Beneficial effects of diazepin-quinazolin-amine derivative (BIX-01294) on preimplantation development and molecular characteristics of cloned mouse embryos

2017 ◽  
Vol 29 (6) ◽  
pp. 1260 ◽  
Author(s):  
Yanfang Huang ◽  
Xiaohong Jiang ◽  
Miao Yu ◽  
Rongfu Huang ◽  
Jianfeng Yao ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Specific Inhibitor ◽  
Epigenetic Modifications ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Molecular Characteristics ◽  
Amine Derivative ◽  
Beneficial Effects ◽  
Small Nuclear Ribonucleoprotein

Somatic cell nuclear transfer is frequently associated with abnormal epigenetic modifications that may lead to the developmental failure of cloned embryos. BIX-01294 (a diazepine–quinazoline–amine derivative) is a specific inhibitor of the histone methyltransferase G9a. The aim of the present study was to investigate the effects of BIX-01294 on development, dimethylation of histone H3 at lysine 9 (H3K9), DNA methylation and the expression of imprinted genes in cloned mouse preimplantation embryos. There were no significant differences in blastocyst rates of cloned embryos treated with or without 0.1 μM BIX-01294. Relative to clone embryos treated without 0.1 μM BIX-01294, exposure of embryos to BIX-01294 decreased histone H3K9 dimethylation and DNA methylation in cloned embryos to levels that were similar to those of in vivo-fertilised embryos at the 2-cell and blastocyst stages. Cloned embryos had lower expression of octamer-binding transcription factor 4 (Oct4) and small nuclear ribonucleoprotein N (Snrpn), but higher expression of imprinted maternally expressed transcript (non-protein coding) (H19) and growth factor receptor-bound protein 10 (Grb10) compared with in vivo-fertilised counterparts. The addition of 0.1 μM BIX-01294 to the activation and culture medium resulted in lower H19 expression and higher cyclin dependent kinase inhibitor 1C (Cdkn1c) and delta-like 1 homolog (Dlk1) expression, but had no effect on the expression of Oct4, Snrpn and Grb10. The loss of methylation at the Grb10 cytosine–phosphorous–guanine (CpG) islands in cloned embryos was partially corrected by BIX-01294. These results indicate that BIX-01294 treatment of cloned embryos has beneficial effects in terms of correcting abnormal epigenetic modifications, but not on preimplantation development.

scholarly journals The histone variant H2A.W and linker histone H1 co-regulate heterochromatin accessibility and DNA methylation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Pierre Bourguet ◽  
Colette L. Picard ◽  
Ramesh Yelagandula ◽  
Thierry Pélissier ◽  
Zdravko J. Lorković ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Histone H1 ◽  
Epigenetic Modifications ◽  
Flowering Plants ◽  
Linker Histone ◽  
Histone Variant ◽  
Null Mutant ◽  
Chromatin Compaction ◽  
Linker Histone H1

AbstractIn flowering plants, heterochromatin is demarcated by the histone variant H2A.W, elevated levels of the linker histone H1, and specific epigenetic modifications, such as high levels of DNA methylation at both CG and non-CG sites. How H2A.W regulates heterochromatin organization and interacts with other heterochromatic features is unclear. Here, we create a h2a.w null mutant via CRISPR-Cas9, h2a.w-2, to analyze the in vivo function of H2A.W. We find that H2A.W antagonizes deposition of H1 at heterochromatin and that non-CG methylation and accessibility are moderately decreased in h2a.w-2 heterochromatin. Compared to H1 loss alone, combined loss of H1 and H2A.W greatly increases accessibility and facilitates non-CG DNA methylation in heterochromatin, suggesting co-regulation of heterochromatic features by H2A.W and H1. Our results suggest that H2A.W helps maintain optimal heterochromatin accessibility and DNA methylation by promoting chromatin compaction together with H1, while also inhibiting excessive H1 incorporation.

scholarly journals Methylmercury Epigenetics

Toxics ◽  
2019 ◽  
Vol 7 (4) ◽  
pp. 56 ◽  
Author(s):  
Megan Culbreth ◽  
Michael Aschner
Keyword(s):  
Dna Methylation ◽  
Mirna Expression ◽  
Epigenetic Modifications ◽  
Nervous System Development ◽  
Gene Promoters ◽  
Toxic Response ◽  
The Impact ◽  
The Brain

Methylmercury (MeHg) has conventionally been investigated for effects on nervous system development. As such, epigenetic modifications have become an attractive mechanistic target, and research on MeHg and epigenetics has rapidly expanded in the past decade. Although, these inquiries are a recent advance in the field, much has been learned in regards to MeHg-induced epigenetic modifications, particularly in the brain. In vitro and in vivo controlled exposure studies illustrate that MeHg effects microRNA (miRNA) expression, histone modifications, and DNA methylation both globally and at individual genes. Moreover, some effects are transgenerationally inherited, as organisms not directly exposed to MeHg exhibited biological and behavioral alterations. miRNA expression generally appears to be downregulated consequent to exposure. Further, global histone acetylation also seems to be reduced, persist at distinct gene promoters, and is contemporaneous with enhanced histone methylation. Moreover, global DNA methylation appears to decrease in brain-derived tissues, but not in the liver; however, selected individual genes in the brain are hypermethylated. Human epidemiological studies have also identified hypo- or hypermethylated individual genes, which correlated with MeHg exposure in distinct populations. Intriguingly, several observed epigenetic modifications can be correlated with known mechanisms of MeHg toxicity. Despite this knowledge, however, the functional consequences of these modifications are not entirely evident. Additional research will be necessary to fully comprehend MeHg-induced epigenetic modifications and the impact on the toxic response.

scholarly journals Effect of BIX-01294 on H3K9me2 levels and the imprinted gene Snrpn in mouse embryonic fibroblast cells

2015 ◽  
Vol 35 (5) ◽  
Author(s):  
Peng Chen ◽  
Jian-Feng Yao ◽  
Rong-Fu Huang ◽  
Fang-Fang Zheng ◽  
Xiao-Hong Jiang ◽  
...  
Keyword(s):  
Epigenetic Regulation ◽  
Biological Effects ◽  
Mouse Embryonic Fibroblast ◽  
Epigenetic Modifications ◽  
Fibroblast Cells ◽  
Imprinted Gene ◽  
Amine Derivative ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein ◽  
Embryonic Fibroblast

BIX-01294 (a diazepin-quinazolin-amine derivative) has important biological effects and its epigenetic regulation at imprinting control regions is highly complex. BIX-01294 may reduce global H3K9me2 levels and affect epigenetic modifications of small nuclear ribonucleoprotein N (Snrpn) in MEFs.

scholarly journals Characterization of Novel Parent-Specific Epigenetic Modifications Upstream of the Imprinted Mouse H19Gene

1998 ◽  
Vol 18 (11) ◽  
pp. 6767-6776 ◽  
Author(s):  
Piroska E. Szabó ◽  
Gerd P. Pfeifer ◽  
Jeffrey R. Mann
Keyword(s):  
Dna Methylation ◽  
Embryonic Stem ◽  
Dnase I ◽  
Epigenetic Modifications ◽  
Monoallelic Expression ◽  
Maternal Allele ◽  
Dnase I Footprinting ◽  
Allele Specific ◽  
Ccaat Box

ABSTRACT Genomic imprinting results in parent-specific monoallelic expression of a small number of genes in mammals. The identity of imprints is unknown, but much evidence points to a role for DNA methylation. The maternal alleles of the imprinted H19 gene are active and hypomethylated; the paternal alleles are inactive and hypermethylated. Roles for other epigenetic modifications are suggested by allele-specific differences in nuclease hypersensitivity at particular sites. To further analyze the possible epigenetic mechanisms determining monoallelic expression of H19, we have conducted in vivo dimethylsulfate and DNase I footprinting of regions upstream of the coding sequence in parthenogenetic and androgenetic embryonic stem cells. These cells carry only maternally and paternally derived alleles, respectively. We observed the presence of maternal-allele-specific dimethylsulfate and DNase I footprints at the promoter indicative of protein-DNA interactions at a CCAAT box and at binding sites for transcription factors Sp1 and AP-2. Also, at the boundary of a region further upstream for which existent differential methylation has been suggested to constitute an imprint, we observed a number of strand-specific dimethylsulfate reactivity differences specific to the maternal allele, along with an unusual chromatin structure in that both strands of maternally derived DNA were strongly hypersensitive to DNase I cutting over a distance of 100 nucleotides. We therefore reveal the existence of novel parent-specific epigenetic modifications, which in addition to DNA methylation, could constitute imprints or maintain monoallelic expression of H19.

scholarly journals Essential roles of Hdac1 and 2 in lineage development and genome-wide DNA methylation during mouse preimplantation development

2019 ◽  
Author(s):  
Panpan Zhao ◽  
Huanan Wang ◽  
Han Wang ◽  
Yanna Dang ◽  
Lei Luo ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Histone Modifications ◽  
Hippo Pathway ◽  
Epigenetic Modifications ◽  
Preimplantation Development ◽  
Global Dna Methylation ◽  
Lineage Specification ◽  
Pluripotent Cells ◽  
Global Methylation

AbstractEpigenetic modifications, including DNA methylation and histone modifications, are reprogrammed considerably following fertilization during mammalian early embryonic development. Incomplete epigenetic reprogramming is a major factor leading to poor developmental outcome in embryos generated by assisted reproductive technologies, such as somatic cell nuclear transfer. However, the role of histone modifications in preimplantation development is poorly understood. Here, we show that co-knockdown (cKD) of Hdac1 and 2 (but not individually) resulted in developmental failure during the morula to blastocyst transition. This outcome was also confirmed with the use of small-molecule Hdac1/2-specific inhibitor FK228. We observed reduced cell proliferation and increased incidence of apoptosis in cKD embryos, which were likely caused by increased acetylation of Trp53. Importantly, both RNA-seq and immunostaining analysis revealed a failure of lineage specification to generate trophectoderm and pluripotent cells. Among many gene expression changes, a substantial decrease of Cdx2 may be partly accounted for by the aberrant Hippo pathway occurring in cKD embryos. In addition, we observed an increase in global DNA methylation, consistent with increased DNA methyltransferases and Uhrf1. Interestingly, deficiency of Rbbp4 and 7 (both are core components of several Hdac1/2-containing epigenetic complexes) results in similar phenotypes as those of cKD embryos. Overall, Hdac1 and 2 play redundant functions required for lineage specification, cell viability and accurate global DNA methylation, each contributing to critical developmental programs safeguarding a successful preimplantation development.SignificanceSubstantial changes to epigenetic modifications occur during preimplantation development and can be detrimental when reprogrammed incompletely. However, little is known about the role of histone modifications in early development. Co-knockdown of Hdac1 and 2, but not individually, resulted in developmental arrest during morula to blastocyst transition, which was accompanied by reduced cell number per embryo and increased incidence of apoptosis. Additionally, we observed a failure of first lineage specification to generate trophectoderm and pluripotent cells, which were associated with reduced expression of key lineage-specific genes and aberrant Hippo pathway. Moreover, an increase in global DNA methylation was found with upregulated Dnmts and Uhrf1. Thus, Hdac1 and 2 play overlapping roles in lineage development, apoptosis, and global methylation during preimplantation development.

scholarly journals Maternal Ezh1/2 deficiency in oocyte delays H3K27me2/3 restoration and impairs epiblast development responsible for embryonic sub-lethality in mouse

2021 ◽  
Author(s):  
Yinan Zhao ◽  
Dan Zhang ◽  
Mengying Liu ◽  
Yingpu Tian ◽  
Jinhua Lu ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Gene Knockout ◽  
Epigenetic Modifications ◽  
Preimplantation Embryos ◽  
Polycomb Repressive Complex 2 ◽  
H3k27 Methylation ◽  
Early Embryos ◽  
Gene Knockout Mouse ◽  
Core Components

Mammalian embryonic development is a complex process regulated by various epigenetic modifications. Recently, maternal histone H3 methylations were found to be inherited and reprogrammed in early embryos to regulate embryonic development. The enhancer of zest homolog 1 and 2 (Ezh1 and Ezh2) belong to the core components of Polycomb repressive complex 2 (PRC2) and are the histone methyltransferase of histone 3 lysine 27 (H3K27). How maternal Ezh1 and Ezh2 function on H3K27 methylation in in vivo preimplantation embryos and embryonic development are not clear. Here, we deleted Ezh1 or/and Ezh2 in growing oocytes using gene knockout mouse models, and found that H3K27me3 in oocytes was disappeared by loss of Ezh2 alone while H3K27me2 was absent upon deletion of both Ezh1 and Ezh2. The effects of Ezh1/2 were inherited in maternal knockout zygotes and early embryos, in which restoration of H3K27me3 was delayed until late blastocyte by loss of Ezh2 alone and H3K27me2 was reestablished until morulae by deletion of Ezh1 and Ezh2. However, the ablation of both Ezh1 and Ezh2, but not single Ezh1 or Ezh2, led to significantly decreased litter size due to growth retardation during post-implantation. Furthermore, maternal Ezh1/2 deficiency caused compromised H3K27me3 and pluripotent epiblast cells in late blastocyst, followed by defective development of epiblast. These results demonstrate that in oocytes, Ezh2 is indispensable for H3K27me3 while Ezh1 complements Ezh2 in H3K27me2. Also, maternal Ezh1/2-H3K27 methylation is inherited in descendant embryos and has a critical effect on fetus and placenta development. Thus, this work sheds light on maternal epigenetic modifications during embryonic development.

38 DNA METHYLATION IN PORCINE PREIMPLANTATION EMBRYOS DEVELOPED IN VIVO AND PRODUCED BY IN VITRO FERTILIZATION, PARTHENOGENETIC ACTIVATION, AND SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 125
Author(s):  
R. S. Deshmukh ◽  
O. Oestrup ◽  
E. Oestrup ◽  
M. Vejlsted ◽  
H. Niemann ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Mass ◽  
Epifluorescence Microscopy ◽  
Preimplantation Embryos ◽  
Parthenogenetic Activation

DNA de- and re-methylation are crucial for reprogramming of the differentiated parental/somatic genome in the ooplasm. The presented research was aimed at analysis of the DNA methylation dynamics in porcine preimplantation embryos developed in vivo (IV) and produced in vitro by IVF, somatic cell nuclear transfer (SCNT), and parthenogenetic activation (PA). Embryos of early and late 1-cell, 2-, 4-, and 8-cell, and early and late blastocysts stages obtained by the mentioned methods were fixed in 4% paraformaldehyde and subjected to immunocytochemistry using anti-5MetC (Mouse monoclonal, Abcam, Cambridge, MA, USA) antibody. DNA was labelled using Hoechst 33258 (Sigma, Copenhagen, Denmark). Epifluorescence microscopy (Leica Microsystems, Wetzlar, Germany) images were subjected to NIH imageJ software to measure the DNA methylation/DNA content signal by manually outlining the nuclei (n = 2003) of the embryos. The data were analysed using PROC-GLM statistical procedure in SAS 9.1 (SAS Institute Inc., Cary, NC, USA), least square means were compared and P-values were used to decide the significant differences within and between different groups of embryos. The 1-cell stages lacked active demethylation of paternal genome in IV and IVF embryos. Embryos produced under in vitro conditions presented higher levels of DNA methylation than IV. A lineage specific DNA methylation (hypermethylation of inner cell mass and hypomethylation of trophectoderm) observed in porcine IV late blastocysts was absent in PA and SCNT blastocysts despite the occurrence of de novo methylation in early blastocysts. SCNT early (50%) and late (14%) blastocysts presented DNA methylation pattern similar to IV early and late blastocysts, respectively. Concluding, DNA methylation patterns are strongly impaired under in vitro conditions in porcine preimplantation embryos.

Distribution of tubulointerstitial nephritis antigen-like 1 and structural matrix proteins in mouse embryos during preimplantation development in vivo and in vitro

Zygote ◽  
2012 ◽  
Vol 22 (2) ◽  
pp. 259-265 ◽  
Author(s):  
Masahiro Sakurai ◽  
Yusuke Sato ◽  
Kuniaki Mukai ◽  
Makoto Suematsu ◽  
Emiko Fukui ◽  
...  
Keyword(s):  
Tubulointerstitial Nephritis ◽  
Preimplantation Development ◽  
Matrix Proteins ◽  
Preimplantation Embryos ◽  
Collagen Type Iv ◽  
Matricellular Protein ◽  
Ecm Proteins ◽  
Structural Matrix

SummaryTubulointerstitial nephritis antigen-like 1 (TINAGL1) is a novel matricellular protein that interacts with structural matrix proteins and promotes cell adhesion and spreading. We have previously reported unique localization of TINAGL1 to the trophectoderm (TE) of mouse blastocysts. TINAGL1 was found to be upregulated in implantation-competent blastocysts after estrogen treatment using progesterone-treated delayed-implantation models. Moreover, colocalization of TINAGL1 and extracellular matrix (ECM) protein laminin 1 was detected in the Reichert membrane on embryonic days 6.5 and 7.5. Although these data suggested a role for TINAGL1 in the embryo development at postimplantation, its relevance to other ECM proteins during preimplantation development is not clear. In this study, we examined the expression of TINAGL1 and its relevance to other ECM proteins fibronectin (FN) and collagen type IV (ColIV) during in vivo development of preimplantation embryos, particularly at blastocyst stage in detail. Localizations of TINAGL1, FN, and ColIV were similar. In 1-cell to 8-cell embryos, they were expressed in cytoplasm of blastomeres, and in morulae they were localized in the outer cells. FN and ColIV were expressed primarily on outer surface of the cells. In blastocysts, FN and ColIV were distributed in the cytoplasm of TE, but, just prior to implantation, they became localized uniquely to the blastocoelic surface of TE. In in vitro fertilized (IVF) blastocysts, expression levels of TINAGL1 and FN were lower than in in vivo blastocysts. These results suggest that, during preimplantation development, TINAGL1 may be involved in roles of structural matrix proteins, whose expression in blastocysts may be affected by in vitro culture.

scholarly journals Involvement of Nlrp9a/b/c in mouse preimplantation development

Reproduction ◽  
2020 ◽  
Vol 160 (2) ◽  
pp. 181-191 ◽  
Author(s):  
Satoko Kanzaki ◽  
Shiori Tamura ◽  
Toshiaki Ito ◽  
Mizuki Wakabayashi ◽  
Koji Saito ◽  
...  
Keyword(s):  
Asymmetric Cell Division ◽  
Culture Conditions ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Dependent Manner ◽  
Blastocyst Development ◽  
Triple Mutant ◽  
Cell Stage

Nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing proteins (NRLPs) are central components of the inflammasome. Accumulating evidence has shown that a reproductive clade of NRLPs is predominantly expressed in oocyte to cleavage stage embryos and participates in mammalian preimplantation development as a component of a multiprotein complex known as the subcortical maternal complex (SCMC). Nlrp9s belong to the reproductive class of NLRPs; Nlrp9b is unique in acting as an inflammasome against rotavirus in intestines. Here we generated mice carrying mutations in all three members of the Nlrp9a/b/c gene (Nlrp9 triple mutant (TMut) mice). When crossed with WT males, the Nlrp9 TMut females were fertile, but deliveries with fewer pups were increased in the mutants. Consistent with this, blastocyst development was retarded and lethality to the preimplantation embryos increased in the Nlrp9 TMut females in vivo. Under in vitro culture conditions, the fertilized eggs from the Nlrp9 TMut females exhibited developmental arrest at the two-cell stage, accompanied by asymmetric cell division. By contrast, double-mutant (DMut) oocytes (any genetic combination) did not exhibit the two-cell block in vitro, showing the functional redundancy of Nlrp9a/b/c. Finally, Nlrp9 could bind to components of the SCMC. These results show that Nlrp9 functions as an immune or reproductive NLRP in a cell-type-dependent manner.

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