Epigenetic regulations follow cell cycle progression during differentiation of human pluripotent stem cells

AbstractMost mammalian stem cells undergo cellular division during their differentiation to produce daughter cells with a new cellular identity. However, the cascade of epigenetic events and molecular mechanisms occurring between successive cell divisions upon differentiation have not yet been described in detail due to technical limitations. Here, we address this question by taking advantage of the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) reporter to develop a culture system allowing the differentiation of human Embryonic Stem Cells (hESCs) synchronised for their cell cycle. Using this approach, we have assessed the epigenome and transcriptome dynamics during the first two divisions leading to definitive endoderm. We first observed that transcription of key markers of differentiation occurs before division suggesting that differentiation is initiated during the progression of cell cycle. Furthermore, ATAC-seq shows a major decrease in chromatin accessibility after pluripotency exit indicating that the first event of differentiation is the inhibition of alternative cell fate. In addition, using digital genomic footprinting we identified novel cell cycle-specific transcription factors with regulatory potential in endoderm specification. Of particular interest, Activator protein 1 (AP-1) controlled p38/MAPK signalling seems to be necessary for blocking endoderm shifting cell fate toward mesoderm lineage. Finally, histone modifications analyses suggest a temporal order between different marks. We can also conclude that enhancers are dynamically and rapidly established / decommissioned between different cell cycle upon differentiation. Overall, these data not only reveal key the successive interplays between epigenetic modifications during differentiation but also provide a valuable resource to investigate novel mechanisms in germ layer specification.

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Transcriptional networks are dynamically regulated during cell cycle progression in human Pluripotent Stem Cells

10.1101/2020.06.14.150748 ◽

2020 ◽

Author(s):

Anna Osnato ◽

Ludovic Vallier

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Cell Cycle Progression ◽

Embryonic Stem ◽

Chromatin Accessibility ◽

Model Systems ◽

Transcriptional Networks ◽

Reporter System ◽

Cycle Progression ◽

Sequence Of Events

AbstractCell cycle progression follows a precise sequence of events marked by different phases and check points which are associated with specific chromatin organisation. Whilst these changes have been extensively studied, their consequences on transcriptional networks remain to be fully uncovered, especially in dynamic model systems such as stem cells. Here, we take advantage of the FUCCI reporter system to show that chromatin accessibility, gene expression and key transcription factors binding change during cell cycle progression in human Embryonic Stem Cells (hESCs). These analyses reveal that core pluripotency factors such as OCT4, NANOG and SOX2 but also chromatin remodelers such as CTCF and RING1B bind the genome at specific phases of the cell cycle. Importantly, this binding pattern allows differentiation in the G1 phase while preserving pluripotency in the S/G2/M. Our results highlight the importance of studying transcriptional and epigenetic regulations in the dynamic context of the cell cycle.

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Babam2 Regulates Cell Cycle Progression and Pluripotency in Mouse Embryonic Stem Cells as Revealed by Induced DNA Damage

Biomedicines ◽

10.3390/biomedicines8100397 ◽

2020 ◽

Vol 8 (10) ◽

pp. 397

Author(s):

Cheuk Yiu Tenny Chung ◽

Paulisally Hau Yi Lo ◽

Kenneth Ka Ho Lee

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Dna Damage ◽

Gamma Irradiation ◽

Cellular Senescence ◽

Cell Cycle Regulation ◽

Cell Cycle Progression ◽

Embryonic Stem ◽

Cycle Progression ◽

Cycle Regulation

BRISC and BRCA1-A complex member 2 (Babam2) plays an essential role in promoting cell cycle progression and preventing cellular senescence. Babam2-deficient fibroblasts show proliferation defect and premature senescence compared with their wild-type (WT) counterpart. Pluripotent mouse embryonic stem cells (mESCs) are known to have unlimited cell proliferation and self-renewal capability without entering cellular senescence. Therefore, studying the role of Babam2 in ESCs would enable us to understand the mechanism of Babam2 in cellular aging, cell cycle regulation, and pluripotency in ESCs. For this study, we generated Babam2 knockout (Babam2−/−) mESCs to investigate the function of Babam2 in mESCs. We demonstrated that the loss of Babam2 in mESCs leads to abnormal G1 phase retention in response to DNA damage induced by gamma irradiation or doxorubicin treatments. Key cell cycle regulators, CDC25A and CDK2, were found to be degraded in Babam2−/− mESCs following gamma irradiation. In addition, Babam2−/− mESCs expressed p53 strongly and significantly longer than in control mESCs, where p53 inhibited Nanog expression and G1/S cell cycle progression. The combined effects significantly reduced developmental pluripotency in Babam2−/− mESCs. In summary, Babam2 maintains cell cycle regulation and pluripotency in mESCs in response to induced DNA damage.

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Molecular Mechanisms of Spindle Checkpoint-Apoptosis Linkage and Karyotypic Stability in Stem Cells.

Blood ◽

10.1182/blood.v110.11.3361.3361 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3361-3361

Author(s):

Charlie Mantel ◽

Sara Rhorabough ◽

Ying Guo ◽

Man-Ryul Lee ◽

Myung-Kwan Han ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Cell Fate ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Ex Vivo ◽

Spindle Checkpoint ◽

Cyclin B1 ◽

Polyploid Cell ◽

Mitotic Slippage

Abstract Ex-vivo expansion of human HSC prior to bone marrow transplantation is still an unrealized goal that could greatly extend the usefulness of this mainstay strategy for treating numerous human hematologic diseases. The safety of this process for potential use in humans depends in large part on the maintenance of karyotypic stability of HSC during expansion, a lack of which could contribute to serious, even fatal, complications such as cancer, and could also contribute to engraftment failure. The spindle checkpoint and its linkage to apoptosis initiation is one of the most important cellular processes that helps maintain chromosomal stability in rapidly proliferating cell populations by removing aneuploid and karyotypically abnormal cells via activation of cell death programs. Detailed understanding of the molecular regulation of this vital cell cycle checkpoint is important to maximize safety of in-vitro HSC expansion techniques. It is widely accepted that mammalian cells enter the next G1-phase with 4N DNA after slippage from prolonged drug-induced mitotic block caused by activation of the transient spindle checkpoint that it is from this state that polyploid/aneuploid cells initiate apoptosis. However, definitive biochemical evidence for G1 is scarce or unconvincing; in part because of methods of protein extraction required for immunoblot analysis that cannot take into account the cell cycle heterogeneity of cell cultures. We used single-cell-intracellular-flow-cytometric analysis to define important factors determining cell fate after mitotic slippage. Results from human and mouse embryonic stem cells that reenter polyploid cell cycles are compared to human somatic hematopoietic cells that die after MS. We now report for the first time that phosphorylation status of pRb, p53, CDK1, and cyclin B1 levels are important for cell fate/apoptosis decision in mitotic-slippage cells, which occurs in a unique, intervening, non-G1, tetraploid subphase. Hyperphosphorylated Rb was extremely abundant in mitotic-slippage cells, a cell signaling event usually associated with early G1-S phase transition. P53 was phosphorylated at sites known to be associated with apoptosis regulation. Cyclin A and B1 were undetectable in mitotic slippage cells; yet, CDK1 was phosphorylated at sites typically associated with its activation. Evidence is also presented raising the possibility of cyclin B1-independent CDK1 activity in mitotic-slippage cells. These findings challenge the current models of spindle checkpoint-apoptosis linkages. Our new model could have important implications for methods to maintain karyotypic stability during ex-vivo HSC expansion.

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Genetic control of pluripotency epigenome determines differentiation bias in mouse embryonic stem cells

10.1101/2021.01.15.426861 ◽

2021 ◽

Author(s):

Candice Byers ◽

Catrina Spruce ◽

Haley J. Fortin ◽

Anne Czechanski ◽

Steven C. Munger ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Genetic Regulation ◽

Embryonic Stem ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Embryoid Bodies ◽

Cell Fate Decisions

AbstractGenetically diverse pluripotent stem cells (PSCs) display varied, heritable responses to differentiation cues in the culture environment. By harnessing these disparities through derivation of embryonic stem cells (ESCs) from the BXD mouse genetic reference panel, along with C57BL/6J (B6) and DBA/2J (D2) parental strains, we demonstrate genetically determined biases in lineage commitment and identify major regulators of the pluripotency epigenome. Upon transition to formative pluripotency using epiblast-like cells (EpiLCs), B6 quickly dissolves naïve networks adopting gene expression modules indicative of neuroectoderm lineages; whereas D2 retains aspects of naïve pluripotency with little bias in differentiation. Genetic mapping identifies 6 major trans-acting loci co-regulating chromatin accessibility and gene expression in ESCs and EpiLCs, indicating a common regulatory system impacting cell state transition. These loci distally modulate occupancy of pluripotency factors, including TRIM28, P300, and POU5F1, at hundreds of regulatory elements. One trans-acting locus on Chr 12 primarily impacts chromatin accessibility in ESCs; while in EpiLCs the same locus subsequently influences gene expression, suggesting early chromatin priming. Consequently, the distal gene targets of this locus are enriched for neurogenesis genes and were more highly expressed when cells carried B6 haplotypes at this Chr 12 locus, supporting genetic regulation of biases in cell fate. Spontaneous formation of embryoid bodies validated this with B6 showing a propensity towards neuroectoderm differentiation and D2 towards definitive endoderm, confirming the fundamental importance of genetic variation influencing cell fate decisions.

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B-MYB Is Essential for Normal Cell Cycle Progression and Chromosomal Stability of Embryonic Stem Cells

PLoS ONE ◽

10.1371/journal.pone.0002478 ◽

2008 ◽

Vol 3 (6) ◽

pp. e2478 ◽

Cited By ~ 67

Author(s):

Kirill V. Tarasov ◽

Yelena S. Tarasova ◽

Wai Leong Tam ◽

Daniel R. Riordon ◽

Steven T. Elliott ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Normal Cell ◽

Cell Cycle Progression ◽

Embryonic Stem ◽

Cycle Progression ◽

Chromosomal Stability ◽

Normal Cell Cycle

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BRPF3-HUWE1-mediated regulation of MYST2 is required for differentiation and cell-cycle progression in embryonic stem cells

Cell Death and Differentiation ◽

10.1038/s41418-020-0577-1 ◽

2020 ◽

Vol 27 (12) ◽

pp. 3273-3288

Author(s):

Hye In Cho ◽

Min Seong Kim ◽

Jina Lee ◽

Byong Chul Yoo ◽

Kyung Hee Kim ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Embryonic Development ◽

Cell Cycle Progression ◽

Histone Acetyltransferase ◽

Embryoid Body ◽

Embryonic Stem ◽

Negative Regulator ◽

Cycle Progression

AbstractBrpf-histone acetyltransferase (HAT) complexes have important roles in embryonic development and regulating differentiation in ESCs. Among Brpf family, Brpf3 is a scaffold protein of Myst2 histone acetyltransferase complex that plays crucial roles in gene regulation, DNA replication, development as well as maintaining pluripotency in embryonic stem cells (ESCs). However, its biological functions in ESCs are not elucidated. In this study, we find out that Brpf3 protein level is critical for Myst2 stability and E3 ligase Huwe1 functions as a novel negative regulator of Myst2 via ubiquitin-mediated degradation. Importantly, Brpf3 plays an antagonistic role in Huwe1-mediated degradation of Myst2, suggesting that protein–protein interaction between Brpf3 and Myst2 is required for retaining Myst2 stability. Further, Brpf3 overexpression causes the aberrant upregulation of Myst2 protein levels which in turn induces the dysregulated cell-cycle progression and also delay of early embryonic development processes such as embryoid-body formation and lineage commitment of mouse ESCs. The Brpf3 overexpression-induced phenotypes can be reverted by Huwe1 overexpression. Together, these results may provide novel insights into understanding the functions of Brpf3 in proper differentiation as well as cell-cycle progression of ESCs via regulation of Myst2 stability by obstructing Huwe1-mediated ubiquitination. In addition, we suggest that this is a useful report which sheds light on the function of an unknown gene in ESC field.

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Pancreatic Progenitor Commitment Is Marked by an Increase in Ink4a/Arf Expression

Biomolecules ◽

10.3390/biom11081124 ◽

2021 ◽

Vol 11 (8) ◽

pp. 1124

Author(s):

Elena Montano ◽

Alessandra Pollice ◽

Valeria Lucci ◽

Geppino Falco ◽

Ornella Affinito ◽

...

Keyword(s):

Stem Cells ◽

Cell Fate ◽

Molecular Mechanisms ◽

Kinase Inhibitor ◽

Embryonic Stem ◽

Cellular Systems ◽

Cell Cycle Regulators ◽

Pancreatic Progenitor ◽

Cell Fate Decisions ◽

Lineage Differentiation

The identification of the molecular mechanisms controlling early cell fate decisions in mammals is of paramount importance as the ability to determine specific lineage differentiation represents a significant opportunity for new therapies. Pancreatic Progenitor Cells (PPCs) constitute a regenerative reserve essential for the maintenance and regeneration of the pancreas. Besides, PPCs represent an excellent model for understanding pathological pancreatic cellular remodeling. Given the lack of valid markers of early endoderm, the identification of new ones is of fundamental importance. Both products of the Ink4a/Arf locus, in addition to being critical cell-cycle regulators, appear to be involved in several disease pathologies. Moreover, the locus’ expression is epigenetically regulated in ES reprogramming processes, thus constituting the ideal candidates to modulate PPCs homeostasis. In this study, starting from mouse embryonic stem cells (mESCs), we analyzed the early stages of pancreatic commitment. By inducing mESCs commitment to the pancreatic lineage, we observed that both products of the Cdkn2a locus, Ink4a and Arf, mark a naïve pancreatic cellular state that resembled PPC-like specification. Treatment with epi-drugs suggests a role for chromatin remodeling in the CDKN2a (Cycline Dependent Kinase Inhibitor 2A) locus regulation in line with previous observations in other cellular systems. Our data considerably improve the comprehension of pancreatic cellular ontogeny, which could be critical for implementing pluripotent stem cells programming and reprogramming toward pancreatic lineage commitment.

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