scholarly journals Strand-specific single-cell methylomics reveals distinct modes of DNA demethylation dynamics during early mammalian development

2019 ◽  
Author(s):  
Maya Sen ◽  
Dylan Mooijman ◽  
Jean-Charles Boisset ◽  
Alex Chialastri ◽  
Mina Popovic ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Embryonic Stem ◽  
Cost Effective ◽  
Dna Demethylation ◽  
Preimplantation Embryos ◽  
Mammalian Development ◽  
Cell Stage ◽  
Stage Of Development ◽  
Genome Wide

AbstractDNA methylation (5mC) is central to cellular identity and the global erasure of 5mC from the parental genomes during preimplantation mammalian development is critical to reset the methylome of terminally differentiated gametes to the pluripotent cells in the blastocyst. While active and passive modes of demethylation have both been suggested to play a role in this process, the relative contribution of these two mechanisms to genome-wide 5mC erasure remains unclear. Here, we report a new high-throughput single-cell method (scMspJI-seq) that enables strand-specific quantification of 5mC, thereby allowing us to systematically probe the dynamics of global demethylation. First, when applied to hybrid mouse embryonic stem cells, we identified substantial cell-to-cell strand-specific 5mC heterogeneity, with a small group of cells displaying asymmetric levels of 5mCpG between the two DNA strands of a chromosome suggesting loss of maintenance methylation. Next, using scMspJI-seq in preimplantation mouse embryos, we discovered that methylation maintenance is active till the 16-cell stage followed by passive demethylation in a fraction of cells within the early blastocyst at the 32-cell stage of development. Finally, we found that human preimplantation embryos qualitatively show temporally delayed yet similar demethylation dynamics as mouse preimplantation embryos. Collectively, these results demonstrate that scMspJI-seq is a sensitive and cost-effective method to map the strand-specific genome-wide patterns of 5mC in single cells, thereby enabling quantitative investigation of methylation dynamics in developmental systems.

scholarly journals Strand-specific single-cell methylomics reveals distinct modes of DNA demethylation dynamics during early mammalian development

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Maya Sen ◽  
Dylan Mooijman ◽  
Alex Chialastri ◽  
Jean-Charles Boisset ◽  
Mina Popovic ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Embryonic Stem ◽  
Cost Effective ◽  
Dna Demethylation ◽  
Mouse Embryos ◽  
Preimplantation Embryos ◽  
Mammalian Development ◽  
Cell Stage ◽  
Stage Of Development

AbstractDNA methylation (5mC) is central to cellular identity. The global erasure of 5mC from the parental genomes during preimplantation mammalian development is critical to reset the methylome of gametes to the cells in the blastocyst. While active and passive modes of demethylation have both been suggested to play a role in this process, the relative contribution of these two mechanisms to 5mC erasure remains unclear. Here, we report a single-cell method (scMspJI-seq) that enables strand-specific quantification of 5mC, allowing us to systematically probe the dynamics of global demethylation. When applied to mouse embryonic stem cells, we identified substantial cell-to-cell strand-specific 5mC heterogeneity, with a small group of cells displaying asymmetric levels of 5mCpG between the two DNA strands of a chromosome suggesting loss of maintenance methylation. Next, in preimplantation mouse embryos, we discovered that methylation maintenance is active till the 16-cell stage followed by passive demethylation in a fraction of cells within the early blastocyst at the 32-cell stage of development. Finally, human preimplantation embryos qualitatively show temporally delayed yet similar demethylation dynamics as mouse embryos. Collectively, these results demonstrate that scMspJI-seq is a sensitive and cost-effective method to map the strand-specific genome-wide patterns of 5mC in single cells.

scholarly journals A genome-wide transcriptomic analysis of embryos fathered by obese males in a murine model of diet-induced obesity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Laura Bernhardt ◽  
Marcus Dittrich ◽  
Rabih El-Merahbi ◽  
Antoine-Emmanuel Saliba ◽  
Tobias Müller ◽  
...  
Keyword(s):  
Gene Expression ◽  
Negative Impact ◽  
Preimplantation Embryos ◽  
High Fat ◽  
Cell Stage ◽  
Genetic Transmission ◽  
Diet Induced Obesity ◽  
Genome Wide ◽  
A Genome ◽  
Paternal Obesity

AbstractPaternal obesity is known to have a negative impact on the male’s reproductive health as well as the health of his offspring. Although epigenetic mechanisms have been implicated in the non-genetic transmission of acquired traits, the effect of paternal obesity on gene expression in the preimplantation embryo has not been fully studied. To this end, we investigated whether paternal obesity is associated with gene expression changes in eight-cell stage embryos fathered by males on a high-fat diet. We used single embryo RNA-seq to compare the gene expression profile of embryos generated by males on a high fat (HFD) versus control (CD) diet. This analysis revealed significant upregulation of the Samd4b and Gata6 gene in embryos in response to a paternal HFD. Furthermore, we could show a significant increase in expression of both Gata6 and Samd4b during differentiation of stromal vascular cells into mature adipocytes. These findings suggest that paternal obesity may induce changes in the male germ cells which are associated with the gene expression changes in the resulting preimplantation embryos.

scholarly journals Retinoic acid signaling is critical during the totipotency window in early mammalian development

2021 ◽  
Author(s):  
Ane Iturbide ◽  
Mayra L. Ruiz Tejeda Segura ◽  
Camille Noll ◽  
Kenji Schorpp ◽  
Ina Rothenaigner ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Regulatory Networks ◽  
Es Cells ◽  
Embryonic Stem ◽  
Mammalian Development ◽  
Cell Stage ◽  
Cell Potential ◽  
Cellular Models ◽  
Early Embryos ◽  
Genome Activation

AbstractTotipotent cells hold enormous potential for regenerative medicine. Thus, the development of cellular models recapitulating totipotent-like features is of paramount importance. Cells resembling the totipotent cells of early embryos arise spontaneously in mouse embryonic stem (ES) cell cultures. Such ‘2-cell-like-cells’ (2CLCs) recapitulate 2-cell-stage features and display expanded cell potential. Here, we used 2CLCs to perform a small-molecule screen to identify new pathways regulating the 2-cell-stage program. We identified retinoids as robust inducers of 2CLCs and the retinoic acid (RA)-signaling pathway as a key component of the regulatory circuitry of totipotent cells in embryos. Using single-cell RNA-seq, we reveal the transcriptional dynamics of 2CLC reprogramming and show that ES cells undergo distinct cellular trajectories in response to RA. Importantly, endogenous RA activity in early embryos is essential for zygotic genome activation and developmental progression. Overall, our data shed light on the gene regulatory networks controlling cellular plasticity and the totipotency program.

scholarly journals Simultaneous deletion of the methylcytosine oxidases Tet1 and Tet3 increases transcriptome variability in early embryogenesis

2015 ◽  
Vol 112 (31) ◽  
pp. E4236-E4245 ◽  
Author(s):  
Jinsuk Kang ◽  
Matthias Lienhard ◽  
William A. Pastor ◽  
Ashu Chawla ◽  
Mark Novotny ◽  
...  
Keyword(s):  
Single Cells ◽  
Cholesterol Biosynthesis ◽  
Early Embryogenesis ◽  
Dna Demethylation ◽  
Cell Stage ◽  
Tet Proteins ◽  
Genes Encoding ◽  
Variable Gene ◽  
Intracellular Pathways ◽  
Tet Enzymes

Dioxygenases of the TET (Ten-Eleven Translocation) family produce oxidized methylcytosines, intermediates in DNA demethylation, as well as new epigenetic marks. Here we show data suggesting that TET proteins maintain the consistency of gene transcription. Embryos lacking Tet1 and Tet3 (Tet1/3 DKO) displayed a strong loss of 5-hydroxymethylcytosine (5hmC) and a concurrent increase in 5-methylcytosine (5mC) at the eight-cell stage. Single cells from eight-cell embryos and individual embryonic day 3.5 blastocysts showed unexpectedly variable gene expression compared with controls, and this variability correlated in blastocysts with variably increased 5mC/5hmC in gene bodies and repetitive elements. Despite the variability, genes encoding regulators of cholesterol biosynthesis were reproducibly down-regulated in Tet1/3 DKO blastocysts, resulting in a characteristic phenotype of holoprosencephaly in the few embryos that survived to later stages. Thus, TET enzymes and DNA cytosine modifications could directly or indirectly modulate transcriptional noise, resulting in the selective susceptibility of certain intracellular pathways to regulation by TET proteins.

TRIM28 maintains genome imprints and regulates development of porcine SCNT embryos

Reproduction ◽  
2021 ◽  
Vol 161 (4) ◽  
pp. 411-424
Author(s):  
Yanhui Zhai ◽  
Meng Zhang ◽  
Xinglan An ◽  
Sheng Zhang ◽  
Xiangjie Kong ◽  
...  
Keyword(s):  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Transcript Abundance ◽  
Dna Demethylation ◽  
Epigenetic Variability ◽  
Promoter Regions ◽  
Cell Stage ◽  
Genome Wide ◽  
Developmental Progression ◽  
Imprinting Gene

Pre-implantation embryos undergo genome-wide DNA demethylation, however certain regions, like imprinted loci remain methylated. Further, the mechanisms ensuring demethylation resistance by TRIM28 in epigenetic reprogramming remain poorly understood. Here, TRIM28 was knocked down in oocytes, and its effects on porcine somatic cell nuclear transfer (SCNT) embryo development was examined. Our results showed that SCNT embryos constructed from TRIM28 knockdown oocytes had significantly lower cleavage (53.9 ± 3.4% vs 64.8 ± 2.7%) and blastocyst rates (12.1 ± 4.3% vs 19.8 ± 1.9%) than control-SCNT embryos. The DNA methylation levels at the promoter regions of the imprinting gene IGF2 and H19 were significantly decreased in the 4-cell stage, and the transcript abundance of other imprinting gene was substantially increased. We also identified an aberrant two-fold decrease in the expression of CXXC1and H3K4me3 methyltransferase (ASH2L and MLL2), and the signal intensity of H3K4me3 had a transient drop in SCNT 2-cell embryos. Our results indicated that maternal TRIM28 knockdown disrupted the genome imprints and caused epigenetic variability in H3K4me3 levels, which blocked the transcription activity of zygote genes and affected the normal developmental progression of porcine SCNT embryos.

102. THE EFFECT OF INSULIN ON EMBRYONIC STEM CELL PROGENITOR CELLS IN THE MOUSE BLASTOCYST

2009 ◽  
Vol 21 (9) ◽  
pp. 21
Author(s):  
J. M. Campbell ◽  
I. Vassiliev ◽  
M. B. Nottle ◽  
M. Lane
Keyword(s):  
Progenitor Cells ◽  
Cell Fate ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Number ◽  
Human Embryos ◽  
Cell Stage ◽  
Stage Of Development

Human ESCs are produced from embryos donated at the mid-stage of pre-implantation development. This cryostorage reduced viability. However, it has been shown that this can be improved by the addition of growth factors to culture medium. The aim of the present study was to examine whether the addition of insulin to embryo culture medium from the 8-cell stage of development increases the number of ES cell progenitor cells in the epiblast in a mouse model. In vivo produced mouse zygotes (C57Bl6 strain) were cultured in G1 medium for 48h to the 8-cell stage, followed by culture in G2 supplemented with insulin (0, 0.17, 1.7 and 1700pM) for 68h, at 37 o C , in 5% O2, 6%CO2, 89% N2 . The number of cells in the inner cell mass (ICM) and epiblast was determined by immunohistochemical staining for Oct4 and Nanog. ICM cells express Oct4, epiblast cells express both Oct4 and Nanog. The addition of insulin at the concentrations examined did not increase the ICM. However, at 1.7pM insulin increased the number of epiblast cells (6.6±0.5 cells vs 4.1±0.5, P=0.001) in the ICM, which increased the proportion of the ICM that was epiblast (38.9±3.7% compared to 25.8±3.4% in the control P=0.01). This indicates that the increase in the epiblast is brought about by a shift in cell fate as opposed to an increase in cell division. The effect of insulin on the proportion of cells in the epiblast was investigated using inhibitors of phosphoinositide3-kinase (PI3K) (LY294002, 50µM); one of insulin's main second messengers, and p53 (pifithrin-α, 30µg/ml); a pro-apoptotic protein inactivated by PI3K. Inhibition of PI3K eliminated the increase caused by insulin (4.5±0.3 cells versus 2.2±0.3 cells, P<0.001), while inhibition of p53 increased the epiblast cell number compared to the control (7.1±0.8 and 4.1±0.7 respectively P=0.001). This study shows that insulin increases epiblast cell number through the activation of PI3K and the inhibition of p53, and may be a strategy for improving ESC isolation from human embryos.

scholarly journals The Influence of Interspecies Somatic Cell Nuclear Transfer on Epigenetic Enzymes Transcription in Early Embryos

2016 ◽  
Vol 39 (2) ◽  
pp. 209-217 ◽  
Author(s):  
Martin Morovic ◽  
Matej Murin ◽  
Frantisek Strejcek ◽  
Michal Benc ◽  
Dusan Paál ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Coming Out ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Dna Methyltransferase ◽  
Dna Methyltransferases ◽  
Preimplantation Embryos ◽  
Mammalian Development ◽  
Cell Stage ◽  
Early Embryos

AbstractOne of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription in oocytes and early embryos of several species including bovine and porcine zygotes is species-dependent process and the incomplete DNA methylation correlates with the nuclear transfer failure rate in mammals. In this study the transcription of DNA methyltransferase 1 and 3a (DNMT1, DNMT3a) genes in early embryonic stages of interspecies (bovine, porcine) nuclear transfer embryos (iSCNT) by RT-PCR were analyzed. Coming out from the diverse timing of embryonic genome activation (EGA) in porcine and bovine preimplantation embryos, the intense effect of ooplasm on transferred somatic cell nucleus was expected. In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly influenced by the ooplasmic environment.

scholarly journals Dosage sensitivity of X-linked genes in human embryonic single cells

2018 ◽  
Author(s):  
Jian-Rong Yang ◽  
Xiaoshu Chen
Keyword(s):  
Single Cells ◽  
Embryonic Stem ◽  
Housekeeping Genes ◽  
Human Embryos ◽  
Cell Stage ◽  
Expression Ratio ◽  
Gene Expression Noise ◽  
Expression Levels ◽  
Cell Transcriptome ◽  
Dosage Sensitivity

ABSTRACTFifty years ago, Susumu Ohno proposed that the expression levels of X-linked genes have doubled as dosage compensation for autosomal genes due to degeneration of Y-linked homologs during evolution of mammalian sex chromosomes. Recent studies have nevertheless shown that the X to autosome expression ratio equals ~1 in haploid human parthenogenetic embryonic stem (pES) cells and ~0.5 in diploid pES cells, thus refuting Ohno’s hypothesis. Here, by reanalyzing a RNA-seq-based single-cell transcriptome dataset of human embryos (Petropoulos, et al. 2016), we found that from the 8-cell stage until the time-point just prior to implantation, the expression levels of X-linked genes are not two-fold upregulated in male cells and gradually decrease from two-fold in female cells. This observation suggests that the expression levels of X-linked genes are imbalanced, with autosomal genes starting from the early 8-cell stage, and that the dosage conversion is fast, such that the X:AA expression ratio reaches ~0.5 in no more than a week. Additional analyses of gene expression noise further suggest that the dosage sensitivity of X-linked genes is weaker than that of autosomal genes in differentiated female cells, which contradicts a key assumption of Ohno’s hypothesis. Moreover, the dosage-sensitive housekeeping genes are preferentially located on autosomes, implying selection against X-linkage for dosage-sensitive genes. Our results collectively suggest an alternative to Ohno’s hypothesis that X-linked genes are less likely to be dosage sensitive than autosomal genes.

scholarly journals Single cell profiling of total RNA using Smart-seq-total

2020 ◽  
Author(s):  
Alina Isakova ◽  
Norma Neff ◽  
Stephen R. Quake
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Embryoid Bodies ◽  
High Yield ◽  
Rna Seq ◽  
Mcf7 Cells ◽  
Total Rna ◽  
Non Coding Rna

ABSTRACTThe ability to interrogate total RNA content of single cells would enable better mapping of the transcriptional logic behind emerging cell types and states. However, current RNA-seq methods are unable to simultaneously monitor both short and long, poly(A)+ and poly(A)-transcripts at the single-cell level, and thus deliver only a partial snapshot of the cellular RNAome. Here, we describe Smart-seq-total, a method capable of assaying a broad spectrum of coding and non-coding RNA from a single cell. Built upon the template-switch mechanism, Smart-seq-total bears the key feature of its predecessor, Smart-seq2, namely, the ability to capture full-length transcripts with high yield and quality. It also outperforms current poly(A)–independent total RNA-seq protocols by capturing transcripts of a broad size range, thus, allowing us to simultaneously analyze protein-coding, long non-coding, microRNA and other non-coding RNA transcripts from single cells. We used Smart-seq-total to analyze the total RNAome of human primary fibroblasts, HEK293T and MCF7 cells as well as that of induced murine embryonic stem cells differentiated into embryoid bodies. We show that simultaneous measurement of non-coding RNA and mRNA from the same cell enables elucidation of new roles of non-coding RNA throughout essential processes such as cell cycle or lineage commitment. Moreover, we show that cell types can be distinguished based on the abundance of non-coding transcripts alone.

scholarly journals Single-cell quantification of a broad RNA spectrum reveals unique noncoding patterns associated with cell types and states

2021 ◽  
Vol 118 (51) ◽  
pp. e2113568118
Author(s):  
Alina Isakova ◽  
Norma Neff ◽  
Stephen R. Quake
Keyword(s):  
Single Cell ◽  
Noncoding Rna ◽  
Single Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Embryoid Bodies ◽  
Rna Seq ◽  
Total Rna ◽  
Rna Transcripts ◽  
Rna Content

The ability to interrogate total RNA content of single cells would enable better mapping of the transcriptional logic behind emerging cell types and states. However, current single-cell RNA-sequencing (RNA-seq) methods are unable to simultaneously monitor all forms of RNA transcripts at the single-cell level, and thus deliver only a partial snapshot of the cellular RNAome. Here we describe Smart-seq-total, a method capable of assaying a broad spectrum of coding and noncoding RNA from a single cell. Smart-seq-total does not require splitting the RNA content of a cell and allows the incorporation of unique molecular identifiers into short and long RNA molecules for absolute quantification. It outperforms current poly(A)-independent total RNA-seq protocols by capturing transcripts of a broad size range, thus enabling simultaneous analysis of protein-coding, long-noncoding, microRNA, and other noncoding RNA transcripts from single cells. We used Smart-seq-total to analyze the total RNAome of human primary fibroblasts, HEK293T, and MCF7 cells, as well as that of induced murine embryonic stem cells differentiated into embryoid bodies. By analyzing the coexpression patterns of both noncoding RNA and mRNA from the same cell, we were able to discover new roles of noncoding RNA throughout essential processes, such as cell cycle and lineage commitment during embryonic development. Moreover, we show that independent classes of short-noncoding RNA can be used to determine cell-type identity.

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