Cdk2 Inhibition Prolongs G1 Phase Progression in Mouse Embryonic Stem Cells

Koledová, Zuzana; Kafková, Leona Rašková; Calabkova, Lenka; Kryštof, Vladimír; Doležel, Petr; Divoký, Vladimír

doi:10.1089/scd.2009.0065

Cited by 58 publications

(60 citation statements)

References 35 publications

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“…1A to C). We enriched cultures for mitotic cells by treating them with nocodazole (200 ng/ml, 16 h), a widely used synchronizing agent that has been successfully employed to synchronize ESCs (19)(20)(21)(22). Cells in G 1 were purified 1 h after release of the nocodazole block.…”

Section: Resultsmentioning

confidence: 99%

Genome-Wide Studies Reveal that H3K4me3 Modification in Bivalent Genes Is Dynamically Regulated during the Pluripotent Cell Cycle and Stabilized upon Differentiation

Grandy

Whitfield

et al. 2016

Molecular and Cellular Biology

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dStem cell phenotypes are reflected by posttranslational histone modifications, and this chromatin-related memory must be mitotically inherited to maintain cell identity through proliferative expansion. In human embryonic stem cells (hESCs), bivalent genes with both activating (H3K4me3) and repressive (H3K27me3) histone modifications are essential to sustain pluripotency. Yet, the molecular mechanisms by which this epigenetic landscape is transferred to progeny cells remain to be established. By mapping genomic enrichment of H3K4me3/H3K27me3 in pure populations of hESCs in G 2 , mitotic, and G 1 phases of the cell cycle, we found striking variations in the levels of H3K4me3 through the G 2 -M-G 1 transition. Analysis of a representative set of bivalent genes revealed that chromatin modifiers involved in H3K4 methylation/demethylation are recruited to bivalent gene promoters in a cell cycle-dependent fashion. Interestingly, bivalent genes enriched with H3K4me3 exclusively during mitosis undergo the strongest upregulation after induction of differentiation. Furthermore, the histone modification signature of genes that remain bivalent in differentiated cells resolves into a cell cycle-independent pattern after lineage commitment. These results establish a new dimension of chromatin regulation important in the maintenance of pluripotency. Human embryonic stem cells (hESCs) are an increasingly powerful tool for regenerative medicine. They recapitulate, in vitro, the molecular phenomena that take place during the first stages of embryonic development. Like their in vivo counterparts, ESCs proliferate rapidly and are able to form the three embryonic germ layers (1). This highly self-renewing and pluripotent state is sustained by a unique epigenetic landscape, consisting of transcription factors, chromatin remodeling complexes, and histone modifications that provide the transcriptional plasticity required for rapid response to differentiation cues (2).Histone H3 lysine 4 and 27 trimethylations (H3K4me3 and H3K27me3, respectively) are key histone modifications that are involved in transcriptional regulation (3, 4). H3K4me3 near transcriptional start sites (TSSs) marks regions of active transcription or transcriptional readiness (5). H3K27me3 modification, in contrast, is a well-established negative regulator of gene expression that repels transcriptional activators and attracts chromatin repressors that promote chromatin compaction (6). Genomic regions that host both histone marks, so-called bivalent domains, were first observed in ESCs, primarily near promoters of genes with developmental functions (7-9). Significant effort has gone into understanding the biological role of bivalency; the consensus is that, in ESCs, it represses transcription but poises genes for rapid expression during lineage commitment (10). Although this proposition is not yet supported with direct evidence, it has become clear that bivalent domains are essential for maintaining ESC pluripotency and self-renewing capacity (10). Despite the extensive...

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Section: Resultsmentioning

confidence: 99%

Genome-Wide Studies Reveal that H3K4me3 Modification in Bivalent Genes Is Dynamically Regulated during the Pluripotent Cell Cycle and Stabilized upon Differentiation

Grandy

Whitfield

et al. 2016

Molecular and Cellular Biology

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“…Nevertheless, they are potentially useful because they produce acute effects, and the dose-response range can easily be explored. Several groups have previously used small molecules (e.g., roscovitine) targeted to CDKs but with a host of off-target effects on ESCs and found drastically different results on promoting differentiation (17,20). To explore the effects of these small molecule inhibitors further, we applied both roscovitine and CVT-313, a more specific CDK2 inhibitor, to ESCs.…”

Section: Knockdown Of Cdk2 or Cyclin A With Sirna Reduces The Prolifementioning

confidence: 99%

“…Furthermore, it was later shown that knockout mice of CDK2, the main CDK thought to be responsible for G1/S progression in ESCs, were viable and therefore presumably did not prematurely differentiate their stem cells (18,19). In contrast, it was recently reported that olomoucine II, another CDK inhibitor, could induce mRNA up-regulation of differentiation markers in mouse ESCs and that CDK2 siRNA could induce morphology changes characteristic of differentiation (20). In human ESCs exposure to roscovitine and other CDK inhibitors sometimes led to a loss of pluripotency markers but other times did not (21)(22)(23)(24).…”

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confidence: 99%

Gap 1 phase length and mouse embryonic stem cell self-renewal

Ballabeni

Kirschner

2012

Proc. Natl. Acad. Sci. U.S.A.

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In somatic cells, the length of the G1 phase of the cell cycle is tightly linked to differentiation, and its elongation can drive differentiation in many cases. Although it has been suggested that the situation is very similar in embryonic stem cells (ESCs), where a rapid cell cycle and a short G1 phase maintain the pluripotent state, evidence has been contradictory. Here we show that, in murine ESCs, elongation of the cell cycle and elongation of G1 are compatible with their pluripotent state. Multiple methods that lengthen the cell cycle and that target cyclin-dependent kinase, retinoblastoma protein, and E2F activity all fail to induce differentiation on their own or even to facilitate differentiation. The resistance of murine ESCs to differentiation induced by lengthening G1 and/or the cell cycle could allow for separate control of these events and provide new opportunities for investigation and application.stemness | proliferation | decoupling

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“…Functional studies addressing the interaction between cell cycle phase and differentiation in ESCs have been based primarily on manipulations of genes associated with cell cycle regulation, particularly CDK2 [10][11][12], p21 [13,14], p27 [15], and hTERT [16] (reviewed in [17]). Constitutive activity of CDK2 and CyclinE [7,11,12] in ESCs is important for their shortened G1 phase.…”

Section: Introductionmentioning

confidence: 99%

“…Constitutive activity of CDK2 and CyclinE [7,11,12] in ESCs is important for their shortened G1 phase. Several studies in mouse and human ESCs showed that inhibition of CDK2 leads to cell cycle arrest in G1 and subsequent differentiation to extraembryonic lineages [11][12][13]. This differentiation, however, does not necessarily reflect increased propensity to differentiate in G1.…”

Section: Introductionmentioning

confidence: 99%

Human Embryonic Stem Cells Exhibit Increased Propensity to Differentiate During the G1 Phase Prior to Phosphorylation of Retinoblastoma Protein

et al. 2012

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While experimentally induced arrest of human embryonic stem cells (hESCs) in G1 has been shown to stimulate differentiation, it remains unclear whether the unperturbed G1 phase in hESCs is causally related to differentiation. Here, we use centrifugal elutriation to isolate and investigate differentiation propensities of hESCs in different phases of their cell cycle. We found that isolated G1 cells exhibit higher differentiation propensity compared with S and G2 cells, and they differentiate at low cell densities even under self-renewing conditions. This differentiation of G1 cells was partially prevented in dense cultures of these cells and completely abrogated in coculture with S and G2 cells. However, coculturing without cell-to-cell contact did not rescue the differentiation of G1 cells. Finally, we show that the subset of G1 hESCs with reduced phosphorylation of retinoblastoma has the highest propensity to differentiate and that the differentiation is preceded by cell cycle arrest. These results provide direct evidence for increased propensity of hESCs to differentiate in G1 and suggest a role for neighboring cells in preventing differentiation of hESCs as they pass through a differentiation sensitive, G1 phase.

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Cdk2 Inhibition Prolongs G1 Phase Progression in Mouse Embryonic Stem Cells

Cited by 58 publications

References 35 publications

Genome-Wide Studies Reveal that H3K4me3 Modification in Bivalent Genes Is Dynamically Regulated during the Pluripotent Cell Cycle and Stabilized upon Differentiation

Genome-Wide Studies Reveal that H3K4me3 Modification in Bivalent Genes Is Dynamically Regulated during the Pluripotent Cell Cycle and Stabilized upon Differentiation

Gap 1 phase length and mouse embryonic stem cell self-renewal

Human Embryonic Stem Cells Exhibit Increased Propensity to Differentiate During the G1 Phase Prior to Phosphorylation of Retinoblastoma Protein

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