation of the CycD1 Thr286 site after Harmine addition (Figure 3D) suggests that CycD1 might be directly phosphorylated by Dyrk1a. To test this, we first used coimmunoprecipitation to examine whether Dyrk1a interacts with CycD1. As shown in Figure 3F, FLAG-tagged Dyrk1a pulls down mCit-fused CycD1. This interaction was bidirectional, as recombinant GST-CycD1 was able to pull down endogenous Dyrk1a, supporting the interpretation that Dyrk1a directly interacts with CycD1 (Figure 3G). To test for a direct phosphorylation of CycD1 by Dyrk1a, we conducted an in vitro kinase assay using recombinant CycD1 as a substrate. As shown in Figure 3H, the WT Dyrk1a, but not the KR mutant, phosphorylated CycD1 in vitro. Furthermore, the Dyrk1a activity toward the T286A mutant of CycD1 was dramatically reduced (Figures 3H and S3A), while a construct with the T288A mutation on CycD1 retained a similar level of phosphorylation (Figure 3H). The latter result points to a role of Dyrk1a in regulating CycD1 Thr286, contrasting with a previous study showing selective phosphorylation on the Thr288 site by Myrk/Dyrk1b, a kinase with sequence homology to Dyrk1a (Takahashi-Yanaga et al., 2006Takahashi-Yanaga F. Mori J. Matsuzaki E. Watanabe Y. Hirata M. Miwa Y. Morimoto S. Sasaguri T. Involvement of GSK-3beta and DYRK1B in differentiation-inducing factor-3-induced phosphorylation of cyclin D1 in HeLa cells.J. Biol. Chem. 2006; 281: 38489-38497Crossref PubMed Scopus (42) Google Scholar). We next examined whether CycD1 Thr286 phosphorylation is sufficient to explain Dyrk1a’s dosage effect on CycD1 protein level and cell cycle entry. To test this hypothesis, HA-tagged CycD1 WT or CycD1 T286A constructs were stably introduced into the tet-mCit-Dyrk1a reporter cell line (Figure 2). Upon Dyrk1a induction, the mutant CycD1 T286A protein remained at a high level (Figure S3B) and did not undergo Dyrk1a dosage-dependent degradation in contrast to the WT CycD1 (Figure S3C). Time-lapse imaging and single-cell analysis further suggested that CycD1 T286A suppresses the Dyrk1a-mediated increase of the fraction of noncycling cells (Figures 3I and S3D) and decreases the fraction of cells in S phase (Figure 3J). Overall, these experiments demonstrate that Dyrk1a controls the rate of CycD1 degradation by directly phosphorylating CycD1 at Thr286 and thereby regulates the fraction of cycling cells. The activity of CycD1/CDK4/6 complexes is not only regulated by the concentration of CycD1, but also by the concentration of CDK inhibitors (CKIs) that keep CDK/Cyclin complexes inactive or active (Sherr and Roberts, 1999Sherr C.J. Roberts J.M. CDK inhibitors: positive and negative regulators of G1-phase progression.Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5130) Google Scholar). We therefore tested whether Dyrk1a may have an additional role in regulating the CDK inhibitor, p21, which can be tightly associated with CycD/CDK4/6 complexes. Surprisingly, Dyrk1a knockdown or kinase inhibition not only increased CycD1 levels but also increased p21 (Figures 3E, S4A, and S4B). This upregulation of p21 was likely due to an increase in protein stability, as it was lost upon proteasome inhibition (Figure 3E). This raised the question whether Dyrk1a directly regulates p21 stability or if the upregulation is indirectly caused by CycD1 upregulation. To differentiate between these two possibilities, we treated cells with Harmine in control or CycD1 knockdown cells (with CycD3 co-knockdown to prevent compensation). As expected, addition of Harmine resulted in an increase in p21 levels (Figure 4A, top). However, the increase was lost when Dyrk1a inhibition was combined with CycD knockdown (Figure 4A, bottom). Thus, p21 levels are not directly controlled by Dyrk1a but change instead indirectly as a result of the Dyrk1a-mediated change in CycD1 level. The CycD1-dependent increase in p21 led us to hypothesize that CycD1 and p21 stabilize each other. Indeed, knockdown of either p21 or CycD1 reduced the other accordingly (Figures S4C and S4D). This may reflect a mechanism whereby p21 and CycD1 degradation is suppressed when a CycD1/CDK4/p21 complex is formed, while the noncomplexed proteins can readily be degraded. To better understand this interdependence between p21 and CycD1, we visualized the respective levels of the two proteins in each cell. We then plotted the data in a density plot that shows the fraction of cells within the population that have a given level of p21 and CycD1. This single-cell analysis showed a significant overall correlation between p21 and CycD1 levels (R = 0.72) that became increasingly stronger as the level of CycD1 and p21 increased (Figure 4B, si-Ctrl). Cells in which we depleted CycD1 or p21 lost the correlation (Figure 4B). In contrast, in cells in which we depleted Dyrk1a, CycD1 levels increased and the correlation between p21 and CycD1 became stronger (R = 0.83, Figure 4B). As an additional test of this hypothesis, we considered that an increase in p21 should also lead to an increase in CycD1 levels. Indeed, a p21 increase induced by Nutlin (an Mdm2 inhibitor that stabilizes the p21 activator p53) or Etopside (a DNA damage inducer that causes the upregulation of p21) both led to a clear increase in CycD1 levels (Figure 4C). To validate whether the correlation in the total expression levels of p21 and CycD1 can be explained by costabilization, we tested whether the shape of the observed distribution can be recreated by a quantitative model, assuming that CycD1 and p21 are protected from degradation when they assemble in a p21/CycD1/CDK4 complex (Figure 4D). Figure 4E shows model calculations for the single-cell distribution of total p21 and CycD1 levels assuming a constant average level of free p21, a variable level of free CycD1, and a previously reported level of protein expression noise. Indeed, this simplified model recreated the codependence in the two-dimensional (2D) p21-CycD1 map (Figure 4F). Similar to the experimental perturbations, the simulation also recapitulated the curved codependent increase of total p21 and CycD1 protein levels when we modeled either an increased synthesis of CycD1 in the context of constant free p21 or an increased synthesis of p21 in the context of constant free CycD1 (Figures 4F and S4E). While costabilization explains the curved shape of the p21-CycD1 distribution, it does not explain why two distinct subpopulations of cells exist in Dyrk1a knockdown cells (Figure 4B). Bimodal distributions typically result from positive or double-negative feedback loops. Based on previous reports, a double-negative feedback may exist in this system involving p21 suppression of CDK/cyclin activity and CDK/cyclin-mediated degradation of p21 (Bornstein et al., 2003Bornstein G. Bloom J. Sitry-Shevah D. Nakayama K. Pagano M. Hershko A. Role of the SCFSkp2 ubiquitin ligase in the degradation of p21Cip1 in S phase.J. Biol. Chem. 2003; 278: 25752-25757Crossref PubMed Scopus (392) Google Scholar, Hengst et al., 1998Hengst L. Göpfert U. Lashuel H.A. Reed S.I. Complete inhibition of Cdk/cyclin by one molecule of p21(Cip1).Genes Dev. 1998; 12: 3882-3888Crossref PubMed Scopus (98) Google Scholar) (Figure 5A, left; activity could be either cyclin E/CDK2 or cyclin D/CDK4). When incorporating this double-negative feedback in the costabilization model, analysis of the stability of steady states reveals a critical saddle point (SP) where cells either reduce or increase p21 levels (Figure 5A, right). Moreover, this model can explain the observed codependence and bimodality for the p21-CycD1 map observed in the Dyrk1a knockdown cells as well as the unimodal distributions in control and CycD1 knockdown cells (Figure 5B). We then tested whether the bimodality of the CycD1-to-p21 distribution in Figure 4B and Figure 5B causes different cell fate outcomes. To measure the G1 CDK activity in single cells, we used phosphorylation of the tumor suppressor retinoblastoma (Rb) at Ser807/Ser811 as a marker for cell cycle entry (Ren and Rollins, 2004Ren S. Rollins B.J. Cyclin C/cdk3 promotes Rb-dependent G0 exit.Cell. 2004; 117: 239-251Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Rb is a well-known dual cyclin D-CDK4/6 and cyclin E-CDK2 substrate that requires phosphorylation to be inactivated and thereby promotes cell cycle entry. Unphosphorylated (or hypophosphorylated) Rb is associated with reversible or permanent exit from the cell cycle (Sherr, 1996Sherr C.J. Cancer cell cycles.Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4960) Google Scholar). Using immunofluorescence and automated image analysis, we quantified the levels of p21, CycD1, phospho-Rb, and the total DNA content in each cell (Figure 5C). We then created a 2D plot where we overlaid a color code for the percent of phospho-Rb-positive cells (% p-Rb) for different CycD1 versus p21 levels. Strikingly, the bimodal distribution of CycD1 and p21 levels in Dyrk1a knockdown cells closely matched the decision to either enter or exit the cell cycle (Figure 5D). This argued that the costabilization between CycD1 and p21 leads to cell-cycle arrest. In addition, the same representation in control cells shows a clear demarcation line separating arrested and cycling cells in which only cells that have relatively higher CycD1 compared to p21 levels are phospho-Rb positive (Figure 5D, green line). This boundary remained at a similar location when we examined control cells or cells treated with siRNAs against CycD1 or Dyrk1a (Figure 5D). This suggests the existence of two types of cell-cycle arrest profiles, one with high CycD1 levels and high p21 (Figure 5D, top right of the maps) and the other with low CycD1 levels and low to intermediate levels of p21 (Figure 5D, bottom of the maps). We hypothesized that cells with relatively low CycD1 levels undergo a more reversible cell-cycle arrest whereas cells with high levels of p21 and CycD1 ultimately proceed toward a more persistent cell cycle exit. This notion was supported by measurement the duration of G1 followed by immunostaining of p21 and CycD1 in Dyrk1a-knocked down cells (Figure 5E). Indeed, cells that had spent long periods in G1 (>41 hr) were nearly exclusively located in the high p21-CycD1 region (Figure 5E). Furthermore, when we put cells into a reversible cell-cycle arrest by serum starvation, we found that quiescent, phosho-Rb-negative cells were characterized by low levels of p21 and CycD1 (Figure 5F). These cells can readily re-enter the cell cycle upon serum addition. Together, our results show that Dyrk1a controls cycling and noncycling cell fates by changing the expression level of CycD1, which in turn can regulate p21 levels. Even in the presence of growth factor, this leads to a three-way decision process whereby a cell either (1) enters the cell cycle if CycD1 levels are higher than p21 levels, (2) transiently arrests if CycD1 levels remain low, or (3) undergoes a more persistent cell cycle exit if the levels of CycD1 and p21 costabilize each other at high levels. To further confirm the model that the high p21-CycD1 region in the 2D map reflects a more persistent exit state, we examined how cells alter their relative CycD1 and p21 levels when cells are forced to persistently arrest by aberrant oncogene activation (also known as oncogene-induced senescence) (Serrano et al., 1997Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a.Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3941) Google Scholar). BJ-5ta cells were infected by lentiviruses carrying the oncogenic HRasG12V allele or empty vector, and the levels of p21, CycD1, and phospho-Rb were measured. Markedly, oncogenic HRasG12V activation induced a clear bifurcation in cell fate in the p21-CycD1 signaling map (Figure 5G), analogous to the change induced by knockdown of Dyrk1a (the HRasG12V expression was confirmed by western blot in Figure S5A). Similar to Dyrk1a knockdown, cells arrested in response to oncogenic HRasG12V expression were almost exclusively located in the high p21-CycD1 region but not in the low CycD1 region (Figure 5G, quantification shown in Figure S5C). As a result, the already high CycD1 protein levels in the high p21-CycD1 region did not undergo further increase after Harmine addition (comparing Figures 5G and S5B, also see Figure S5C). Nevertheless, the group of cells with low CycD1 still responded to Harmine and increased CycD1 to increase phospho-Rb and consequent proliferation (monitored by percent in S phase, Figure S5D). The proliferation increase upon Harmine treatment is consistent with the previously reported role of Dyrk1a in oncogenic Ras-induced growth arrest (Litovchick et al., 2011Litovchick L. Florens L.A. Swanson S.K. Washburn M.P. DeCaprio J.A. DYRK1A protein kinase promotes quiescence and senescence through DREAM complex assembly.Genes Dev. 2011; 25: 801-813Crossref PubMed Scopus (175) Google Scholar). In addition, the Litovchick study suggested that Lin52 is a substrate of Dyrk1a with a role in regulating quiescence and senescence. Interestingly, knockdown of Lin52 mediated an increase in both CycD1 and p21 (Figures S5E and S5F), suggesting a potential parallel regulatory pathway. However, in contrast to the increase in CycD1 and p21 mediated by Dyrk1a knockdown, the population shift was more biased toward p21 than toward CycD1 in the 2D signaling map (Figures S5G and S5H). As a consequence, Harmine treatment only resulted in a moderate proliferation increase in the Lin52 knockdown background (Figure S5I). In a corollary experiment where we stably expressed HA-tagged Lin52-WT or Lin52-S28A (Figure S6A), neither Lin52 WT nor Lin52 S28A was able to rescue the Dyrk1a-dependent increase in noncycling cells (Figures S6B and S6C). To avoid potential compensation mechanisms that may occur in stably expressed cells, we also transiently expressed GFP-tagged Dyrk1a together with Lin52 WT or Lin52 S28A. These experiments showed a partial rescue of the Dyrk1a-dependent decrease in S phase entry in the Lin52 WT as well as S28A-expressing cells (Figure S6D). While this did not provide support for the hypothesis that Dyrk1a regulates the cell cycle by phosphorylating Lin52, it supports the previous result that Lin52 has a longer-term role in regulating quiescence and senescence. Since DS adult patients have a much lower incidence o" @default.
• W2012258557 created "2016-06-24" @default.
• W2012258557 creator A5012379493 @default.
• W2012258557 creator A5016961938 @default.
• W2012258557 creator A5021268165 @default.
• W2012258557 creator A5085144258 @default.
• W2012258557 date "2013-10-01" @default.
• W2012258557 modified "2023-10-17" @default.
• W2012258557 title "Dosage of Dyrk1a Shifts Cells within a p21-Cyclin D1 Signaling Map to Control the Decision to Enter the Cell Cycle" @default.
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