scholarly journals DNA methylation. Inhibition of de novo and maintenance methylation in vitro by RNA and synthetic polynucleotides.

1984 ◽  
Vol 259 (20) ◽  
pp. 12437-12443 ◽  
Author(s):  
A Bolden ◽  
C Ward ◽  
J A Siedlecki ◽  
A Weissbach
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Maintenance Methylation

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 Variants ofDNMT3Acause transcript-specific DNA methylation patterns and affect hematopoiesis

2018 ◽  
Vol 1 (6) ◽  
pp. e201800153 ◽  
Author(s):  
Tanja Božić ◽  
Joana Frobel ◽  
Annamarija Raic ◽  
Fabio Ticconi ◽  
Chao-Chung Kuo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem ◽  
Acute Myeloid

De novo DNA methyltransferase 3A (DNMT3A) plays pivotal roles in hematopoietic differentiation. In this study, we followed the hypothesis that alternative splicing ofDNMT3Ahas characteristic epigenetic and functional sequels. SpecificDNMT3Atranscripts were either down-regulated or overexpressed in human hematopoietic stem and progenitor cells, and this resulted in complementary and transcript-specific DNA methylation and gene expression changes. Functional analysis indicated that, particularly, transcript 2 (coding for DNMT3A2) activates proliferation and induces loss of a primitive immunophenotype, whereas transcript 4 interferes with colony formation of the erythroid lineage. Notably, in acute myeloid leukemia expression of transcript 2 correlates with its in vitro DNA methylation and gene expression signatures and is associated with overall survival, indicating thatDNMT3Avariants also affect malignancies. Our results demonstrate that specificDNMT3Avariants have a distinct epigenetic and functional impact. Particularly, DNMT3A2 triggers hematopoietic differentiation and the corresponding signatures are reflected in acute myeloid leukemia.

The action of the Cu2+, Ag+ and time inducing the in vitro anther culture-derived barley regenerants

2020 ◽  
Author(s):  
Piotr Tomasz Bednarek ◽  
Renata Orłowska
Keyword(s):  
Dna Methylation ◽  
Tissue Culture ◽  
De Novo ◽  
Culture Conditions ◽  
Ion Concentration ◽  
Tissue Cultures ◽  
Regeneration Medium ◽  
Green Plants ◽  
Anther Cultures

Abstract BackgroundPlant regeneration via anther cultures is a world-wide approach as it allows for the regeneration of uniform and homozygous double haploids. Recent studies have shown that in vitro cultures are the origin of the so-called tissue culture-induced variation (TCIV) that may lead to off-type regenerants. Moreover, the regeneration of green plants may be limited by the presence of albinos. It was demonstrated that the presence of Cu2+ and Ag+ ions in the regeneration medium might increase the number of green plants.ResultsDArTseqMet markers were evaluated based on regenerants and donor plants derived via in vitro anther cultures of barley. The regenerants were obtained under varying Cu2+ and Ag+ ion concentration in the regeneration medium during distinct time conditions of the tissue cultures. The DArTseqMet markers were quantified using a semi-quantitative MSAP approach delivering data on CG and CHG sequence contexts de novo methylation and demethylation. Under each tissue culture conditions, the number of regenerated green plants per 100 anthers was evaluated. Conditional moderation analysis was applied to test for the role of Cu2+ and Ag+ ions in the medium. Moreover, the importance of the time of in vitro anther cultures were analyzed.ConclusionsOur data demonstrate that DNA de novo methylation and demethylation affecting CG and CXG DNA sequence contexts is moderated by the presence of Cu2+ and Ag+ ions in the medium conditional on the time of in vitro tissue cultures. The level of de novo methylation and demethylation and the difference between the two is essential for the understanding of moderation. Moreover, Cu2+ and Ag+ play in concert moderating DNA methylation changes. For the in vitro tissue culture purposes, the lower the delta value equal to de novo methylation less demethylation and the higher the value of the (Cu+Ag) predictor conditional on time, the higher the number of green plants should be evaluated. Moreover, evaluation of GPs is even more probable under positive delta and higher (Cu+Ag) values. Our data are congruent with the putative function of these ions in the ethylene and DNA methylation pathways.

scholarly journals Perturbation of maintenance and de novo DNA methylation in vitro by UVB (280-340 nm)-induced pyrimidine photodimers.

1985 ◽  
Vol 82 (18) ◽  
pp. 6055-6059 ◽  
Author(s):  
F. F. Becker ◽  
P. Holton ◽  
M. Ruchirawat ◽  
J. N. Lapeyre
Keyword(s):  
Dna Methylation ◽  
De Novo

scholarly journals Retroviral Expression in Embryonic Stem Cells and Hematopoietic Stem Cells

2000 ◽  
Vol 20 (20) ◽  
pp. 7419-7426 ◽  
Author(s):  
Sara R. Cherry ◽  
D. Biniszkiewicz ◽  
L. van Parijs ◽  
D. Baltimore ◽  
R. Jaenisch
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Transcriptional Repression ◽  
Fluorescent Protein ◽  
De Novo ◽  
Es Cells ◽  
Embryonic Stem ◽  
Hematopoietic Stem

ABSTRACT Achieving long-term retroviral expression in primary cells has been problematic. De novo DNA methylation of infecting proviruses has been proposed as a major cause of this transcriptional repression. Here we report the development of a mouse stem cell virus (MSCV) long terminal repeat-based retroviral vector that is expressed in both embryonic stem (ES) cells and hematopoietic stem (HS) cells. Infected HS cells and their differentiated descendants maintained long-term and stable retroviral expression after serial adoptive transfers. In addition, retrovirally infected ES cells showed detectable expression level of the green fluorescent protein (GFP). Moreover, GFP expression of integrated proviruses was maintained after in vitro differentiation of infected ES cells. Long-term passage of infected ES cells resulted in methylation-mediated silencing, while short-term expression was methylation independent. Tissues of transgenic animals, which we derived from ES cells carrying the MSCV-based provirus, did not express GFP. However, treatment with the demethylating agent 5-azadeoxycytidine reactivated the silent provirus, demonstrating that DNA methylation is involved in the maintenance of retroviral repression. Our results indicate that retroviral expression in ES cells is repressed by methylation-dependent as well as methylation-independent mechanisms.

Altered chromatin condensation of heat-stressed spermatozoa perturbs the dynamics of DNA methylation reprogramming in the paternal genome after in vitro fertilisation in cattle

2014 ◽  
Vol 26 (8) ◽  
pp. 1107 ◽  
Author(s):  
Mohammad Bozlur Rahman ◽  
Md. Mostofa Kamal ◽  
Tom Rijsselaere ◽  
Leen Vandaele ◽  
Mohammed Shamsuddin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Chromatin Condensation ◽  
Dna Demethylation ◽  
In Vitro Fertilisation ◽  
Paternal Genome ◽  
Time Points ◽  
Fertilisation Rate ◽  
De Novo Methylation

Shortly after penetration of the oocyte, sperm DNA is actively demethylated, which is required for totipotent zygotic development. Aberrant DNA methylation is thought to be associated with altered chromatin condensation of spermatozoa. The objectives of this study were to investigate the dynamics of DNA methylation reprogramming in the paternal pronucleus and subsequent fertilisation potential of heat-stressed bull spermatozoa having altered chromatin condensation. Hence, bovine zygotes (n = 1239) were collected at three different time points (12, 18 and 24 h post insemination, hpi), and stained with an antibody against 5-methylcytosine. Fluorescence intensities of paternal and maternal pronuclei were measured by ImageJ. DNA methylation patterns in paternal pronuclei derived from heat-stressed spermatozoa did not differ between time points (P > 0.05), whereas control zygotes clearly showed demethylation and de novo methylation at 18 and 24 hpi, respectively. Moreover, heat-stressed spermatozoa showed a highly reduced (P < 0.01) fertilisation rate compared with non-heat-stressed or normal control spermatozoa (53.7% vs 70.2% or 81.5%, respectively). Our data show that the normal pattern of active DNA demethylation followed by de novo methylation in the paternal pronucleus is perturbed when oocytes are fertilised with heat-stressed spermatozoa, which may be responsible for decreased fertilisation potential.

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.

Corrigendum to: Decreased expression of DNA methyltransferases in the testes of patients with non-obstructive azoospermia leads to changes in global DNA methylation levels

2019 ◽  
Vol 31 (8) ◽  
pp. 1424
Author(s):  
Fatma Uysal ◽  
Gokhan Akkoyunlu ◽  
Saffet Ozturk
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Testicular Biopsy ◽  
Round Spermatid ◽  
Dna Methyltransferases ◽  
Global Dna Methylation ◽  
Obstructive Azoospermia ◽  
Cellular Localisation ◽  
Cpg Dinucleotide ◽  
Maintenance Methylation

DNA methylation plays key roles in epigenetic regulation during mammalian spermatogenesis. DNA methyltransferases (DNMTs) function in de novo and maintenance methylation processes by adding a methyl group to the fifth carbon atom of the cytosine residues within cytosine–phosphate–guanine (CpG) and non-CpG dinucleotide sites. Azoospermia is one of the main causes of male infertility, and is classified as obstructive (OA) or non-obstructive (NOA) azoospermia based on histopathological characteristics. The molecular background of NOA is still largely unknown. DNA methylation performed by DNMTs is implicated in the transcriptional regulation of spermatogenesis-related genes. The aim of the present study was to evaluate the cellular localisation and expression levels of the DNMT1, DNMT3A and DNMT3B proteins, as well as global DNA methylation profiles in testicular biopsy samples obtained from men with various types of NOA, including hypospermatogenesis (hyposperm), round spermatid (RS) arrest, spermatocyte (SC) arrest and Sertoli cell-only (SCO) syndrome. In the testicular biopsy samples, DNMT1 expression and global DNA methylation levels decreased gradually from the hyposperm to SCO groups (PPP

R882H DNMT3A Causes Dominant-Negative Inhibition Of WT DNMT3A

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3812-3812
Author(s):  
David A. Russler-Germain ◽  
David H Spencer ◽  
Margaret A. Young ◽  
Tamara Lamprecht ◽  
Chris Miller ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Catalytic Domain ◽  
De Novo ◽  
Dominant Negative ◽  
Size Exclusion ◽  
Dominant Negative Effect ◽  
Methyltransferase Activity ◽  
Negative Effect

Abstract Mutations in DNMT3A (encoding one of two mammalian de novo DNA methyltransferases) are found in >30% of normal karyotype AML cases and correlate with poor clinical outcomes. Most DNMT3A mutations occur at position R882 within the catalytic domain (most commonly R882H) and are virtually always heterozygous. This over-representation suggests that mutations at R882 may result in gain-of-function or dominant-negative activity that contributes to leukemogenesis. However, how DNA methylation might be altered in DNMT3A-mutant cases of AML remains unclear, and no published study to date has addressed the effects of mixing wild-type (WT) and R882H DNMT3A. Importantly, mouse HSPCs deficient in Dnmt3a dramatically expand over time and have a concurrent defect in differentiation (Challen, GA et al. Nat Genet, 2011). Mice haploinsufficient for Dnmt3a, on the other hand, do not have a measurable defect in hematopoiesis. Collectively, these data suggest that the heterozygous R882 mutations probably cause more than a simple loss-of-function phenotype. We purified full-length, human WT and R882H DNMT3A using a mammalian tissue culture system to produce recombinant proteins for biochemical modeling of the de novo methylation potential of a DNMT3A-mutant AML cell. rhR882H DNMT3A exhibits roughly 10-20% of the de novo DNA methyltransferase activity of rhWT DNMT3A, similar to observations by other groups. We added increasing amounts of R882H DNMT3A to a fixed amount of WT DNMT3A and observed a linear increase in the net enzymatic activity, reflecting the summed activity of the two forms of DNMT3A in these 4-hour in vitro reactions. In contrast, 12-hour in vitro DNA methylation assays with mixed WT and R882H DNMT3A demonstrated net methylation less than the predicted summed activity of the two enzymes, suggesting that a dominant-negative effect of R882H DNMT3A may occur with a long equilibration time. To better simulate an AML cell with a heterozygous R882H mutation, we co-transfected HEK293T cells with equal amounts of poly-His-tagged WT and R882H DNMT3A expression vectors. Subsequently co-purified (i.e. in vivo-mixed) WT and R882H DNMT3A exhibited a striking reduction in methyltransferase activity, with total activity similar to R882H DNMT3A alone (Figure 1A). TSQ mass spectrometry allowed us to verify the presence and quantify the relative concentration of WT and R882H DNMT3A in our co-purified samples. We exploited a novel tryptic cleavage site in DNMT3A produced by the R882H mutation to generate standard concentration curves using recombinant peptides distinguishing the two protein forms. Our co-purified enzyme preparations had WT:R882H ratios ranging from 0.79 to 1.60; all demonstrated the dominant-negative effect of R882H. DNMT3A is a processive enzyme, catalyzing multiple methyl-group transfers before dissociating from target DNA. This is dependent on the ability of WT DNMT3A to form homo-oligomers (tetramers and larger), which was recently shown to be disrupted by the R882H mutation using the catalytic domain of DNMT3A produced in E.coli (Holz-Schietinger, C et al. JBC, 2012). We therefore postulated that the dominant-negative effect of R882H may be due to the disruption of WT DNMT3A oligomerization. Using a Superose 6 size exclusion column, we confirmed the tetramerization defect of R882H DNMT3A relative to WT DNMT3A. Notably, in vivo-mixed (co-purified) WT and R882H DNMT3A complexes exhibited a pattern of oligomerization identical to R882H DNMT3A alone. However, WT and R882H DNMT3A mixed in vitro exhibited a distribution of oligomers corresponding to the expected average of the WT and R882H curves (Figure 1B). These data demonstrate that production of equal amounts of WT and R882H DNMT3A within the same cell provides an environment where R882H DNMT3A can exert a potent dominant-negative effect on WT DNMT3A. Furthermore, our data suggest that this effect is associated with diminished formation of tetramers when WT and R882H DNMT3A are complexed together. Thus, the R882H mutation has two distinct consequences that affect DNMT3A activity in AML cells: 1) it severely reduces its own de novo methyltransferase activity, and 2) it disrupts the ability of WT DNMT3A to form functional tetramers. These two effects severely reduce total DNMT3A activity in AML cells, and may explain why this mutation is virtually always heterozygous in AML samples, since homozygosity would not further reduce DNMT3A activity. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document