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G0phase

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Mitosis in ananimal cell(phases ordered counter-clockwise), with G0labeled at left.
Many mammalcells,such as this 9x Hneuron,remain permanently or semipermanently in G0.

TheG0phasedescribes a cellular state outside of the replicativecell cycle.Classically[when?],cells were thought to enter G0primarily due to environmental factors, like nutrient deprivation, that limited the resources necessary for proliferation. Thus it was thought of as aresting phase.G0is now known to take different forms and occur for multiple reasons. For example, most adultneuronalcells, among the most metabolically active cells in the body, are fully differentiated and reside in a terminal G0phase. Neurons reside in this state, not because of stochastic or limited nutrient supply, but as a part of their developmental program.

G0was first suggested as a cell state based on early cell cycle studies. When the first studies defined the four phases of the cell cycle using radioactive labeling techniques, it was discovered that not all cells in a populationproliferateat similar rates.[1]A population's "growth fraction" – or the fraction of the population that was growing – was actively proliferating, but other cells existed in a non-proliferative state. Some of these non-proliferating cells could respond to extrinsic stimuli and proliferate by re-entering the cell cycle.[2]Early contrasting views either considered non-proliferating cells to simply be in an extendedG1phaseor in a cell cycle phase distinct from G1– termed G0.[3]Subsequent research pointed to arestriction point(R-point) in G1where cells can enter G0before the R-point but are committed to mitosis after the R-point.[4]These early studies provided evidence for the existence of a G0state to which access is restricted. These cells that do not divide further exit G1phase to enter an inactive stage calledquiescent stage.

Diversity of G0states

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Schematickaryogramof the human chromosomes, showing their usual state in the G0and G1phase of the cell cycle. At top center it also shows the chromosome 3 pair after having undergoneDNA synthesis,occurring in theS phase(annotated as S) of the cell cycle. This interval includes theG2phaseandmetaphase(annotated as "Meta." ).
Further information:Karyotype

Three G0states exist and can be categorized as either reversible (quiescent) or irreversible (senescentanddifferentiated). Each of these three states can be entered from the G1phase before the cell commits to the next round of the cell cycle. Quiescence refers to a reversible G0state where subpopulations of cells reside in a 'quiescent' state before entering the cell cycle after activation in response to extrinsic signals. Quiescent cells are often identified by lowRNAcontent, lack of cell proliferation markers, and increased label retention indicating low cell turnover.[5][6]Senescence is distinct from quiescence because senescence is an irreversible state that cells enter in response to DNA damage or degradation that would make a cell's progeny nonviable. SuchDNA damagecan occur fromtelomere shorteningover many cell divisions as well as reactive oxygen species (ROS) exposure, oncogene activation, and cell-cell fusion. While senescent cells can no longer replicate, they remain able to perform many normal cellular functions.[7][8][9][10]Senescence is often a biochemical alternative to the self-destruction of such a damaged cell byapoptosis.In contrast to cellular senescence, quiescence is not a reactive event but part of the core programming of several different cell types. Finally, differentiated cells are stem cells that have progressed through a differentiation program to reach a mature – terminally differentiated – state. Differentiated cells continue to stay in G0and perform their main functions indefinitely.

Characteristics of quiescent stem cells

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Transcriptomes

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Thetranscriptomesof several types of quiescent stem cells, such ashematopoietic,muscle, and hair follicle, have been characterized throughhigh-throughputtechniques, such asmicroarrayandRNA sequencing.Although variations exist in their individual transcriptomes, most quiescent tissue stem cells share a common pattern of gene expression that involves downregulation of cell cycle progression genes, such ascyclin A2,cyclin B1,cyclin E2,andsurvivin,and upregulation of genes involved in the regulation of transcription and stem cell fate, such asFOXO3andEZH1.Downregulation of mitochondrialcytochrome Calso reflects the low metabolic state of quiescent stem cells.[11]

Epigenetic

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Many quiescent stem cells, particularlyadult stem cells,also share similarepigeneticpatterns. For example,H3K4me3andH3K27me3,are two majorhistonemethylation patterns that form a bivalent domain and are located near transcription initiation sites. These epigenetic markers have been found to regulate lineage decisions in embryonic stem cells as well as control quiescence in hair follicle and muscle stem cells viachromatinmodification.[11]

Regulation of quiescence

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Cell cycle regulators

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Functionaltumor suppressor genes,particularlyp53andRb gene,are required to maintain stem cell quiescence and prevent exhaustion of theprogenitor cellpool through excessive divisions. For example, deletion of all three components of the Rb family of proteins has been shown to halt quiescence in hematopoietic stem cells. Lack of p53 has been shown to prevent differentiation of these stem cells due to the cells' inability to exit the cell cycle into the G0phase. In addition to p53 and Rb,cyclin dependent kinaseinhibitors (CKIs), such asp21,p27,andp57,are also important for maintaining quiescence. In mouse hematopoietic stem cells, knockout of p57 and p27 leads to G0exit through nuclear import ofcyclin D1and subsequentphosphorylationof Rb. Finally, theNotch signaling pathwayhas been shown to play an important role in maintenance of quiescence.[11]

Post-transcriptional regulation

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Post-transcriptional regulation of gene expression viamiRNAsynthesis has been shown to play an equally important role in the maintenance of stem cell quiescence. miRNA strands bind to the 3′ untranslated region (3′ UTR) of targetmRNAs,preventing their translation into functional proteins. The length of the 3′ UTR of a gene determines its ability to bind to miRNA strands, thereby allowing regulation of quiescence. Some examples of miRNA's in stem cells include miR-126, which controls thePI3K/AKT/mTOR pathwayin hematopoietic stem cells, miR-489, which suppresses the DEKoncogenein muscle stem cells, and miR-31, which regulatesMyf5in muscle stem cells. miRNA sequestration of mRNA withinribonucleoproteincomplexes allows quiescent cells to store the mRNA necessary for quick entry into theG1 phase.[11]

Response to stress

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Stem cells that have been quiescent for a long time often face various environmental stressors, such asoxidative stress.However, several mechanisms allow these cells to respond to such stressors. For example, theFOXOtranscription factors respond to the presence ofreactive oxygen species(ROS) whileHIF1AandLKB1respond tohypoxicconditions. In hematopoietic stem cells,autophagyis induced to respond to metabolic stress.[11]

Examples of reversible G0phase

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Tissue stem cells

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Stem cells are cells with the unique ability to produce differentiateddaughter cellsand to preserve their stem cell identity through self-renewal.[12] In mammals, most adult tissues containtissue-specific stem cellsthat reside in the tissue and proliferate to maintain homeostasis for the lifespan of the organism. These cells can undergo immense proliferation in response to tissue damage before differentiating and engaging in regeneration. Some tissue stem cells exist in a reversible, quiescent state indefinitely until being activated by external stimuli. Many different types of tissue stem cells exist, includingmuscle stem cells(MuSCs),neural stem cells(NSCs),intestinal stem cells(ISCs), and many others.

Stem cell quiescence has been recently suggested to be composed of two distinct functional phases, G0and an 'alert' phase termed GAlert.[13] Stem cells are believed to actively and reversibly transition between these phases to respond to injury stimuli and seem to gain enhanced tissue regenerative function in GAlert.Thus, transition into GAlerthas been proposed as an adaptive response that enables stem cells to rapidly respond to injury or stress by priming them for cell cycle entry. In muscle stem cells,mTORC1activity has been identified to control the transition from G0into GAlertalong with signaling through theHGFreceptorcMet.[13]

Mature hepatocytes

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While a reversible quiescent state is perhaps most important for tissue stem cells to respond quickly to stimuli and maintain proper homeostasis and regeneration, reversible G0phases can be found in non-stem cells such as mature hepatocytes.[14]Hepatocytes are typically quiescent in normal livers but undergo limited replication (less than 2 cell divisions) during liver regeneration after partial hepatectomy. However, in certain cases, hepatocytes can experience immense proliferation (more than 70 cell divisions) indicating that their proliferation capacity is not hampered by existing in a reversible quiescent state.[14]

Examples of irreversible G0phase

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Senescent cells

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Often associated with aging and age-related diseases in vivo, senescent cells can be found in many renewable tissues, including thestroma,vasculature,hematopoietic system,and manyepithelialorgans. Resulting from accumulation over many cell divisions, senescence is often seen in age-associated degenerative phenotypes. Senescent fibroblasts in models of breast epithelial cell function have been found to disrupt milk protein production due to secretion of matrixmetalloproteinases.[15] Similarly, senescent pulmonary artery smooth muscle cells caused nearby smooth muscle cells to proliferate and migrate, perhaps contributing to hypertrophy of pulmonary arteries and eventually pulmonary hypertension.[16]

Differentiated muscle

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During skeletalmyogenesis,cycling progenitor cells known asmyoblastsdifferentiate and fuse together into non-cycling muscle cells called myocytes that remain in a terminal G0phase.[17]As a result, the fibers that make upskeletal muscle(myofibers) are cells with multiple nuclei, referred to as myonuclei, since each myonucleus originated from a single myoblast. Skeletal muscle cells continue indefinitely to provide contractile force through simultaneous contractions of cellular structures calledsarcomeres.Importantly, these cells are kept in a terminal G0phase since disruption of muscle fiber structure after myofiber formation would prevent proper transmission of force through the length of the muscle. Muscle growth can be stimulated by growth or injury and involves the recruitment of muscle stem cells – also known as satellite cells – out of a reversible quiescent state. These stem cells differentiate and fuse to generate new muscle fibers both in parallel and in series to increase force generation capacity.

Cardiac muscleis also formed through myogenesis but instead of recruiting stem cells to fuse and form new cells, heart muscle cells – known ascardiomyocytes– simply increase in size as the heart grows larger. Similarly to skeletal muscle, if cardiomyocytes had to continue dividing to add muscle tissue the contractile structures necessary for heart function would be disrupted.

Differentiated bone

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Of the four major types of bone cells,osteocytesare the most common and also exist in a terminal G0phase. Osteocytes arise from osteoblasts that are trapped within a self-secreted matrix. While osteocytes also have reduced synthetic activity, they still serve bone functions besides generating structure. Osteocytes work through various mechanosensory mechanisms to assist in the routine turnover over bony matrix.

Differentiated nerve

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Outside of a fewneurogenicniches in the brain, mostneuronsare fully differentiated and reside in a terminal G0phase. These fully differentiated neurons formsynapseswhere electrical signals are transmitted byaxonsto thedendritesof nearby neurons. In this G0state, neurons continue functioning until senescence or apoptosis. Numerous studies have reported accumulation ofDNA damagewith age, particularlyoxidative damage,in the mammalianbrain.[18]

Mechanism of G0entry

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Role of Rim15

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Rim15 was first discovered to play a critical role in initiatingmeiosisindiploidyeast cells. Under conditions of low glucose and nitrogen, which are key nutrients for the survival of yeast, diploid yeast cells initiate meiosis through the activation of early meiotic-specific genes (EMGs). The expression of EMGs is regulated by Ume6. Ume6 recruits thehistone deacetylases,Rpd3and Sin3, to repress EMG expression when glucose and nitrogen levels are high, and it recruits the EMG transcription factor Ime1 when glucose and nitrogen levels are low. Rim15, named for its role in the regulation of an EMG called IME2, displaces Rpd3 and Sin3, thereby allowing Ume6 to bring Ime1 to the promoters of EMGs for meiosis initiation.[19]

In addition to playing a role in meiosis initiation, Rim15 has also been shown to be a critical effector for yeast cell entry into G0in the presence of stress. Signals from several different nutrient signaling pathways converge on Rim15, which activates the transcription factors, Gis1, Msn2, and Msn4. Gis1 binds to and activates promoters containing post-diauxic growthshift (PDS) elements while Msn2 and Msn4 bind to and activate promoters containing stress-response elements(STREs). Although it is not clear how Rim15 activates Gis1 and Msn2/4, there is some speculation that it may directly phosphorylate them or be involved in chromatin remodeling. Rim15 has also been found to contain aPAS domainat itsN terminal,making it a newly discovered member of the PASkinasefamily. The PAS domain is a regulatory unit of the Rim15 protein that may play a role in sensing oxidative stress in yeast.[19]

Nutrient signaling pathways

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Glucose

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Yeast grows exponentially throughfermentationof glucose. When glucose levels drop, yeast shift from fermentation tocellular respiration,metabolizing the fermentative products from their exponential growth phase. This shift is known as the diauxic shift after which yeast enter G0.When glucose levels in the surroundings are high, the production ofcAMPthrough the RAS-cAMP-PKA pathway (acAMP-dependent pathway) is elevated, causingprotein kinase A(PKA) to inhibit its downstream target Rim15 and allow cell proliferation. When glucose levels drop, cAMP production declines, lifting PKA's inhibition of Rim15 and allowing the yeast cell to enter G0.[19]

Nitrogen

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In addition to glucose, the presence of nitrogen is crucial for yeast proliferation. Under low nitrogen conditions, Rim15 is activated to promote cell cycle arrest through inactivation of the protein kinasesTORC1and Sch9. While TORC1 and Sch9 belong to two separate pathways, namely the TOR and Fermentable Growth Medium induced pathways respectively, both protein kinases act to promote cytoplasmic retention of Rim15. Under normal conditions, Rim15 is anchored to the cytoplasmic14-3-3 protein,Bmh2, via phosphorylation of its Thr1075. TORC1 inactivates certainphosphatasesin the cytoplasm, keeping Rim15 anchored to Bmh2, while it is thought that Sch9 promotes Rim15 cytoplasmic retention through phosphorylation of another 14-3-3 binding site close to Thr1075. When extracellular nitrogen is low, TORC1 and Sch9 are inactivated, allowing dephosphorylation of Rim15 and its subsequent transport to the nucleus, where it can activate transcription factors involved in promoting cell entry into G0.It has also been found that Rim15 promotes its own export from the nucleus throughautophosphorylation.[19]

Phosphate

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Yeast cells respond to low extracellular phosphate levels by activating genes that are involved in the production and upregulation of inorganic phosphate. The PHO pathway is involved in the regulation of phosphate levels. Under normal conditions, the yeastcyclin-dependent kinase complex,Pho80-Pho85, inactivates thePho4transcription factor through phosphorylation. However, when phosphate levels drop, Pho81 inhibits Pho80-Pho85, allowing Pho4 to be active. When phosphate is abundant, Pho80-Pho85 also inhibits the nuclear pool of Rim 15 by promoting phosphorylation of its Thr1075 Bmh2 binding site. Thus, Pho80-Pho85 acts in concert with Sch9 and TORC1 to promote cytoplasmic retention of Rim15 under normal conditions.[19]

Mechanism of G0exit

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Cyclin C/Cdk3 and Rb

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The transition from G1toS phaseis promoted by the inactivation of Rb through its progressivehyperphosphorylationby theCyclin D/Cdk4andCyclin E/Cdk2 complexes in late G1.An early observation that loss of Rb promoted cell cycle re-entry in G0cells suggested that Rb is also essential in regulating the G0to G1transition in quiescent cells.[20]Further observations revealed that levels ofcyclin CmRNA are highest when human cells exit G0,suggesting that cyclin C may be involved in Rb phosphorylation to promote cell cycle re-entry of G0arrested cells.Immunoprecipitationkinase assays revealed that cyclin C has Rb kinase activity. Furthermore, unlike cyclins D and E, cyclin C's Rb kinase activity is highest during early G1and lowest during late G1and S phases, suggesting that it may be involved in the G0to G1transition. The use offluorescence-activated cell sortingto identify G0cells, which are characterized by a high DNA to RNA ratio relative to G1cells, confirmed the suspicion that cyclin C promotes G0exit as repression of endogenous cyclin C byRNAiin mammalian cells increased the proportion of cells arrested in G0.Further experiments involving mutation of Rb at specific phosphorylation sites showed that cyclin C phosphorylation of Rb at S807/811 is necessary for G0exit. It remains unclear, however, whether this phosphorylation pattern is sufficient for G0exit. Finally, co-immunoprecipitation assays revealed thatcyclin-dependent kinase 3(cdk3) promotes G0exit by forming a complex with cyclin C to phosphorylate Rb at S807/811. Interestingly, S807/811 are also targets of cyclin D/cdk4 phosphorylation during the G1to S transition. This might suggest a possible compensation of cdk3 activity by cdk4, especially in light of the observation that G0exit is only delayed, and not permanently inhibited, in cells lacking cdk3 but functional in cdk4. Despite the overlap of phosphorylation targets, it seems that cdk3 is still necessary for the most effective transition from G0to G1.[21]

Rb and G0exit

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Studies suggest that Rb repression of theE2Ffamily of transcription factors regulates the G0to G1transition just as it does the G1to S transition. Activating E2F complexes are associated with the recruitment ofhistone acetyltransferases,which activate gene expression necessary for G1entry, whileE2F4complexes recruit histone deacetylases, which repress gene expression. Phosphorylation of Rb by Cdk complexes allows its dissociation from E2F transcription factors and the subsequent expression of genes necessary for G0exit. Other members of the Rbpocket protein family,such as p107 and p130, have also been found to be involved in G0arrest. p130 levels are elevated in G0and have been found to associate with E2F-4 complexes to repress transcription of E2F target genes. Meanwhile, p107 has been found to rescue the cell arrest phenotype after loss of Rb even though p107 is expressed at comparatively low levels in G0cells. Taken together, these findings suggest that Rb repression of E2F transcription factors promotes cell arrest while phosphorylation of Rb leads to G0exit via derepression of E2F target genes.[20]In addition to its regulation of E2F, Rb has also been shown to suppressRNA polymerase IandRNA polymerase III,which are involved inrRNAsynthesis. Thus, phosphorylation of Rb also allows activation of rRNA synthesis, which is crucial for protein synthesis upon entry into G1.[21]

References

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