scholarly journals Dnmt1 has de novo activity targeted to transposable elements

2021 ◽  
Author(s):  
Chuck Haggerty ◽  
Helene Kretzmer ◽  
Christina Riemenschneider ◽  
Abhishek Sampath Kumar ◽  
Alexandra L. Mattei ◽  
...  
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Critical Role ◽  
Embryonic Stem ◽  
Genetically Engineered ◽  
Genomic Recruitment ◽  
Methylation Activity ◽  
De Novo Methylation

AbstractDNA methylation plays a critical role during development, particularly in repressing retrotransposons. The mammalian methylation landscape is dependent on the combined activities of the canonical maintenance enzyme Dnmt1 and the de novo Dnmts, 3a and 3b. Here, we demonstrate that Dnmt1 displays de novo methylation activity in vitro and in vivo with specific retrotransposon targeting. We used whole-genome bisulfite and long-read Nanopore sequencing in genetically engineered methylation-depleted mouse embryonic stem cells to provide an in-depth assessment and quantification of this activity. Utilizing additional knockout lines and molecular characterization, we show that the de novo methylation activity of Dnmt1 depends on Uhrf1, and its genomic recruitment overlaps with regions that enrich for Uhrf1, Trim28 and H3K9 trimethylation. Our data demonstrate that Dnmt1 can catalyze DNA methylation in both a de novo and maintenance context, especially at retrotransposons, where this mechanism may provide additional stability for long-term repression and epigenetic propagation throughout development.

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

2003 ◽  
Vol 23 (16) ◽  
pp. 5594-5605 ◽  
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.

scholarly journals DNA Methylation Density Influences the Stability of an Epigenetic Imprint and Dnmt3a/b-Independent De Novo Methylation

2002 ◽  
Vol 22 (21) ◽  
pp. 7572-7580 ◽  
Author(s):  
Matthew C. Lorincz ◽  
Dirk Schübeler ◽  
Shauna R. Hutchinson ◽  
David R. Dickerson ◽  
Mark Groudine
Keyword(s):  
Dna Methylation ◽  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
De Novo ◽  
Embryonic Stem ◽  
Dna Methyltransferases ◽  
The Stability ◽  
Murine Erythroleukemia ◽  
De Novo Methylation

ABSTRACT DNA methylation plays an important role in transcriptional repression. To gain insight into the dynamics of demethylation and de novo methylation, we introduced a proviral reporter, premethylated at different densities, into a defined chromosomal site in murine erythroleukemia cells and monitored the stability of the introduced methylation and reporter gene expression. A high density of methylation was faithfully propagated in vivo. In contrast, a low level of methylation was not stable, with complete demethylation and associated transcriptional activation or maintenance-coupled de novo methylation and associated silencing occurring with equal probability. Deletion of the proviral enhancer increased the probability of maintenance-coupled de novo methylation, suggesting that this enhancer functions in part to antagonize such methylation. The DNA methyltransferases (MTases) Dnmt3a and Dnmt3b are thought to be the sole de novo MTases in the mammalian genome. To determine whether these enzymes are responsible for maintenance-coupled de novo methylation, the unmethylated or premethylated proviral reporter was introduced into DNA MTase-deficient embryonic stem cells. These studies revealed the presence of a Dnmt3a/Dnmt3b-independent de novo methyltransferase activity that is stimulated by the presence of preexisting methylation.

scholarly journals Disabling de novo DNA methylation in embryonic stem cells allows an illegitimate fate trajectory

2021 ◽  
Vol 118 (38) ◽  
pp. e2109475118
Author(s):  
Masaki Kinoshita ◽  
Meng Amy Li ◽  
Michael Barber ◽  
William Mansfield ◽  
Sabine Dietmann ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Fate ◽  
De Novo ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dna Methyltransferases ◽  
Bhlh Transcription Factor ◽  
Mammalian Development

Genome remethylation is essential for mammalian development but specific reasons are unclear. Here we examined embryonic stem (ES) cell fate in the absence of de novo DNA methyltransferases. We observed that ES cells deficient for both Dnmt3a and Dnmt3b are rapidly eliminated from chimeras. On further investigation we found that in vivo and in vitro the formative pluripotency transition is derailed toward production of trophoblast. This aberrant trajectory is associated with failure to suppress activation of Ascl2. Ascl2 encodes a bHLH transcription factor expressed in the placenta. Misexpression of Ascl2 in ES cells provokes transdifferentiation to trophoblast-like cells. Conversely, Ascl2 deletion rescues formative transition of Dnmt3a/b mutants and improves contribution to chimeric epiblast. Thus, de novo DNA methylation safeguards against ectopic activation of Ascl2. However, Dnmt3a/b-deficient cells remain defective in ongoing embryogenesis. We surmise that multiple developmental transitions may be secured by DNA methylation silencing potentially disruptive genes.

scholarly journals In Vivo Activity of Murine De Novo Methyltransferases, Dnmt3a and Dnmt3b

1999 ◽  
Vol 19 (12) ◽  
pp. 8211-8218 ◽  
Author(s):  
Chih-Lin Hsieh
Keyword(s):  
De Novo ◽  
Leukemia Virus ◽  
Human Cells ◽  
Moloney Murine Leukemia Virus ◽  
Methyltransferase Activity ◽  
Metaphase Chromosomes ◽  
Methylation Activity ◽  
De Novo Methylation

ABSTRACT The putative de novo methyltransferases, Dnmt3a and Dnmt3b, were reported to have weak methyltransferase activity in methylating the 3′ long terminal repeat of Moloney murine leukemia virus in vitro. The activity of these enzymes was evaluated in vivo, using a stable episomal system that employs plasmids as targets for DNA methylation in human cells. De novo methylation of a subset of the CpG sites on the stable episomes is detected in human cells overexpressing the murine Dnmt3a or Dnmt3b1 protein. This de novo methylation activity is abolished when the cysteine in the P-C motif, which is the catalytic site of cytosine methyltransferases, is replaced by a serine. The pattern of methylation on the episome is nonrandom, and different regions of the episome are methylated to different extents. Furthermore, Dnmt3a also methylates the sequence methylated by Dnmt3a on the stable episome in the corresponding chromosomal target. Overexpression of human DNMT1 or murine Dnmt3b does not lead to the same pattern or degree of de novo methylation on the episome as overexpression of murine Dnmt3a. This finding suggests that these three enzymes may have different targets or requirements, despite the fact that weak de novo methyltransferase activity has been demonstrated in vitro for all three enzymes. It is also noteworthy that both Dnmt3a and Dnmt3b proteins coat the metaphase chromosomes while displaying a more uniform pattern in the nucleus. This is the first evidence that Dnmt3a and Dnmt3b have de novo methyltransferase function in vivo and the first indication that the Dnmt3a and Dnmt3b proteins may have preferred target sites.

scholarly journals Targeting de novo lipogenesis and the Lands cycle induces ferroptosis in KRAS-mutant lung cancer

2021 ◽  
Author(s):  
Caterina Bartolacci ◽  
Cristina Andreani ◽  
Goncalo Dias do Vale ◽  
Stefano Berto ◽  
Margherita Melegari ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Fatty Acid ◽  
Metabolic Networks ◽  
Fatty Acid Synthase ◽  
De Novo ◽  
Genetically Engineered ◽  
De Novo Lipogenesis ◽  
Main Process

Mutant KRAS (KM) is the most common oncogene in lung cancer (LC). KM regulates several metabolic networks, but their role in tumorigenesis is still not sufficiently characterized to be exploited in cancer therapy. To identify metabolic networks specifically deregulated in KMLC, we characterized the lipidome of genetically engineered LC mice, cell lines, patient derived xenografts and primary human samples. We also determined that KMLC, but not EGFR-mutant (EGFR-MUT) LC, is enriched in triacylglycerides (TAG) and phosphatidylcholines (PC). We also found that KM upregulates fatty acid synthase (FASN), a rate-limiting enzyme in fatty acid (FA) synthesis promoting the synthesis of palmitate and PC. We determined that FASN is specifically required for the viability of KMLC, but not of LC harboring EGFR-MUT or wild type KRAS. Functional experiments revealed that FASN inhibition leads to ferroptosis, a reactive oxygen species (ROS)-and iron-dependent cell death. Consistently, lipidomic analysis demonstrated that FASN inhibition in KMLC leads to accumulation of PC with polyunsaturated FA (PUFA) chains, which are the substrate of ferroptosis. Integrating lipidomic, transcriptome and functional analyses, we demonstrated that FASN provides saturated (SFA) and monounsaturated FA (MUFA) that feed the Lands cycle, the main process remodeling oxidized phospholipids (PL), such as PC. Accordingly, either inhibition of FASN or suppression of the Lands cycle enzymes PLA2 and LPCAT3, promotes the intracellular accumulation of lipid peroxides and ferroptosis in KMLC both in vitro and in vivo. Our work supports a model whereby the high oxidative stress caused by KM dictates a dependency on newly synthesized FA to repair oxidated phospholipids, establishing a targetable vulnerability. These results connect KM oncogenic signaling, FASN induction and ferroptosis, indicating that FASN inhibitors already in clinical trial in KMLC patients (NCT03808558) may be rapidly deployed as therapy for KMLC.

MicroRNAs: Crucial Players in the Differentiation of Human Pluripotent and Multipotent Stem Cells into Functional Hepatocyte-Like Cells

2021 ◽  
Vol 16 ◽  
Author(s):  
Hao Xu ◽  
Liying Wu ◽  
Guojia Yuan ◽  
Xiaolu Liang ◽  
Xiaoguang Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Liver Disease ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Critical Role ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
The Body

: Hepatic disease negatively impacts liver function and metabolism. Primary human hepatocytes are the gold standard for the prediction and successful treatment of liver disease. However, the sources of hepatocytes for drug toxicity testing and disease modeling are limited. To overcome this issue, pluripotent stem cells (PSCs) have emerged as an alternative strategy for liver disease therapy. Human PSCs, including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can self-renew and give rise to all cells of the body. Human PSCs are attractive cell sources for regenerative medicine, tissue engineering, drug discovery, and developmental studies. Several recent studies have shown that mesenchymal stem cells (MSCs) can also differentiate (or trans-differentiate) into hepatocytes. Differentiation of human PSCs and MSCs into functional hepatocyte-like cells (HLCs) opens new strategies to study genetic diseases, hepatotoxicity, infection of hepatotropic viruses, and analyze hepatic biology. Numerous in vitro and in vivo differentiation protocols have been established to obtain human PSCs/MSCs-derived HLCs and mimic their characteristics. It was recently discovered that microRNAs (miRNAs) play a critical role in controlling the ectopic expression of transcription factors and governing the hepatocyte differentiation of human PSCs and MSCs. In this review, we focused on the role of miRNAs in the differentiation of human PSCs and MSCs into hepatocytes.

84 MARKERS OF DNA METHYLATION IN OVINE UTERO–PLACENTAL TISSUES DURING EARLY PREGNANCY: EFFECTS OF ASSISTED REPRODUCTIVE TECHNOLOGY

2012 ◽  
Vol 24 (1) ◽  
pp. 154
Author(s):  
A. T. Grazul-Bilska ◽  
M. L. Johnson ◽  
P. P. Borowicz ◽  
D. A. Redmer ◽  
L. P. Reynolds
Keyword(s):  
Dna Methylation ◽  
Assisted Reproductive Technology ◽  
Early Pregnancy ◽  
De Novo ◽  
Reproductive Technology ◽  
The Other ◽  
Gravid Uterus ◽  
Natural Breeding ◽  
De Novo Methylation

Compromised pregnancies can be caused by genetic, epigenetic, environmental and/or other factors. Assisted reproductive technology (ART) may have profound effects on placental and fetal development, leading eventually to compromised pregnancy. DNA methylation, regulated by DNA methyltransferases (Dnmt) and other factors, plays an important role during embryonic, including placental, development. Altered DNA methylation in the trophoblast and, subsequently, the placenta has been reported for compromised pregnancies and may contribute to embryonic/fetal loss. Little is known, however, about DNA methylation processes in placental tissues during early stages of normal or compromised pregnancies in any species. Thus, we hypothesised that ART would affect the expression of 5 methylcytosine (5mC; a marker of global methylation) and mRNA for Dnmt1, 3a and 3b in utero-placental tissues during early pregnancy in sheep. Pregnancies (n = 7 per group) were achieved through natural breeding (NAT, control), or transfer of embryos generated through natural breeding (NAT-ET), in vitro fertilization (IVF) or in vitro activation (IVA; parthenogenetic clones). On Day 22 of pregnancy, caruncle (CAR; maternal placenta) and fetal membranes (FM; fetal placenta) were snap-frozen separately for RNA extraction followed by quantitative real-time PCR. In addition, cross sections of gravid uterus were fixed and then used for immunohistochemical detection and image analysis of 5 mC in FM. In FM, expression of mRNA for Dnmt3a was ∼2-fold greater (P < 0.01) in IVA compared with the other groups and was similar in NAT, NAT-ET and IVF groups. Expression of 5 mC was ∼2- to 3-fold greater (P < 0.02) in IVF and IVA than in NAT. In CAR, mRNA expression for Dnmt1 was ∼1.5-fold greater (P < 0.04) in IVA compared with the other groups, but Dnmt3a expression was less (P < 0.04) in NAT-ET and IVA than NAT. Expression of mRNA for Dnmt1 in FM and 3b in FM and CAR was similar in all groups. In IVA and/or IVF pregnancy, increased expression of Dnmt3a mRNA and/or 5 mC in FM may indicate de novo methylation in the fetal placenta. Furthermore, in pregnancies created through ART, decreased expression of Dnmt3a in CAR may indicate reduced de novo methylation in maternal placenta. Thus, in sheep, ART may have specific effects on growth and function of utero-placental and fetal tissues through regulation of DNA methylation and likely other mechanisms. These data provide a foundation for determining the basis for altered DNA methylation of specific genes in placental and embryonic tissues in compromised pregnancies. In addition, these data will help us to better understand placental regulatory mechanisms in compromised pregnancies and to identify strategies for rescuing such pregnancies. Supported by Hatch Project ND01712; USDA grant 2007-01215 to LPR and ATGB, NIH grant HL64141 to LPR and DAR and NSF-MRI-ARRA grant to ATGB.

scholarly journals In Vitro Organotypic Systems to Model Tumor Microenvironment in Human Papillomavirus (HPV)-Related Cancers

Cancers ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1150 ◽  
Author(s):  
Vincenza De Gregorio ◽  
Francesco Urciuolo ◽  
Paolo Antonio Netti ◽  
Giorgia Imparato
Keyword(s):  
Human Papillomavirus ◽  
Tumor Microenvironment ◽  
Critical Role ◽  
Genetically Engineered ◽  
Cancer Disease ◽  
Raft Culture ◽  
Squamous Epithelia

Despite the well-known role of chronic human papillomavirus (HPV) infections in causing tumors (i.e., all cervical cancers and other human malignancies from the mucosal squamous epithelia, including anogenital and oropharyngeal cavity), its persistence is not sufficient for cancer development. Other co-factors contribute to the carcinogenesis process. Recently, the critical role of the underlying stroma during the HPV life cycle and HPV-induced disease have been investigated. The tumor stroma is a key component of the tumor microenvironment (TME), which is a specialized entity. The TME is dynamic, interactive, and constantly changing—able to trigger, support, and drive tumor initiation, progression, and metastasis. In previous years, in vitro organotypic raft cultures and in vivo genetically engineered mouse models have provided researchers with important information on the interactions between HPVs and the epithelium. Further development for an in-depth understanding of the interaction between HPV-infected tissue and the surrounding microenvironment is strongly required. In this review, we critically describe the HPV-related cancers modeled in vitro from the simplified ‘raft culture’ to complex three-dimensional (3D) organotypic models, focusing on HPV-associated cervical cancer disease platforms. In addition, we review the latest knowledge in the field of in vitro culture systems of HPV-associated malignancies of other mucosal squamous epithelia (anogenital and oropharynx), as well as rare cutaneous non-melanoma associated cancer.

De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells

Development ◽  
1996 ◽  
Vol 122 (10) ◽  
pp. 3195-3205 ◽  
Author(s):  
H. Lei ◽  
S.P. Oh ◽  
M. Okano ◽  
R. Juttermann ◽  
K.A. Goss ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Mammalian Cells ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Embryonic Stem ◽  
Cytosine Methyltransferase ◽  
Methyl Cytosine ◽  
Methylation Activity ◽  
De Novo Methylation

It has been a controversial issue as to how many DNA cytosine methyltransferase mammalian cells have and whether de novo methylation and maintenance methylation activities are encoded by a single gene or two different genes. To address these questions, we have generated a null mutation of the only known mammalian DNA methyltransferase gene through homologous recombination in mouse embryonic stem cells and found that the development of the homozygous embryos is arrested prior to the 8-somite stage. Surprisingly, the null mutant embryonic stem cells are viable and contain low but stable levels of methyl cytosine and methyltransferase activity, suggesting the existence of a second DNA methyltransferase in mammalian cells. Further studies indicate that de novo methylation activity is not impaired by the mutation as integrated provirus DNA in MoMuLV-infected homozygous embryonic stem cells become methylated at a similar rate as in wild-type cells. Differentiation of mutant cells results in further reduction of methyl cytosine levels, consistent with the de novo methylation activity being down regulated in differentiated cells. These results provide the first evidence that an independently encoded DNA methyltransferase is present in mammalian cells which is capable of de novo methylating cellular and viral DNA in vivo.

QSER1 protects DNA methylation valleys from de novo methylation

Science ◽  
2021 ◽  
Vol 372 (6538) ◽  
pp. eabd0875 ◽  
Author(s):  
Gary Dixon ◽  
Heng Pan ◽  
Dapeng Yang ◽  
Bess P. Rosen ◽  
Therande Jashari ◽  
...  
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Embryonic Stem ◽  
Central Question ◽  
Mammalian Development ◽  
Uncharacterized Gene ◽  
Pathological Conditions ◽  
Genome Wide ◽  
A Genome ◽  
De Novo Methylation

DNA methylation is essential to mammalian development, and dysregulation can cause serious pathological conditions. Key enzymes responsible for deposition and removal of DNA methylation are known, but how they cooperate to regulate the methylation landscape remains a central question. Using a knockin DNA methylation reporter, we performed a genome-wide CRISPR-Cas9 screen in human embryonic stem cells to discover DNA methylation regulators. The top screen hit was an uncharacterized gene, QSER1, which proved to be a key guardian of bivalent promoters and poised enhancers of developmental genes, especially those residing in DNA methylation valleys (or canyons). We further demonstrate genetic and biochemical interactions of QSER1 and TET1, supporting their cooperation to safeguard transcriptional and developmental programs from DNMT3-mediated de novo methylation.

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