Strain-specific transgene methylation occurs early in mouse development and can be recapitulated in embryonic stem cells

A murine transgene, HRD, is methylated only when carried in certain inbred strain backgrounds. A locus on distal chromosome 4, Ssm1 (strain-specific modifier), controls this phenomenon. In order to characterize the activity of Ssm1, we have investigated developmental acquisition of methylation over the transgene. Analysis of postimplantation embryos revealed that strain-specific methylation is initiated prior to embryonic day (E) 6.5. Strain-specific transgene methylation is all-or-none in pattern and occurs exclusively in the primitive ectoderm lineage. A strain-independent pattern of partial methylation occurs in the primitive endoderm and trophectoderm lineages. To examine earlier stages, embryonic stem (ES) cells were derived from E3.5 blastocysts and examined for transgene methylation before and after differentiation. Though the transgene had already acquired some methylation in undifferentiated ES cells, differentiation induced further, de novo methylation in a strain-dependent manner. Analysis of methylation in ES cultures suggests that the transgene and endogenous genes (such as immunoglobulin genes) are synchronously methylated during early development. These results are interpreted in the context of a model in which Ssm1-like modifier genes produce alterations in chromatin structure during and/or shortly after implantation, thereby marking target loci for de novo methylation with the rest of the genome during gastrulation.

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Establishment and Maintenance of Genomic Methylation Patterns in Mouse Embryonic Stem Cells by Dnmt3a and Dnmt3b

Molecular and Cellular Biology ◽

10.1128/mcb.23.16.5594-5605.2003 ◽

2003 ◽

Vol 23 (16) ◽

pp. 5594-5605 ◽

Cited By ~ 473

Author(s):

Taiping Chen ◽

Yoshihide Ueda ◽

Jonathan E. Dodge ◽

Zhenjuan Wang ◽

En Li

Keyword(s):

Dna Methylation ◽

De Novo ◽

Es Cells ◽

Embryonic Stem ◽

Single Copy ◽

Dna Methyltransferases ◽

Methylation Patterns ◽

Genomic Methylation ◽

De Novo Methylation

ABSTRACT We have previously shown that the DNA methyltransferases Dnmt3a and Dnmt3b carry out de novo methylation of the mouse genome during early postimplantation development and of maternally imprinted genes in the oocyte. In the present study, we demonstrate that Dnmt3a and Dnmt3b are also essential for the stable inheritance, or “maintenance,” of DNA methylation patterns. Inactivation of both Dnmt3a and Dnmt3b in embryonic stem (ES) cells results in progressive loss of methylation in various repeats and single-copy genes. Interestingly, introduction of the Dnmt3a, Dnmt3a2, and Dnmt3b1 isoforms back into highly demethylated mutant ES cells restores genomic methylation patterns; these isoforms appear to have both common and distinct DNA targets, but they all fail to restore the maternal methylation imprints. In contrast, overexpression of Dnmt1 and Dnmt3b3 failed to restore DNA methylation patterns due to their inability to catalyze de novo methylation in vivo. We also show that hypermethylation of genomic DNA by Dnmt3a and Dnmt3b is necessary for ES cells to form teratomas in nude mice. These results indicate that genomic methylation patterns are determined partly through differential expression of different Dnmt3a and Dnmt3b isoforms.

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The Autism Risk Factor CHD8 Is a Chromatin Activator and Functionally Dependent on the ERK-MAPK Pathway Effector ELK1

10.1101/2020.11.10.377010 ◽

2020 ◽

Author(s):

Bahareh Haddad Derafshi ◽

Tamas Danko ◽

Soham Chanda ◽

Pedro Batista ◽

Ulrike Litzenburger ◽

...

Keyword(s):

Binding Sites ◽

De Novo ◽

Cell Model ◽

Mapk Pathway ◽

Es Cells ◽

Embryonic Stem ◽

Chromatin Accessibility ◽

Cooperative Interaction ◽

Dependent Manner ◽

Human Neurons

AbstractThe chromodomain helicase DNA-binding protein CHD8 is among the most frequently found de-novo mutations in autism (1–3). Unlike most other autism-risk genes, CHD8 mutations appear to be fully penetrant (4). Despite its prominent disease involvement, little known about its molecular function. Based on sequence homology, CHD8 is believed to be a chromatin regulator, but mechanisms for its genomic targeting and its role on chromatin are unclear. Here, we developed a human cell model carrying conditional CHD8 loss-of-function alleles. Full knockout CHD8 was required for the viability of undifferentiated human embryonic stem (ES) cells, whereas postmitotic neurons survived following CHD8 depletion. However, chromatin accessibility maps and transcriptional profiling revealed that CHD8 is a potent general chromatin activator, enhancing transcription of its direct target genes, including a large group of autism genes. CHD8’s genomic binding sites in human neurons were significantly enriched for ELK1 (ETS) motifs. Moreover, positive CHD8-dependent chromatin remodeling was enhanced at ELK1 motif-containing CHD8 binding sites. ELK1 was the most prominent ETS factor expressed in human neurons and was necessary for CHD8 to target the sites that contained the ELK1 motif, demonstrating a cooperative interaction between ELK1 and CHD8 on chromatin. We also observed potential role of CHD8 in ELK1 localization on nuclear compartments in a transcription-stage-dependent manner. Finally, inhibition of ELK1 activity or ELK1 knockdown that enhances the neurogenesis from embryonic stem cells (ES) was dependent on the presence of CHD8. In summary, our results establish that CHD8 is a strong activator of chromatin accessibility and transcription in neurons and reveals a role in regulating many high-risk autism genes. Additionally, we show there is molecular and functional interdependence of CHD8 and ELK1 in chromatin binding of CHD8, nuclear interaction of ELK1, and neurogenesis enhancement. These data imply the involvement of the MAPK/ERK pathway effector ELK1 in pathogenesis of autism caused by CHD8 mutations (5).

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Steroidogenic Factor-1 (SF-1)-Driven Differentiation of Murine Embryonic Stem (ES) Cells into a Gonadal Lineage

Endocrinology ◽

10.1210/en.2011-0219 ◽

2011 ◽

Vol 152 (7) ◽

pp. 2870-2882 ◽

Cited By ~ 24

Author(s):

Unmesh Jadhav ◽

J. Larry Jameson

Keyword(s):

Target Genes ◽

De Novo ◽

Embryoid Body ◽

Es Cells ◽

Embryonic Stem ◽

Cell Lineage ◽

Culture Conditions ◽

Steroidogenic Factor 1 ◽

Lineage Differentiation ◽

And Function

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells (SF-1-ES cells) has been shown to prime the cells for steroidogenesis. When provided with exogenous cholesterol substrate, and after treatment with retinoic acid and cAMP, SF-1-ES cells produce progesterone but do not produce other steroids such as cortisol, estradiol, or testosterone. In this study, we explored culture conditions that optimize SF-1-mediated differentiation of ES cells into defined steroidogenic lineages. When embryoid body formation was used to facilitate cell lineage differentiation, SF-1-ES cells were found to be restricted in their differentiation, with fewer cells entering neuronal pathways and a larger fraction entering the steroidogenic lineage. Among the differentiation protocols tested, leukemia inhibitory factor (LIF) removal, followed by prolonged cAMP treatment was most efficacious for inducing steroidogenesis in SF-1-ES cells. In this protocol, a subset of SF-1-ES cells survives after LIF withdrawal, undergoes morphologic differentiation, and recovers proliferative capacity. These cells are characterized by induction of steroidogenic enzyme genes, use of de novo cholesterol, and production of multiple steroids including estradiol and testosterone. Microarray studies identified additional pathways associated with SF-1 mediated differentiation. Using biotinylated SF-1 in chromatin immunoprecipitation assays, SF-1 was shown to bind directly to multiple target genes, with induction of binding to some targets after steroidogenic treatment. These studies indicate that SF-1 expression, followed by LIF removal and treatment with cAMP drives ES cells into a steroidogenic pathway characteristic of gonadal steroid-producing cells.

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TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells

Nature Genetics ◽

10.1038/s41588-017-0002-y ◽

2017 ◽

Vol 50 (1) ◽

pp. 83-95 ◽

Cited By ~ 74

Author(s):

Nipun Verma ◽

Heng Pan ◽

Louis C. Doré ◽

Abhijit Shukla ◽

Qing V. Li ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

De Novo ◽

Embryonic Stem ◽

Tet Proteins ◽

De Novo Methylation

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SUMO conjugation susceptibility of Akt/protein kinase B affects the expression of the pluripotency transcription factor Nanog in embryonic stem cells

PLoS ONE ◽

10.1371/journal.pone.0254447 ◽

2021 ◽

Vol 16 (7) ◽

pp. e0254447

Author(s):

Marcos Francia ◽

Martin Stortz ◽

Camila Vazquez Echegaray ◽

Camila Oses ◽

Paula Verneri ◽

...

Keyword(s):

Transcription Factor ◽

Es Cells ◽

Embryonic Stem ◽

Dependent Manner ◽

Cellular Functions ◽

Cellular Processes ◽

Sumo Conjugation ◽

Heterologous System ◽

Canonical Pathways ◽

The Impact

Akt/PKB is a kinase involved in the regulation of a wide variety of cell processes. Its activity is modulated by diverse post-translational modifications (PTMs). Particularly, conjugation of the small ubiquitin-related modifier (SUMO) to this kinase impacts on multiple cellular functions, such as proliferation and splicing. In embryonic stem (ES) cells, this kinase is key for pluripotency maintenance. Among other functions, Akt is known to promote the expression of Nanog, a central pluripotency transcription factor (TF). However, the relevance of this specific PTM of Akt has not been previously analyzed in this context. In this work, we study the effect of Akt1 variants with differential SUMOylation susceptibility on the expression of Nanog. Our results demonstrate that both, the Akt1 capability of being modified by SUMO conjugation and a functional SUMO conjugase activity are required to induce Nanog gene expression. Likewise, we found that the common oncogenic E17K Akt1 mutant affected Nanog expression in ES cells also in a SUMOylatability dependent manner. Interestingly, this outcome takes places in ES cells but not in a non-pluripotent heterologous system, suggesting the presence of a crucial factor for this induction in ES cells. Remarkably, the two major candidate factors to mediate this induction, GSK3-β and Tbx3, are non-essential players of this effect, suggesting a complex mechanism probably involving non-canonical pathways. Furthermore, we found that Akt1 subcellular distribution does not depend on its SUMOylatability, indicating that Akt localization has no influence on the effect on Nanog, and that besides the membrane localization of E17K Akt mutant, SUMOylation is also required for its hyperactivity. Our results highlight the impact of SUMO conjugation in the function of a kinase relevant for a plethora of cellular processes, including the control of a key pluripotency TF.

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Vascular endothelial growth factor synergistically enhances bone morphogenetic protein-4-dependent lymphohematopoietic cell generation from embryonic stem cells in vitro

Blood ◽

10.1182/blood.v95.7.2275 ◽

2000 ◽

Vol 95 (7) ◽

pp. 2275-2283 ◽

Cited By ~ 65

Author(s):

Naoki Nakayama ◽

Jae Lee ◽

Laura Chiu

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Bone Morphogenetic Protein ◽

Es Cells ◽

Embryonic Stem ◽

Dependent Manner ◽

Vascular Endothelial ◽

Morphogenetic Protein ◽

Endothelial Growth Factor

Abstract The totipotent mouse embryonic stem (ES) cell is known to differentiate into cells expressing the β-globin gene when stimulated with bone morphogenetic protein (BMP)-4. Here, we demonstrate that BMP-4 is essential for generating both erythro-myeloid colony-forming cells (CFCs) and lymphoid (B and NK) progenitor cells from ES cells and that vascular endothelial growth factor (VEGF) synergizes with BMP-4. The CD45+ myelomonocytic progenitors and Ter119+ erythroid cells began to be detected with 0.5 ng/mL BMP-4, and their levels plateaued at approximately 2 ng/mL. VEGF alone weakly elevated the CD34+ cell population though no lymphohematopoietic progenitors were induced. However, when combined with BMP-4, 2 to 20 ng/mL VEGF synergistically augmented the BMP-4-dependent generation of erythro-myeloid CFCs and lymphoid progenitors from ES cells, which were enriched in CD34+ CD31lo and CD34+CD45− cell populations, respectively, in a dose-dependent manner. Furthermore, during the 7 days of in vitro differentiation, BMP-4 was required within the first 4 days, whereas VEGF was functional after the action of BMP-4 (in the last 3 days). Thus, VEGF is a synergistic enhancer for the BMP-4-dependent differentiation processes, and it seems to be achieved by the ordered action of the 2 factors.

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An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein

eLife ◽

10.7554/elife.35069 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 20

Author(s):

Pooran Singh Dewari ◽

Benjamin Southgate ◽

Katrina Mccarten ◽

German Monogarov ◽

Eoghan O'Duibhir ◽

...

Keyword(s):

Stem Cells ◽

Protein Function ◽

Es Cells ◽

Embryonic Stem ◽

Design Tool ◽

Epitope Tagging ◽

Selection Strategies ◽

Mammalian Cell Cultures ◽

Endogenous Genes ◽

Clonal Lines

CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here, we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5–30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain-tumor-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of 60 different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells.

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Developmental regulation of yolk sac hematopoiesis by Krüppel-like factor 6

Blood ◽

10.1182/blood-2005-05-1916 ◽

2006 ◽

Vol 107 (4) ◽

pp. 1357-1365 ◽

Cited By ~ 89

Author(s):

Nobuyuki Matsumoto ◽

Atsushi Kubo ◽

Huixian Liu ◽

Kuniharu Akita ◽

Friedrich Laub ◽

...

Keyword(s):

Developmental Regulation ◽

Es Cells ◽

Embryonic Stem ◽

Early Embryo ◽

Yolk Sac ◽

Homozygous Mutation ◽

Mesoderm Induction ◽

Mrna Levels ◽

Mouse Development

Krüppel-like factor 6 (KLF6) is a member of a growing family of transcription factors that share a common 3 C2H2 zinc finger DNA binding domain and have broad activity in regulating proliferation and development. We have previously established that Klf6 is expressed in neuronal tissue, hindgut, heart, lung, kidney, and limb buds during midgestation. To explore the potential role of Klf6 in mouse development, we analyzed Klf6-/- mice and found that the homozygous mutation is embryonic lethal by embryonic day (E) 12.5 and associated with markedly reduced hematopoiesis and poorly organized yolk sac vascularization. Additionally, mRNA levels of Scl and Gata1 were reduced by approximately 80% in Klf6-/- yolk sacs. To further analyze this phenotype, we generated Klf6-/- embryonic stem (ES) cells by homologous recombination, and compared their capacity to differentiate into the hematopoietic lineage with that of either Klf6+/- or Klf6+/+ ES cells. Consistent with the phenotype in the early embryo, Klf6-/- ES cells displayed significant hematopoietic defects following differentiation into EBs. Prolongation of epiblast-like cells and delays in mesoderm induction were also observed in the Klf6-/- EBs, associated with delayed expression of Brachyury, Klf1, and Gata1. Forced expression of KLF6 using a tet-inducible system enhanced the hematopoietic potential of wild-type EBs. Collectively, these findings implicate Klf6 in ES-cell differentiation and hematopoiesis.

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Mice develop normally in the absence of Smad4 nucleocytoplasmic shuttling

Biochemical Journal ◽

10.1042/bj20061830 ◽

2007 ◽

Vol 404 (2) ◽

pp. 235-245 ◽

Cited By ~ 12

Author(s):

Christine A. Biondi ◽

Debipriya Das ◽

Michael Howell ◽

Ayesha Islam ◽

Elizabeth K. Bikoff ◽

...

Keyword(s):

Transcriptional Activation ◽

Nuclear Export ◽

Transforming Growth Factor ◽

Es Cells ◽

Nuclear Export Signal ◽

Embryonic Stem ◽

Nucleocytoplasmic Shuttling ◽

Mouse Development ◽

Developmental Defects

Smad4 in partnership with R-Smads (receptor-regulated Smads) activates TGF-β (transforming growth factor-β)-dependent signalling pathways essential for early mouse development. Smad4 null embryos die shortly after implantation due to severe defects in cell proliferation and visceral endoderm differentiation. In the basal state, Smad4 undergoes continuous shuttling between the cytoplasm and the nucleus due to the combined activities of an N-terminal NLS (nuclear localization signal) and an NES (nuclear export signal) located in its linker region. Cell culture experiments suggest that Smad4 nucleocytoplasmic shuttling plays an important role in TGF-β signalling. In the present study we have investigated the role of Smad4 shuttling in vivo using gene targeting to engineer two independent mutations designed to eliminate Smad4 nuclear export. As predicted this results in increased levels of Smad4 in the nucleus of homozygous ES cells (embryonic stem cells) and primary keratinocytes, in the presence or absence of ligand. Neither mutation affects Smad4 expression levels nor its ability to mediate transcriptional activation in homozygous cell lines. Remarkably mouse mutants lacking the Smad4 NES develop normally. Smad4 NES mutants carrying one copy of a Smad4 null allele also fail to display developmental defects. The present study clearly demonstrates that Smad4 nucleocytoplasmic shuttling is not required for embryonic development or tissue homoeostasis in normal, healthy adult mice.

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