Cell Cycle Of Embryonic Stem Cells Research Articles

INO80 Is Required for the Cell Cycle Control, Survival, and Differentiation of Mouse ESCs by Transcriptional Regulation

Precise regulation of the cell cycle of embryonic stem cells (ESCs) is critical for their self-maintenance and differentiation. The cell cycle of ESCs differs from that of somatic cells and is different depending on the cell culture conditions. However, the cell cycle regulation in ESCs via epigenetic mechanisms remains unclear. Here, we showed that the ATP-dependent chromatin remodeler Ino80 regulates the cell cycle genes in ESCs under primed conditions. Ino80 loss led to a significantly extended length of the G1-phase in ESCs grown under primed culture conditions. Ino80 directly bound to the transcription start site and regulated the expression of cell cycle-related genes. Furthermore, Ino80 loss induced cell apoptosis. However, the regulatory mechanism of Ino80 in differentiating ESC cycle slightly differed; an extended S-phase was detected in differentiating inducible Ino80 knockout ESCs. RNA-seq analysis of differentiating ESCs revealed that the expression of genes associated with organ development cell cycle is persistently altered in Ino80 knockout cells, suggesting that cell cycle regulation by Ino80 is not limited to undifferentiated ESCs. Therefore, our study establishes the function of Ino80 in ESC cycle via transcriptional regulation, at least partly. Moreover, this Ino80 function may be universal to other cell types.

Tet1 Suppresses p21 to Ensure Proper Cell Cycle Progression in Embryonic Stem Cells.

Ten eleven translocation 1 (Tet1) is a DNA dioxygenase that promotes DNA demethylation by oxidizing 5-methylcytosine. It can also partner with chromatin-activating and repressive complexes to regulate gene expressions independent of its enzymatic activity. Tet1 is highly expressed in embryonic stem cells (ESCs) and regulates pluripotency and differentiation. However, its roles in ESC cell cycle progression and proliferation have not been investigated. Using a series of Tet1 catalytic mutant (Tet1m/m), knockout (Tet1−/−) and wild type (Tet1+/+) mouse ESCs (mESCs), we identified a non-catalytic role of Tet1 in the proper cell cycle progression and proliferation of mESCs. Tet1−/−, but not Tet1m/m, mESCs exhibited a significant reduction in proliferation and delayed progression through G1. We found that the cyclin-dependent kinase inhibitor p21/Cdkn1a was uniquely upregulated in Tet1−/− mESCs and its knockdown corrected the slow proliferation and delayed G1 progression. Mechanistically, we found that p21 was a direct target of Tet1. Tet1 occupancy at the p21 promoter overlapped with the repressive histone mark H3K27me3 as well as with the H3K27 trimethyl transferase PRC2 component Ezh2. A loss of Tet1, but not loss of its catalytic activity, significantly reduced the enrichment of Ezh2 and H3K27 trimethylation at the p21 promoter without affecting the DNA methylation levels. We also found that the proliferation defects of Tet1−/− mESCs were independent of their differentiation defects. Together, these findings established a non-catalytic role for Tet1 in suppressing p21 in mESCs to ensure a rapid G1-to-S progression, which is a key hallmark of ESC proliferation. It also established Tet1 as an epigenetic regulator of ESC proliferation in addition to its previously defined roles in ESC pluripotency and differentiation.

ATP-Dependent Chromatin Remodeler CHD9 Controls the Proliferation of Embryonic Stem Cells in a Cell Culture Condition-Dependent Manner.

Emerging evidence suggests that chromodomain-helicase-DNA-binding (CHD) proteins are involved in stem cell maintenance and differentiation via the coordination of chromatin structure and gene expression. However, the molecular function of some CHD proteins in stem cell regulation is still poorly understood. Herein, we show that Chd9 knockdown (KD) in mouse embryonic stem cells (ESCs) cultured in normal serum media, not in 2i-leukemia inhibitory factor (LIF) media, causes rapid cell proliferation. This is caused by transcriptional regulation related to the cell cycle and the response to growth factors. Our analysis showed that, unlike the serum cultured-Chd9 KD ESCs, the 2i-LIF-cultured-Chd9 KO ESCs displayed elevated levels of critical G1 phase regulators such as p21 and p27. Consistently, the DNA binding sites of CHD9 overlap with some transcription factor DNA motifs that are associated with genes regulating the cell cycle and growth pathways. These transcription factors include the cycle gene homology region (CHR), Arid5a, and LIN54. Collectively, our results provide new insights into CHD9-mediated gene transcription for controlling the cell cycle of ESCs.

Phosphorylation of TFCP2L1 by CDK1 is required for stem cell pluripotency and bladder carcinogenesis

Molecular programs involved in embryogenesis are frequently upregulated in oncogenic dedifferentiation and metastasis. However, their precise roles and regulatory mechanisms remain elusive. Here, we showed that CDK1 phosphorylation of TFCP2L1, a pluripotency‐associated transcription factor, orchestrated pluripotency and cell‐cycling in embryonic stem cells (ESCs) and was aberrantly activated in aggressive bladder cancers (BCs). In murine ESCs, the protein interactome and transcription targets of Tfcp2l1 indicated its involvement in cell cycle regulation. Tfcp2l1 was phosphorylated at Thr177 by Cdk1, which affected ESC cell cycle progression, pluripotency, and differentiation. The CDK1‐TFCP2L1 pathway was activated in human BC cells, stimulating their proliferation, self‐renewal, and invasion. Lack of TFCP2L1 phosphorylation impaired the tumorigenic potency of BC cells in a xenograft model. In patients with BC, high co‐expression of TFCP2L1 and CDK1 was associated with unfavorable clinical characteristics including tumor grade, lymphovascular and muscularis propria invasion, and distant metastasis and was an independent prognostic factor for cancer‐specific survival. These findings demonstrate the molecular and clinical significance of CDK1‐mediated TFCP2L1 phosphorylation in stem cell pluripotency and in the tumorigenic stemness features associated with BC progression.

Cell cycle and pluripotency: Convergence on octamer‑binding transcription factor 4 (Review).

Embryonic stem cells (ESCs) have unlimited expansion potential and the ability to differentiate into all somatic cell types for regenerative medicine and disease model studies. Octamer-binding transcription factor 4 (OCT4), encoded by the POU domain, class 5, transcription factor 1 gene, is a transcription factor vital for maintaining ESC pluripotency and somatic reprogramming. Many studies have established that the cell cycle of ESCs is featured with an abbreviated G1 phase and a prolonged S phase. Changes in cell cycle dynamics are intimately associated with the state of ESC pluripotency, and manipulating cell-cycle regulators could enable a controlled differentiation of ESCs. The present review focused primarily on the emerging roles of OCT4 in coordinating the cell cycle progression, the maintenance of pluripotency and the glycolytic metabolism in ESCs.

Cellular retinoic acid binding protein I mediates rapid non-canonical activation of ERK1/2 by all-trans retinoic acid

All-trans retinoic acid (atRA), one of the active ingredients of vitamin A, exerts canonical activities to regulate gene expression mediated by nuclear RA receptors (RARs). AtRA could also elicit certain non-canonical activities including, mostly, rapid activation of extracellular signal regulated kinase 1/2 (ERK1/2); but the mechanism was unclear. In this study, we have found that cellular retinoic acid binding protein I (CRABPI) mediates the non-canonical, RAR- and membrane signal-independent activation of ERK1/2 by atRA in various cellular backgrounds. In the context of embryonic stem cells (ESCs), atRA/CRABPI-dependent ERK1/2 activation rapidly affects ESC cell cycle, specifically to expand the G1 phase. This is mediated by ERK stimulation resulting in dephosphorylation of nuclear p27, which elevates nuclear p27 protein levels to block G1 progression to S phase. This is the first study to identify CRABPI as the mediator for non-canonical activation of ERK1/2 by atRA, and demonstrate a new functional role for CRABPI in modulating ESC cell cycle progression.

Role of the Epigenetic Regulator HP1γ in the Control of Embryonic Stem Cell Properties

The unique properties of embryonic stem cells (ESC) rely on long-lasting self-renewal and their ability to switch in all adult cell type programs. Recent advances have shown that regulations at the chromatin level sustain both ESC properties along with transcription factors. We have focused our interest on the epigenetic modulator HP1γ (Heterochromatin Protein 1, isoform γ) that binds histones H3 methylated at lysine 9 (meH3K9) and is highly plastic in its distribution and association with the transcriptional regulation of specific genes during cell fate transitions. These characteristics of HP1γ make it a good candidate to sustain the ESC flexibility required for rapid program changes during differentiation. Using RNA interference, we describe the functional role of HP1γ in mouse ESC. The analysis of HP1γ deprived cells in proliferative and in various differentiating conditions was performed combining functional assays with molecular approaches (RT-qPCR, microarray). We show that HP1γ deprivation slows down the cell cycle of ESC and decreases their resistance to differentiating conditions, rendering the cells poised to differentiate. In addition, HP1γ depletion hampers the differentiation to the endoderm as compared with the differentiation to the neurectoderm or the mesoderm. Altogether, our results reveal the role of HP1γ in ESC self-renewal and in the balance between the pluripotent and the differentiation programs.

Opposing microRNA families regulate self-renewal in mouse embryonic stem cells

When embryonic stem cells (ESCs) differentiate, they must both silence the ESC self-renewal program as well as activate new tissue specific programs. In the absence of DGCR8 (Dgcr8 -/-), a protein required for microRNA (miRNA) biogenesis, mouse ESCs are unable to silence self-renewal. Here, we find that the introduction of let-7 miRNAs, a family of miRNAs highly expressed in somatic cells, can suppress self-renewal in Dgcr8 -/-, but not wild-type ESCs. Introduction of ESC cell cycle regulating (ESCC) miRNAs into the Dgcr8 -/- ESCs, blocks the capacity of let-7 to suppress self-renewal. Profiling and bioinformatic analyses show that let-7 inhibits while ESCC miRNAs indirectly activate numerous self-renewal genes. Furthermore, inhibition of the let-7 family promotes de-differentiation of somatic cells to induced pluripotent stem (iPS) cells. Together, these findings show how the ESCC and let-7 miRNAs act through common pathways to alternatively stabilize the self-renewing versus differentiated cell fates.