The monoallelic parent of origin-specific expression of imprinted genes in mammals is regulated by differentially DNA methylated imprinting control regions (ICRs). In contrast to most of the genome, ...ICRs must maintain their DNA methylation and parental identity despite extensive epigenetic reprogramming that takes place after fertilization. Previous work demonstrated that the Krüppel-associated box (KRAB)-containing zinc finger protein (KZFP) ZFP57 protects select ICRs from demethylation and preserves parental identity. However, some loci are unaffected in
-null mice. Thus, it has been speculated that other KZFPs must be involved in this process. Takahashi and colleagues (pp. 49-54) now report one such KZFP: ZFP445.
Assisted reproductive technologies (ART) have enabled millions of couples with compromised fertility to conceive children. Nevertheless, there is a growing concern regarding the safety of these ...procedures due to an increased incidence of imprinting disorders, premature birth, and low birth weight in ART-conceived offspring. An integral aspect of ART is the oxygen concentration used during in vitro development of mammalian embryos, which is typically either atmospheric (~20%) or reduced (5%). Both oxygen tension levels have been widely used, but 5% oxygen improves preimplantation development in several mammalian species, including that of humans. To determine whether a high oxygen tension increases the frequency of epigenetic abnormalities in mouse embryos subjected to ART, we measured DNA methylation and expression of several imprinted genes in both embryonic and placental tissues from concepti generated by in vitro fertilization (IVF) and exposed to 5% or 20% oxygen during culture. We found that placentae from IVF embryos exhibit an increased frequency of abnormal methylation and expression profiles of several imprinted genes, compared to embryonic tissues. Moreover, IVF-derived placentae exhibit a variety of epigenetic profiles at the assayed imprinted genes, suggesting that these epigenetic defects arise by a stochastic process. Although culturing embryos in both of the oxygen concentrations resulted in a significant increase of epigenetic defects in placental tissues compared to naturally conceived controls, we did not detect significant differences between embryos cultured in 5% and those cultured in 20% oxygen. Thus, further optimization of ART should be considered to minimize the occurrence of epigenetic errors in the placenta.
Although widely used, assisted reproductive technologies (ARTs) are associated with adverse perinatal outcomes. To elucidate their underlying causes, we have conducted a longitudinal analysis of ...placental development and fetal growth using a mouse model to investigate the effects of individual ART procedures: hormone stimulation,
fertilization (IVF), embryo culture and embryo transfer. We demonstrate that transfer of blastocysts naturally conceived without hormone stimulation and developed
prior to transfer can impair early placentation and fetal growth, but this effect normalizes by term. In contrast, embryos cultured
before transfer do not exhibit this compensation but rather display placental overgrowth, reduced fetal weight, reduced placental DNA methylation and increased levels of sFLT1, an anti-angiogenic protein implicated in causing the maternal symptoms of preeclampsia in humans. Increases in sFLT1 observed in this study suggest that IVF procedures could increase the risk for preeclampsia. Moreover, our results indicate that embryo culture is the major factor contributing to most placental abnormalities and should therefore be targeted for optimization.
Allele-specific expression of imprinted gene clusters is governed by gametic DNA methylation at master regulators called imprinting control regions (ICRs). Non-gametic or secondary differentially ...methylated regions (DMRs) at promoters and exonic regions reinforce monoallelic expression but do not control an entire cluster. Here, we unveil an unconventional secondary DMR that is indispensable for tissue-specific imprinting of two previously unlinked genes, Grb10 and Ddc. Using polymorphic mice, we mapped an intronic secondary DMR at Grb10 with paternal-specific CTCF binding (CBR2.3) that forms contacts with Ddc. Deletion of paternal CBR2.3 removed a critical insulator, resulting in substantial shifting of chromatin looping and ectopic enhancer-promoter contacts. Destabilized gene architecture precipitated abnormal Grb10-Ddc expression with developmental consequences in the heart and muscle. Thus, we redefine the Grb10-Ddc imprinting domain by uncovering an unconventional intronic secondary DMR that functions as an insulator to instruct the tissue-specific, monoallelic expression of multiple genes—a feature previously ICR exclusive.
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•Maternal Grb10 suppresses growth, whereas paternal Ddc stimulates heart development•Intronic, maternally methylated CBR2.3 binds CTCF on the paternal allele•CBR2.3 is a tissue-specific and allele-specific CTCF-dependent insulator for a heart enhancer•Paternal CBR2.3 deletion phenocopies the maternal allele, impairing heart growth
Inherited monoallelic gene expression is governed by master regulatory elements known as imprinting control regions. Juan et al. describe an unconventional, developmentally unveiled, differentially methylated region that drives tissue-specific imprinting of Grb10 and Ddc through the assembly of allele-specific CTCF-dependent architecture, thereby eliciting enhancer-promoter interactions in the developing heart.
Abstract
Increasing evidence has demonstrated that exposure to endocrine-disrupting chemicals impacts maternal and fetal health, but the underlying mechanisms are still unclear. We previously showed ...that dietary exposure to 10 µg/kg body weight (bw)/d and 10 mg/kg bw/d of bisphenol A (BPA) during pregnancy induced metabolic abnormalities in F1 male offspring and gestational glucose intolerance in F0 pregnant mice. The aim of this study was to elucidate the underlying etiologies of BPA exposure−induced metabolic disease by analyzing the male fetal liver metabolome. Using the Metabolon Discover HD4 Platform, our laboratory identified metabolic pathways that were altered by BPA exposure, including biochemicals in lipid and amino acid metabolism. Specifically, primary and secondary bile acids were increased in liver from BPA-exposed embryonic day 18.5 male fetuses. We subsequently showed that increased bile acid was associated with a defective farnesoid X receptor−dependent negative feedback mechanism in BPA-exposed fetuses. In addition, through metabolomics, we observed that BPA-exposed fetuses had elevated tryptophan levels. Independent liquid chromatography and mass spectrometry measurement revealed that BPA-exposed dams also had increased tryptophan levels relative to those of controls. Because several key enzymes in tryptophan catabolism are vitamin B6 dependent and vitamin B6 deficiencies have been linked to gestational diabetes, we tested the impact of vitamin B6 supplementation and showed that it rescued gestational glucose intolerance in BPA-exposed pregnant mice. Our study has therefore identified two pathways (bile acid and tryptophan metabolism) that potentially underlie BPA-induced maternal and fetal metabolic disease.
Analysis of the male fetal liver metabolome revealed that bile acids and tryptophan are metabolites linked to increased risk for metabolic disease in BPA-exposed male offspring and pregnant mice.
In vitro culture of mouse embryos results in loss of imprinting. The aim of the present study was to examine how two of the techniques commonly used during assisted reproduction, namely embryo ...culture and embryo transfer, affect genomic imprinting after implantation in the mouse. F1 hybrid mouse embryos were subjected to three experimental conditions: control (unmanipulated), embryo transfer and in-vitro-culture followed by embryo transfer. Concepti were collected on d9.5 of development and allelic expression determination of ten imprinted genes (H19, Snrpn, Igf2, Kcnq1ot1, Cdkn1c, Kcnq1, Mknr3, Ascl2, Zim1, Peg3) was performed. Although control concepti had monoallelic imprinted gene expression in all tissues, both manipulated groups had aberrant expression of one or more imprinted genes in the yolk sac and placenta. Culture further exacerbated the effects of transfer by increasing the number of genes with aberrant allelic expression in extraembryonic, as well as embryonic tissues. Additionally, placentae of both groups of manipulated concepti exhibited reduced levels of Igf2 mRNA and increased levels of Ascl2 mRNA when compared with their unmanipulated counterparts. Furthermore, we show that biallelic expression of Kcnq1ot1 coincided with loss of methylation on the maternal allele of the KvDMR1 locus, a phenotype often associated with the human syndrome Beckwith–Wiedemann. In conclusion, our results show that even the most basic manipulation used during human-assisted reproduction, namely, embryo transfer, can lead to misexpression of several imprinted genes during post-implantation development. Additionally, our results serve as a cautionary tale for gene expression studies in which embryo transfer is used.
During mammalian spermatogenesis, germ cell chromatin undergoes dramatic histone acetylation-mediated reorganization, whereby 90%–99% of histones are evicted. Given the potential role of retained ...histones in fertility and embryonic development, the genomic location of retained nucleosomes is of great interest. However, the ultimate position and mechanisms underlying nucleosome eviction or retention are poorly understood, including several studies utilizing micrococcal-nuclease sequencing (MNase-seq) methodologies reporting remarkably dissimilar locations. We utilized assay for transposase accessible chromatin sequencing (ATAC-seq) in mouse sperm and found nucleosome enrichment at promoters but also retention at inter- and intragenic regions and repetitive elements. We further generated germ-cell-specific, conditional knockout mice for the key histone acetyltransferase Gcn5, which resulted in abnormal chromatin dynamics leading to increased sperm histone retention and severe reproductive phenotypes. Our findings demonstrate that Gcn5-mediated histone acetylation promotes chromatin accessibility and nucleosome eviction in spermiogenesis and that loss of histone acetylation leads to defects that disrupt male fertility and potentially early embryogenesis.
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•ATAC-seq localizes retained nucleosomes to promoters and repetitive DNA in sperm•Gcn5-mediated histone acetylation is necessary for proper spermiogenesis•Gcn5 loss alters chromatin dynamics leading to increased histone retention in sperm•Gcn5 is necessary for normal sperm formation and male fertility in mice
Luense et al. utilize ATAC-seq to track nucleosomes through male germ cell maturation and report retention at promoters and repetitive elements in sperm. They develop a mouse model, via conditional deletion of the acetyltransferase Gcn5, to study histone hyperacetylation and eviction, resulting in abnormal sperm histone retention and male fertility defects.
Imprinted genes are differentially marked during germ cell development to allow for their eventual parent-of-origin specific expression. A subset of imprinted genes becomes methylated during oocyte ...growth in both mouse and human. However the timing and mechanisms of methylation acquisition are unknown. Here, we examined the methylation of the Snrpn, Igf2r, Peg1 and Peg3 differentially methylated regions in postnatal growing mouse oocytes. Our findings indicate that methylation was acquired asynchronously at these different genes. Further analysis of Snrpn DMR1 revealed that parental alleles retain an epigenetic memory of their origin as the two alleles were recognized in a parental-specific manner in the absence of DNA methylation. In addition, we show that methylation acquisition was probably related to oocyte diameter and coincided with the accumulation of Dnmt3a, Dnmt3b and Dnmt3L transcripts. Methylation of the repetitive retroviral-like intracisternal A particle also occurred during this same window of oocyte growth. These findings contribute to our understanding of the epigenetic mechanisms underlying imprint acquisition during female germ cell development and have implications for the practice of assisted reproductive technologies.
Self-renewal of spermatogonial stem cells is vital to lifelong production of male gametes and thus fertility. However, the underlying mechanisms remain enigmatic. Here, we show that DOT1L, the sole ...H3K79 methyltransferase, is required for spermatogonial stem cell self-renewal. Mice lacking DOT1L fail to maintain spermatogonial stem cells, characterized by a sequential loss of germ cells from spermatogonia to spermatids and ultimately a Sertoli cell only syndrome. Inhibition of DOT1L reduces the stem cell activity after transplantation. DOT1L promotes expression of the fate-determining HoxC transcription factors in spermatogonial stem cells. Furthermore, H3K79me2 accumulates at
and
genes. Our findings identify an essential function for DOT1L in adult stem cells and provide an epigenetic paradigm for regulation of spermatogonial stem cells.
Cohesins, which mediate sister chromatin cohesion, and CTCF, which functions at chromatin boundaries, play key roles in the structural and functional organization of chromosomes. We examined the ...binding of these two factors on the Kaposi's sarcoma‐associated herpesvirus (KSHV) episome during latent infection and found a striking colocalization within the control region of the major latency transcript responsible for expressing LANA (ORF73), vCyclin (ORF72), vFLIP (ORF71), and vmiRNAs. Deletion of the CTCF‐binding site from the viral genome disrupted cohesin binding, and crippled colony formation in 293 cells. Clonal instability correlated with elevated expression of lytic cycle gene products, notably the neighbouring promoter for K14 and vGPCR (ORF74). siRNA depletion of RAD21 from latently infected cells caused an increase in K14 and ORF74, and lytic inducers caused a rapid dissociation of RAD21 from the viral genome. RAD21 and SMC1 also associate with the cellular CTCF sites at mammalian c‐myc promoter and H19/Igf2 imprinting control region. We conclude that cohesin subunits associate with viral and cellular CTCF sites involved in complex gene regulation and chromatin organization.
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