scholarly journals Polycomb Group Proteins Recruit TET1 to Remove Aberrant DNA Methylation in Murine Embryonic Stem Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2571-2571
Author(s):  
Yong Lei ◽  
Margaret A. Goodell
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Dna Methyltransferase ◽  
Molecular Mechanisms ◽  
Embryonic Stem ◽  
Polycomb Group ◽  
Aberrant Methylation ◽  
Wild Type ◽  
Epigenetic Marks ◽  
Aberrant Dna Methylation

Abstract At the top of the hematopoietic hierarchy are hematopoietic stem cells (HSCs), which reside in the bone marrow and are characterized by the ability to self-renew or differentiate into various types of mature blood cells. The self-renewal capacity of HSCs relies on the accurate transmission of epigenetic marks to their progeny. Our lab has shown previously that, despite global hypomethylation, DNA hypermethylation frequently occurs on Polycomb group protein (PcG) target genes and many tumor suppressor genes in aged HSCs (Sun et al.Cell Stem Cell. 2014). At the same time, such epigenetic marks are correctly maintained in young HSCs. These observations indicate the presence of epigenetic maintenance systems that deteriorate with age. Currently, the molecular mechanisms through which aberrant DNA hypermethylation accumulates only with age are unclear. To address this gap in knowledge, we examined the maintenance and clearance of forced DNA methylation in murine embryonic stem cells (ESCs) using a CRISPR/dCas9-based targeted DNA methylation tool, dCas9-MQ1147 (Lei et al, Nature Communication. 2017). We forcibly methylated genes that are bound in ESCs by PcG, including Runx1, Gata2, and Hoxa loci. Surprisingly, we found that the exogenous methylation induced by dCas9-MQ1147 was efficiently removed from the target sites during cell proliferation, indicating that methylation status is predetermined and maintained by local genetic and epigenetic marks. To understand which demethylation mechanism is responsible for the removal of the exogenous aberrant DNA methylation in our model, we treated both human somatic cells and murine ESCs with cell cycle arrest drugs, including selective ATP-competitive inhibitor of CDK1 (Ro-3306), selective Ca2+/calmodulin-dependent protein kinase inhibitor (KN93), and microtubule formation inhibitor (Paclitaxel) after inducing targeted methylation. Following treatment, we observed that cell cycle arrest cannot delay passive demethylation at the hypermethylated sites, indicating the removel of aberrant methylation is DNA duplication independent. To further investigate the demethylation mechanism herein, we next validated the contribution of DNA hydroxymethylation enzyme Ten-eleven Translocation (TET) activity in the Tet1/2/3 triple knockout (TKO) mESCs. Time-dependent experiments showed that TKO mESCs had a significant exogenous methylation retention compared to their wild-type counterparts. These data indicate that TET family proteins are recruited to remove aberrant methylation from the unmethylated PcG binding region via TDG or base excision repair, but not inaccurate maintenance by DNA methyltransferase 1 (DNMT1). To determine which TET protein or proteins contribute to the maintenance of predetermined unmethylation status, we used prokaryote DNA methyltransferase MQ1 wild-type protein to generate genome-wide hypermethylation in wild-type mESCs. We detected that TET1 was the most highly upregulated TET protein, with over 5-fold upregulation, following induced hypermethylation. Furthermore, by applying the novel Degron targeted degradation technique, we specific remove the expressed MQ1 protein and found that the degradation of MQ1 led to the reduction of Tet1 overexpression. These data indicate that TET1 participates in the removal of aberrant DNA methylation in mESCs. Overall, this study suggests that a proofreading mechanism at the PcG-targeted region recognizes aberrant DNA methylation and recruits TET1 to restore its original unmethylated status. The dysregulation of this mechanism in aging HSCs may lead to the accumulation of methylation abnormalities during proliferation. This study sheds light on an important molecular mechanisms responsible for maintaining the epigenetic status in ESCs and provides insight into how aberrant DNA methylation accumulates in these cells over time. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Dysregulation of schizophrenia-associated genes and genome-wide hypomethylation in neurons overexpressing DNMT1

Epigenomics ◽  
2021 ◽  
Author(s):  
Sonal Saxena ◽  
Sumana Choudhury ◽  
Pranay Amruth Maroju ◽  
Anuhya Anne ◽  
Lov Kumar ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Copy Number ◽  
Embryonic Stem ◽  
Wild Type ◽  
Independent Manner ◽  
Transcript Levels ◽  
Genome Wide ◽  
Methylation Patterns ◽  
Increased Activity

Aim: To study the effects of DNMT1 overexpression on transcript levels of genes dysregulated in schizophrenia and on genome-wide methylation patterns. Materials & methods: Transcriptome and DNA methylome comparisons were made between R1 (wild-type) and Dnmt1tet/tet mouse embryonic stem cells and neurons overexpressing DNMT1. Genes dysregulated in both Dnmt1tet/tet cells and schizophrenia patients were studied further. Results & conclusions: About 50% of dysregulated genes in patients also showed altered transcript levels in Tet/Tet neurons in a DNA methylation-independent manner. These neurons unexpectedly showed genome-wide hypomethylation, increased transcript levels of Tet1 and Apobec 1-3 genes and increased activity and copy number of LINE-1 elements. The observed similarities between Tet/Tet neurons and schizophrenia brain samples reinforce DNMT1 overexpression as a risk factor.

New Role of the Regulatory Gene SOX2 in Hematopoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4195-4195
Author(s):  
Elena Levantini ◽  
Francesca Bertolotti ◽  
Francesco Cerisoli ◽  
Anna L. Ferri ◽  
Elisa Brescia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Blood Cell ◽  
Molecular Mechanisms ◽  
Bone Marrow Cells ◽  
Regulatory Gene ◽  
Embryonic Stem ◽  
Mutant Mice ◽  
Wild Type ◽  
Cell Production

Abstract Several genes encoding transcription factors of different families have been implicated in the development and differentiation of multiple cell systems. The Sry-type high-mobility-group box 2 gene (Sox2) encodes a transcription factor that is expressed in very early cells such as embryonic stem cells and neural stem cells, where it plays important functional roles (Genes and Dev.17:126, 2003; Development131:3805, 2004). To investigate whether Sox2 plays a role also in blood cell production, we first analyzed its expression in murine hematopoietic cells. Results indicate that the gene is transcriptionally active at low levels in primitive progenitors. Furthermore, in order to address the functional implication of Sox2 in hematopoiesis we analyzed mature and precursor cells in mutant mice compound heterozygotes for a null Sox2 allele and for the deletion of a Sox2 5′ enhancer, as the complete inactivation of the gene in homozygosis is embryonic lethal. At the peripheral blood level we did not detect significant variations in the mutants. However analysis of bone marrow precursors in clonogenic assays showed that Sox2 knock-down mice exhibited a significant increase in the number of multipotent precursors, as compared to wild type animals. Moreover, bone marrow cells of wild type and mutant mice were analyzed for the expression of a panel of regulatory genes involved in the control of different somatic stem cells. Preliminary evidence suggests that some of these genes are modulated in the mutant cells. These observations support the view that Sox2 plays a role at early stages of blood cell production, providing further evidence that common molecular mechanisms may be involved in the regulation of several different types of multipotent cells.

RNA interference in mammalian DNA methylation1This review is part of Special Issue entitled Asilomar Chromatin and has undergone the Journal’s usual peer review process.

2012 ◽  
Vol 90 (1) ◽  
pp. 70-77 ◽  
Author(s):  
Jeffrey R. Mann ◽  
Deidre M. Mattiske
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Dna Methyltransferase ◽  
Germ Line ◽  
Peer Review Process ◽  
Embryonic Stem ◽  
Cell Types ◽  
Heterochromatin Formation ◽  
Nonspecific Effects

RNAi and Dicer-dependent siRNAs are required for constitutive heterochromatin formation in fission yeast and for establishing DNA methylation at repetitive elements in plants. In the mammalian male germ line, DICER1-independent piRNAs are required for the full establishment of DNA methylation of dispersed repetitive transposable elements. However, in other mammalian cell types, no clear picture has yet emerged of the role of RNAi in establishing heterochromatin and DNA methylation. In mouse embryonic stem cells, which remain viable on loss of DICER1 and ablation of RNAi, while no firm evidence has been obtained for defective heterochromatin formation, there are indications of defective DNA methylation. The latter has been attributed to an indirect effect of reduced DNA methyltransferase (DNMT) activity due to a loss of miRNA-mediated gene regulation. However, it is unclear whether the reductions in DNMT activity were sufficient to affect DNA methylation. We consider it equally likely that the defects in DNA methylation that can be observed in DICER1-deficient embryonic stem cells are the result of nonspecific effects related to RNAi loss aside from reduced DNMT activity.

scholarly journals Effects of TET1 knockdown on gene expression and DNA methylation in porcine induced pluripotent stem cells

Reproduction ◽  
2013 ◽  
Vol 146 (6) ◽  
pp. 569-579 ◽  
Author(s):  
Anran Fan ◽  
Kuiying Ma ◽  
Xinglan An ◽  
Yu Ding ◽  
Peipei An ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Molecular Mechanisms ◽  
Embryonic Stem ◽  
Differentiation Markers ◽  
Expression Of Genes ◽  
Undifferentiated State ◽  
Induced Pluripotent

TET1 is implicated in maintaining the pluripotency of embryonic stem cells. However, its precise effects on induced pluripotent stem cells (iPSCs), and particularly on porcine iPSCs (piPSCs), are not well defined. To investigate the role of TET1 in the pluripotency and differentiation of piPSCs, piPSCs were induced from porcine embryonic fibroblasts by overexpression ofPOU5F1(OCT4),SOX2,KLF4, andMYC(C-MYC). siRNAs targeting toTET1were used to transiently knockdown the expression ofTET1in piPSCs. Morphological abnormalities and loss of the undifferentiated state of piPSCs were observed in the piPSCs after the downregulation ofTET1. The effects ofTET1knockdown on the expression of key stem cell factors and differentiation markers were analyzed to gain insights into the molecular mechanisms underlying the phenomenon. The results revealed that knockdown ofTET1resulted in the downregulated expression of pluripotency-related genes, such asLEFTY2,KLF2, andSOX2, and the upregulated expression of differentiation-related genes includingPITX2,HAND1,GATA6, andLEF1. However,POU5F1,MYC,KLF4, andNANOGwere actually not downregulated. Further analysis showed that the methylation levels of the promoters forPOU5F1andMYCincreased significantly afterTET1downregulation, whereas there were no obvious changes in the promoters ofSOX2,KLF4, andNANOG. The methylation of the whole genome increased, while hydroxymethylation slightly declined. Taken together, these results suggest thatTET1may play important roles in the self-renewal of piPSCs and the maintenance of their characteristics by regulating the expression of genes and the DNA methylation.

DNA methylation and copy number variation analyses of human embryonic stem cell–derived neuroprogenitors after low-dose decabromodiphenyl ether and/or bisphenol A exposure

2017 ◽  
Vol 37 (5) ◽  
pp. 475-485 ◽  
Author(s):  
L Du ◽  
W Sun ◽  
XM Li ◽  
XY Li ◽  
W Liu ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Bisphenol A ◽  
Copy Number ◽  
Neural Differentiation ◽  
Dna Methyltransferase ◽  
Embryonic Stem ◽  
Control Group ◽  
Decabromodiphenyl Ether ◽  
Bde 209

The polybrominated diphenyl ether flame retardants decabromodiphenyl ether (BDE-209) and bisphenol A (BPA) are environmental contaminants that can cross the placenta and exert toxicity in the developing fetal nervous system. Copy number variants (CNVs) play a role in a number of genetic disorders and may be implicated in BDE-209/BPA teratogenicity. In this study, we found that BDE-209 and/or BPA exposure decreased neural differentiation efficiency of human embryonic stem cells (hESCs), although there was a >90% induction of neuronal progenitor cells (NPCs) from exposed hESCs. However, the mean of CNV numbers in the NPCs with BDE-209 + BPA treatment was significantly higher compared to the other groups, whereas DNA methylation was lower and DNA methyltransferase(DNMT1 and DNMT3A) expression were significantly decreased in all of the BDE-209 and/or BPA treatment groups compared with the control groups. The number of CNVs in chromosomes 3, 4, 11, 22, and X in NPCs with BDE-209 and/or BPA exposure was higher compared to the control group. In addition, CNVs in chromosomes 7, 8, 14, and 16 were stable in hESCs and hESCs-derived NPCs irrespective of BDE-209/BPA exposure, and CNVs in chromosomes 20 q11.21 and 16 p13.11 might be induced by neural differentiation. Thus, BDE-209/BPA exposure emerges as a potential source of CNVs distinct from neural differentiation by itself. BDE-209 and/or BPA exposure may cause genomic instability in cultured stem cells via reduced activity of DNA methyltransferase, suggesting a new mechanism of human embryonic neurodevelopmental toxicity caused by this class of environmental toxins.

scholarly journals DNMT3A Regulates Hematopoietic Stem Cell Function Via DNA Methylation-Independent Functions

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 24-24
Author(s):  
Won Kyun Koh ◽  
Hamza Celik ◽  
Jacob Tao ◽  
Jake Fairchild ◽  
Ostap Kukhar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Ectopic Expression ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Independent Functions

Abstract The balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) is strictly regulated to sustain blood production throughout adult life. De novo DNA methyltransferase 3-alpha (DNMT3A) is one of the major epigenetic regulators that is essential for efficient HSC differentiation. DNMT3A mutations are prevalent in myeloid diseases that include acute myeloid leukemia (AML; ~22%) and myelodysplastic syndrome (MDS; ~10%) where they act as initiating events However, the precise molecular mechanisms of how DNMT3A regulates normal hematopoiesis and its mutations prime HSCs for leukemic formation are unclear. Although DNMT3A is described as a DNA methyltransferase enzyme, the lack of consistent correlation between changes in DNA methylation and differential gene expression in Dnmt3a-null HSCs in mouse models, and AML patients with DNMT3A mutations undermine the conventional understanding of DNMT3A's canonical role in hematopoietic cells. Hence, we hypothesized that DNMT3A may have novel functions outside of DNA methylation that regulate HSC fate decisions. To answer this question, we first ectopically expressed GFP-labeled Dnmt3a constructs (wild-type Dnmt3a, Dnmt3aE752A; complete DNA methylation dead, and Dnmt3aR832A; reduced DNA methylation target recognition) and empty vector (negative control) in Dnmt3a-null (Vav-Cre: Dnmt3afl/fl = Dnmt3a-/- in hematopoiesis) bone marrow (BM) cells. The result showed that similar to restoring wild-type Dnmt3a, ectopic expression of Dnmt3aE752A as well as Dnmt3aR832A showed a rescue effect of decreased engraftment of transduced cells in the peripheral blood as well as reduced HSC numbers in the BM. Analysis of DNA methylation by whole-genome bisulfite sequencing (WGBS) in transduced cells showed this phenotypic and functional rescue of the Dnmt3a-/- phenotype occurred in the absence of restored DNA methylation patterns. To study the importance of Dnmt3a-mediated DNA methyltransferase activity in a more physiological system, we generated knock-in mice that have one copy of either wild-type Dnmt3a, Dnmt3aE752A, or Dnmt3aR832A (CAGG-Cre-ER T2 = ER T2-Cre: Dnmt3afl/+, Dnmt3afl/E752A, and Dnmt3afl/R832A) to be compared to the Dnmt3a-null group (ER T2-Cre: Dnmt3afl/-). These mice contain one allele with loxP-flanked Dnmt3a that is deleted by tamoxifen-inducible Cre-mediated recombination and one allele of either wild-type Dnmt3a, Dnmt3aE752A, Dnmt3aR832A, or germline knockout Dnmt3anull. 5-weeks post-tamoxifen (~93% floxed allele recombination), competitive transplantation of 250 phenotypically defined test HSCs against with 2.5x10 5 congenic competitor BM cells was performed. Dnmt3a fl/R832A recipients had higher engraftment (35.6 % +/- 6.1) than Dnmt3afl/+ (28.5% +/- 7.2) and Dnmt3afl/- (10.7% +/- 2.79), while Dnmt3afl/E752A had slightly higherengraftment (12.5% +/- 3) than Dnmt3afl/-. Analysis of the BM 18 weeks post-transplant showed that Dnmt3afl/E752A and Dnmt3afl/R832A HSCs phenocopied the HSC self-renewal potential phenotype of heterozygous Dnmt3a fl/+HSCs (Fig. 1). The absolute count of donor-derived HSCs per mouse after the transplant were: ER T2-Cre control (675.7 +/- 299.3), Dnmt3afl/+ (1870 +/- 961.4), Dnmt3afl/- (3546 +/- 1019), Dnmt3afl/E752A (1130 +/- 362.7), and Dnmt3afl/R832A (1184 +/- 344.5) (mean +/- S.E.M.). While the described clonal expansion of Dnmt3a-null HSCs was observed, HSCs with one copy of full-length Dnmt3a but devoid of its methyltransferase capacity mimicked the heterozygous state rather than the homozygous loss-of-function. This is the first evidence to suggest that DNMT3A potentially regulates HSCs by non-canonical (DNA methylation independent) mechanisms. DNA methylation analysis by WGBS is ongoing to determine if Dnmt3afl/E752A and Dnmt3afl/R832A HSCs show a methylome comparable to Dnmt3a-null HSCs whilst having the functional potential of Dnmt3a-heterozygous HSCs, which will be complemented with other molecular analyses including gene expression. Our study opens new avenues for investigations into the molecular mechanisms of DNMT3A function in HSC biology, which could ultimately benefit clinical practice by identifying new therapeutic approaches for the patients with DNMT3A mutations. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals MicroRNAs and Stem-like Properties: The Complex Regulation Underlying Stemness Maintenance and Cancer Development

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1074
Author(s):  
Giuseppina Divisato ◽  
Silvia Piscitelli ◽  
Mariantonietta Elia ◽  
Emanuela Cascone ◽  
Silvia Parisi
Keyword(s):  
Stem Cells ◽  
Molecular Mechanisms ◽  
Epithelial Mesenchymal Transition ◽  
Embryonic Stem ◽  
Cell Types ◽  
Cancer Development ◽  
Special Focus ◽  
Self Renewal ◽  
Mesenchymal Transition ◽  
Different Cell Types

Embryonic stem cells (ESCs) have the extraordinary properties to indefinitely proliferate and self-renew in culture to produce different cell progeny through differentiation. This latter process recapitulates embryonic development and requires rounds of the epithelial–mesenchymal transition (EMT). EMT is characterized by the loss of the epithelial features and the acquisition of the typical phenotype of the mesenchymal cells. In pathological conditions, EMT can confer stemness or stem-like phenotypes, playing a role in the tumorigenic process. Cancer stem cells (CSCs) represent a subpopulation, found in the tumor tissues, with stem-like properties such as uncontrolled proliferation, self-renewal, and ability to differentiate into different cell types. ESCs and CSCs share numerous features (pluripotency, self-renewal, expression of stemness genes, and acquisition of epithelial–mesenchymal features), and most of them are under the control of microRNAs (miRNAs). These small molecules have relevant roles during both embryogenesis and cancer development. The aim of this review was to recapitulate molecular mechanisms shared by ESCs and CSCs, with a special focus on the recently identified classes of microRNAs (noncanonical miRNAs, mirtrons, isomiRs, and competitive endogenous miRNAs) and their complex functions during embryogenesis and cancer development.

scholarly journals Adhesion and Growth of Neuralized Mouse Embryonic Stem Cells on Parylene-C/SiO2 Substrates

Materials ◽  
2021 ◽  
Vol 14 (12) ◽  
pp. 3174
Author(s):  
Alan F. Murray ◽  
Evangelos Delivopoulos
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Primary Neurons ◽  
Tissue Engineering Scaffolds ◽  
Parylene C ◽  
Unexpected Outcome

Neuronal patterning on microfabricated architectures has developed rapidly over the past few years, together with the emergence of soft biocompatible materials and tissue engineering scaffolds. Previously, we introduced a patterning technique based on serum and the biopolymer parylene-C, achieving highly compliant growth of primary neurons and astrocytes on different geometries. Here, we expanded this technique and illustrated that neuralized cells derived from mouse embryonic stem cells (mESCs) followed stripes of variable widths with conformity equal to or higher than that of primary neurons and astrocytes. Our results indicate the presence of undifferentiated mESCs, which also conformed to the underlying patterns to a high degree. This is an exciting and unexpected outcome, as molecular mechanisms governing cell and ECM protein interactions are different in stem cells and primary cells. Our study enables further investigations into the development and electrophysiology of differentiating patterned neural stem cells.

scholarly journals Reduced Genomic Cytosine Methylation and Defective Cellular Differentiation in Embryonic Stem Cells Lacking CpG Binding Protein

2005 ◽  
Vol 25 (12) ◽  
pp. 4881-4891 ◽  
Author(s):  
Diana L. Carlone ◽  
Jeong-Heon Lee ◽  
Suzanne R. L. Young ◽  
Erika Dobrota ◽  
Jill Sergesketter Butler ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Dna Methyltransferase ◽  
Cellular Differentiation ◽  
Cytosine Methylation ◽  
Epigenetic Modification ◽  
Binding Protein ◽  
Embryonic Stem ◽  
Growth Media ◽  
Methyltransferase Activity

ABSTRACT Cytosine methylation at CpG dinucleotides is a critical epigenetic modification of mammalian genomes. CpG binding protein (CGBP) exhibits a unique DNA-binding specificity for unmethylated CpG motifs and is essential for early murine development. Embryonic stem cell lines deficient for CGBP were generated to further examine CGBP function. CGBP − / − cells are viable but show an increased rate of apoptosis and are unable to achieve in vitro differentiation following removal of leukemia inhibitory factor from the growth media. Instead, CGBP − / − embryonic stem cells remain undifferentiated as revealed by persistent expression of the pluripotent markers Oct4 and alkaline phosphatase. CGBP − / − cells exhibit a 60 to 80% decrease in global cytosine methylation, including hypo-methylation of repetitive elements, single-copy genes, and imprinted genes. Total DNA methyltransferase activity is reduced by 30 to 60% in CGBP − / − cells, and expression of the maintenance DNA methyltransferase 1 protein is similarly reduced. However, de novo DNA methyltransferase activity is normal. Nearly all aspects of the pleiotropic CGBP − / − phenotype are rescued by introduction of a CGBP expression vector. Hence, CGBP is essential for normal epigenetic modification of the genome by cytosine methylation and for cellular differentiation, consistent with the requirement for CGBP during early mammalian development.

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