6 ALTERED CHROMATIN CONFORMATION IN BOVINE SPERMATOZOA PERTURBS DYNAMIC DNA METHYLATION IN THE MALE PRONUCLEUS AFTER FERTILIZATION IN VITRO

2013 ◽  
Vol 25 (1) ◽  
pp. 150 ◽  
Author(s):  
M. B. Rahman ◽  
M. M. Kamal ◽  
T. Rijsselaere ◽  
L. Vandaele ◽  
M. Shamsuddin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Heat Stress ◽  
De Novo ◽  
Chromatin Condensation ◽  
Sperm Chromatin ◽  
Chromatin Conformation ◽  
Epigenetic Marks ◽  
Time Points ◽  
Male Pronucleus ◽  
De Novo Methylation

Soon after fertilization, mammalian zygotes need proper DNA methylation reprogramming, at which time the epigenetic marks that the oocyte and sperm have acquired during gametogenesis are erased to allow totipotent zygotic development. Aberrant epigenetic marks in the paternal genome are thought to be associated with altered chromatin condensation in spermatozoa of suboptimal quality. We have recently reported that heat stress on bulls during germ cell development, especially at the spermiogenesis stage, altered sperm chromatin condensation. The objective of this study was to investigate dynamic DNA methylation reprogramming in the male pronucleus after fertilization of oocytes with sperm known to have altered chromatin conformation. To evaluate dynamic DNA methylation reprogramming, zygotes collected at 3 different time points [i.e. 12, 18, and 24 h post-insemination (hpi)] were immunocytochemically investigated using an antibody against 5-methylcytosine (5mC). The total fluorescence intensity of the male pronuclei (n = 89, ≥25 in each group) was measured by ImageJ and data were analyzed by ANOVA. The DNA methylation pattern in male pronuclei when oocytes were fertilized with heat-stressed sperm did not change between time points (P > 0.05), whereas control zygotes clearly showed demethylation and de novo methylation at 18 and 24 hpi, respectively. The results of this study indicated that dynamic DNA methylation reprogramming patterns such as DNA demethylation followed by de novo methylation in the male pronucleus soon after fertilization were altered when oocytes were fertilized with heat-stressed sperm. In conclusion, altered sperm chromatin conformation due to heat stress perturbs dynamic DNA methylation reprogramming in the male pronucleus, which may hamper nuclear totipotency and embryo survival.

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.

A targeted-replacement system for identification of signals for de novo methylation in Neurospora crassa

1994 ◽  
Vol 14 (11) ◽  
pp. 7059-7067
Author(s):  
V P Miao ◽  
M J Singer ◽  
M R Rountree ◽  
E U Selker
Keyword(s):  
Dna Methylation ◽  
Neurospora Crassa ◽  
De Novo ◽  
Gene Replacement ◽  
Position Effects ◽  
Efficient Gene ◽  
Bona Fide ◽  
Replacement System ◽  
Methylation Signal ◽  
De Novo Methylation

Transformation of eukaryotic cells can be used to test potential signals for DNA methylation. This approach is not always reliable, however, because of chromosomal position effects and because integration of multiple and/or rearranged copies of transforming DNA can influence DNA methylation. We developed a robust system to evaluate the potential of DNA fragments to function as signals for de novo methylation in Neurospora crassa. The requirements of the system were (i) a location in the N. crassa genome that becomes methylated only in the presence of a bona fide methylation signal and (ii) an efficient gene replacement protocol. We report here that the am locus fulfills these requirements, and we demonstrate its utility with the identification of a 2.7-kb fragment from the psi 63 locus as a new portable signal for de novo methylation.

scholarly journals Infection with Human Immunodeficiency Virus Type 1 Upregulates DNA Methyltransferase, Resulting in De Novo Methylation of the Gamma Interferon (IFN-γ) Promoter and Subsequent Downregulation of IFN-γ Production

1998 ◽  
Vol 18 (9) ◽  
pp. 5166-5177 ◽  
Author(s):  
Judy A. Mikovits ◽  
Howard A. Young ◽  
Paula Vertino ◽  
Jean-Pierre J. Issa ◽  
Paula M. Pitha ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Methylation Status ◽  
Immunodeficiency Virus ◽  
Ifn Γ ◽  
Hiv 1 ◽  
De Novo Methylation

ABSTRACT The immune response to pathogens is regulated by a delicate balance of cytokines. The dysregulation of cytokine gene expression, including interleukin-12, tumor necrosis factor alpha, and gamma interferon (IFN-γ), following human retrovirus infection is well documented. One process by which such gene expression may be modulated is altered DNA methylation. In subsets of T-helper cells, the expression of IFN-γ, a cytokine important to the immune response to viral infection, is regulated in part by DNA methylation such that mRNA expression inversely correlates with the methylation status of the promoter. Of the many possible genes whose methylation status could be affected by viral infection, we examined the IFN-γ gene as a candidate. We show here that acute infection of cells with human immunodeficiency virus type 1 (HIV-1) results in (i) increased DNA methyltransferase expression and activity, (ii) an overall increase in methylation of DNA in infected cells, and (iii) the de novo methylation of a CpG dinucleotide in the IFN-γ gene promoter, resulting in the subsequent downregulation of expression of this cytokine. The introduction of an antisense methyltransferase construct into lymphoid cells resulted in markedly decreased methyltransferase expression, hypomethylation throughout the IFN-γ gene, and increased IFN-γ production, demonstrating a direct link between methyltransferase and IFN-γ gene expression. The ability of increased DNA methyltransferase activity to downregulate the expression of genes like the IFN-γ gene may be one of the mechanisms for dysfunction of T cells in HIV-1-infected individuals.

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 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 Differential requirement of MED14 and UVH6 for heterochromatin transcription upon destabilization of silencing

2018 ◽  
Author(s):  
Pierre Bourguet ◽  
Stève de Bossoreille ◽  
Leticia López-González ◽  
Marie-Noëlle Pouch-Pélissier ◽  
Ángeles Gómez-Zambrano ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Heat Stress ◽  
Environmental Changes ◽  
Constitutive Heterochromatin ◽  
Epigenetic Marks ◽  
Two Factors ◽  
Silencing Pathways ◽  
Heterochromatin Silencing ◽  
Insight Into ◽  
Chromatin Patterns

AbstractConstitutive heterochromatin is commonly associated with high levels of repressive epigenetic marks and is stably maintained transcriptionally silent by the concerted action of different, yet convergent, silencing pathways. Reactivation of heterochromatin transcription is generally associated with alterations in levels of these epigenetic marks. However, in mutants for particular epigenetic regulators, or upon particular environmental changes such as heat stress, heterochromatin-associated silencing is destabilized without noticeable changes in epigenetic marks. This suggests that transcription can occur in a non-permissive chromatin context, yet the factors involved remain poorly known. Here, we show that heat stress-induced transcription of heterochromatin depends on the TFIIH component UVH6 and the Mediator subunit MED14. Mutants for these two factors exhibit hypersensitivity to heat stress, and under these conditions, UVH6 and MED14 are required for transcription of a high number of loci. We further show that MED14, but not UVH6, is required for transcription when heterochromatin silencing is destabilized in the absence of stress. In this case, MED14 requires proper chromatin patterns of repressive epigenetic marks for its function. We also uncover that MED14 regulates non-CG DNA methylation at a subset of RNA-directed DNA methylation target loci. These findings provide insight into the control of heterochromatin transcription upon silencing destabilization and identify MED14 as a regulator of DNA methylation.

scholarly journals Inheritance of DNA methylation in Coprinus cinereus

1986 ◽  
Vol 6 (1) ◽  
pp. 195-200
Author(s):  
M E Zolan ◽  
P J Pukkila
Keyword(s):  
Dna Methylation ◽  
Life Cycle ◽  
Homologous Chromosome ◽  
De Novo ◽  
Coprinus Cinereus ◽  
De Novo Methylation

We examined the inheritance of 5-methylcytosine residues at a centromere-linked locus in the basidiomycete Coprinus cinereus. Although methylated and unmethylated tracts were inherited both mitotically and meiotically the lengths of these tracts were variable. This variation was not confined to any one phase of the life cycle of the organism, and it usually involved the simultaneous de novo methylation of at least four HpaII-MspI sites. We also found that the higher levels of methylation at this locus were transmitted through meiosis, regardless of the level of methylation of the homologous chromosome.

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.

De Novo DNA Methylation Is Associated with Granulopoiesis and Megakaryopoiesis but Not Erythropoiesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3868-3868 ◽  
Author(s):  
Jens Lichtenberg ◽  
Elisabeth F. Heuston ◽  
Stacie M. Anderson ◽  
NIH Intramural Sequencing Center ◽  
Cheryl A. Keller ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Promoter Methylation ◽  
De Novo ◽  
Cell Type ◽  
Epigenetic Memory ◽  
Venn Diagrams ◽  
A Cell ◽  
Total Dna ◽  
Methylation Peak ◽  
De Novo Methylation

Abstract Previous research has shown that progressive DNA demethylation is a feature of erythroid differentiation (Hogart et al. 2012, Genome Res., 22:1407-18; Shearstone et al. 2011, Science, 334:799-802), but the epigenetic changes that occur during granulopoiesis and megakaryopoiesis have not been well characterized. To establish a comprehensive map of changes in DNA methylation, we have extended our analysis of DNA methylation to include megakaryocytes (MEG) and their progenitors (CFU-MEG), as well as granulocyte macrophage progenitors (GMP), common myeloid progenitors (CMP) and hematopoietic stem cells (HSC; Lin- Sca-1+, c-kit+). Erythroblasts (ERY), their progenitors (CFU-E), MEG, CFU-MEG, GMP, CMP and HSC were isolated by FACS from adult C57BL/6J mouse bone marrow. We performed whole genome DNA methylation profiling using Methyl Binding Domain 2 (MBD2) pulldown with subsequent high-throughput sequencing to detect regions containing 5 or more methylated CpGs within a 200bp window. Using the reduced representation bilsulfite sequencing data of Shearstone et al. as a benchmark, we identified CCAT (Xu et al. 2010, Bioinformatics, 26:1199-1204) as the best performing peak calling software to detect de novo methylation, and used CCAT to generate DNA methylation profiles for our isolated hematopoietic populations. We confirmed that erythropoiesis undergoes global demethylation (Figure 1). Epigenetic memory is a feature of erythropopiesis, with the majority of methylation peaks detected in erythroblasts also found in their progenitors. For example, 34,427 of the 36,135 CFU-E peaks are also found in HSC. Using Ingenuity Pathway Analysis (IPA), we found that the CFU-E genes with de novo promoter methylation (380 genes) were associated with the "DNA methylation and transcriptional repression signaling pathway" (p=9.6E-5), supporting the model of suppression of DNA methylation and epigenetic memory as a feature of erythropoiesis. In contrast, we found that compared to CFU-E, CFU-MEG undergo de novo methylation. Specifically, we detected 25,531 methylation peaks in CFU-MEG that are not present in HSC or CMP (Figure 1). Genes with de novo promoter methylation in CFU-MEG (1227 genes) were associated with silencing of extracellular signaling pathways, including disruption of MAPK and FAK signaling (p=1.4E-5). We have developed a systems biology data ranch named SBR-Blood (Lichtenberg et al. 2016, Nucleic Acid Res., 44:D925-31) that includes 228 publicly available epigenetic and RNA expression profiling datasets. Using SBR-Blood, we correlated the increased methylation in CFU-MEG with increased expression of the de novo methylation genes Dnmt3A/B genes (6 and 34-fold respectively), and decreased expression of the maintenance methylation gene Dnmt1 (1.5-fold). Compared to HSC, GMP acquired 15,115 de novo methylation peaks and CMP gained 4020 de novo peaks (Figure 1). Promoter-specific de novo methylation in 784 GMP genes was associated with silencing of Endothelin-1 signaling (p=8.1E-5), an observation confirmed using RNASeq expression profiles in SBR-Blood (e.g. AC-complex 20-fold decrease). In summary, we have shown that in contrast to erythropoiesis, megakaryopoiesis and granulopoiesis are associated with specific de novo methylation that defines their respective lineages. Figure 1 Venn diagrams, annotated with total DNA methylation peak counts for a cell type, depicting the intersections between the peaks of DNA methylation profiles in megakaryopoiesis and granulopoiesis, compared to erythropoiesis. Figure 1. Venn diagrams, annotated with total DNA methylation peak counts for a cell type, depicting the intersections between the peaks of DNA methylation profiles in megakaryopoiesis and granulopoiesis, compared to erythropoiesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Arabidopsis RNA Polymerases IV and V Are Required To Establish H3K9 Methylation, but Not Cytosine Methylation, on Geminivirus Chromatin

2016 ◽  
Vol 90 (16) ◽  
pp. 7529-7540 ◽  
Author(s):  
Jamie N. Jackel ◽  
Jessica M. Storer ◽  
Tami Coursey ◽  
David M. Bisaro
Keyword(s):  
Dna Methylation ◽  
Small Rnas ◽  
Cytosine Methylation ◽  
De Novo ◽  
Rna Polymerases ◽  
Epigenetic Marks ◽  
Content Type ◽  
Pol Iv ◽  
Pol V ◽  
Virus Genomes

ABSTRACTIn plants, RNA-directed DNA methylation (RdDM) employs small RNAs to target enzymes that methylate cytosine residues. Cytosine methylation and dimethylation of histone 3 lysine 9 (H3K9me2) are often linked. Together they condition an epigenetic defense that results in chromatin compaction and transcriptional silencing of transposons and viral chromatin. Canonical RdDM (Pol IV-RdDM), involving RNA polymerases IV and V (Pol IV and Pol V), was believed to be necessary to establish cytosine methylation, which in turn could recruit H3K9 methyltransferases. However, recent studies have revealed that a pathway involving Pol II and RNA-dependent RNA polymerase 6 (RDR6) (RDR6-RdDM) is likely responsible for establishing cytosine methylation at naive loci, while Pol IV-RdDM acts to reinforce and maintain it. We used the geminivirusBeet curly top virus(BCTV) as a model to examine the roles of Pol IV and Pol V in establishing repressive viral chromatin methylation. As geminivirus chromatin is formedde novoin infected cells, these viruses are unique models for processes involved in the establishment of epigenetic marks. We confirm that Pol IV and Pol V are not needed to establish viral DNA methylation but are essential for its amplification. Remarkably, however, both Pol IV and Pol V are required for deposition of H3K9me2 on viral chromatin. Our findings suggest that cytosine methylation alone is not sufficient to triggerde novodeposition of H3K9me2 and further that Pol IV-RdDM is responsible for recruiting H3K9 methyltransferases to viral chromatin.IMPORTANCEIn plants, RNA-directed DNA methylation (RdDM) uses small RNAs to target cytosine methylation, which is often linked to H3K9me2. These epigenetic marks silence transposable elements and DNA virus genomes, but how they are established is not well understood. Canonical RdDM, involving Pol IV and Pol V, was thought to establish cytosine methylation that in turn could recruit H3K9 methyltransferases, but recent studies compel a reevaluation of this view. We used BCTV to investigate the roles of Pol IV and Pol V in chromatin methylation. We found that both are needed to amplify, but not to establish, DNA methylation. However, both are required for deposition of H3K9me2. Our findings suggest that cytosine methylation is not sufficient to recruit H3K9 methyltransferases to naive viral chromatin and further that Pol IV-RdDM is responsible.

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