Should I stay or should I go: Protection and maintenance of DNA methylation at imprinted genes

Recent findings shed light on the coordination of two fundamental, yet mechanistically opposing, processes in the early mammalian embryo. During the oocyte-to-embryo transition and early preimplantation development nuclear reprogramming occurs. This resetting of the epigenome in maternal and paternal pronuclei to a ground state is the essential step ensuring totipotency in the zygote, the first embryonic stage. Radical, global DNA demethylation, which occurs actively in the paternal and passively in the maternal genome, is a prominent feature of nuclear reprogramming; yet, this process poses a danger to a subset of methylated sequences that must be preserved for their germline to soma inheritance. Genomic imprinting and its importance were demonstrated three decades ago by a series of experiments generating non-viable mammalian uniparental embryos. Indeed, imprinted loci, gene clusters with parent-of-origin specific gene expression patterns, must retain their differential methylation status acquired during gametogenesis throughout embryogenesis and in adult tissues. It is just recently that the molecular players that protect/maintain imprinting marks during reprogramming in preimplantation embryos have been identified, in particular, an epigenetic modifier complex formed by ZFP57 and TRIM28/KAP1. The interaction of these and other molecules with the newly formed embryonic chromatin and imprinted genes is discussed and highlighted herein.     

Should I stay or should I go: Wnt signals at the synapse

Several extracellular factors, including Wnt proteins, have been reported to induce synapse formation. In this issue, Klassen and Shen (2007) report that Wnt proteins can also act as antisynaptogenic signals to prevent synapse formation in certain parts of the worm Caenorhabditis elegans. The differential response of axon populations to local Wnt proteins may contribute to the patterning of synaptic connections.     

Should I stay or should I go: predator- and conspecific-induced hatching in a marine snail

Predator-induced hatching plasticity has been demonstrated in many species of amphibians. However, animals from other clades (e.g., marine species of molluscs and annelids) also place their embryos in capsules or gelatinous masses and might also exhibit hatching plasticity to predators. To date there is no evidence of predator-induced hatching plasticity from any marine species or a major clade of bilateria animals, the Lophotrochozoa. We studied predator-induced hatching plasticity of Nucella lamellosa, a carnivorous marine snail that deposits embryos in capsules. We used two experiments to investigate the effects of two types of predator, crabs and isopods, on developing embryos. In the first experiment, we quantified proportion of hatched embryos from capsules through time exposed to water-borne chemicals of crabs and isopods. Crabs delayed time-to-hatching, and the effects of predators were additive. In the second experiment, we quantified proportion of hatched embryos from capsules through time, developmental stage, and size of embryos in capsules exposed to water-borne chemicals of crabs and conspecifics. With this experiment, we wanted to answer: (1) whether a delay in hatching corresponded to embryos developing slower, and (2) whether the general products of metabolic waste from organisms can delay hatching. We unexpectedly observed that adult conspecific snails accelerated hatching but not developmental rate—the few past studies on the effects of conspecifics have all demonstrated that conspecifics delay time-to-hatching and rate of development. The results were also inconsistent with metabolic waste in general causing a delay in hatching, although the effect of conspecifics does weaken this inference. This study demonstrates that predators delay time-to-hatching in a marine mollusc, and suggests that predator-induced hatching plasticity is widespread among animals and likely evolved multiple times within the bilateria. In addition, conspecifics accelerated time-to-hatching in a marine mollusc, which suggests that conspecifics, like predators, might commonly influence when embryos hatch.     

Should I stay or should I go: VCP/p97-mediated chromatin extraction in the DNA damage response

The ordered assembly of DNA repair factors on chromatin has been studied in great detail, whereas we are only beginning to realize that selective extraction of proteins from chromatin plays a central role in the DNA damage response. Interestingly, the protein modifier ubiquitin not only regulates the well-documented recruitment of repair proteins, but also governs the temporally and spatially controlled extraction of proteins from DNA lesions. The facilitator of protein extraction is the ubiquitin-dependent ATPase valosin-containing protein (VCP)/p97 complex, which, through its segregase activity, directly extracts ubiquitylated proteins from chromatin. In this review, we summarize recent studies that uncovered this important role of VCP/p97 in the cellular response to genomic insults and discuss how ubiquitin regulates two intuitively counteracting activities at sites of DNA damage.     

Should I stay or should I go? Female brood desertion and male counterstrategy in rock sparrows

Brood desertion involves a series of interactions between themembers of a pair. This process is likely to be based on eithermember's perception of the other's propensity to desert. Wemanipulated this perception in males by experimentally increasingfemale body mass in the rock sparrow (Petronia petronia), aspecies in which females can desert their first brood beforethe nestlings from the first brood leave the nest. We predictedthat the male would either desert the brood first or stay evenif this implied the risk of caring for the brood alone. We foundthat males mated to loaded females did not leave but stayedand significantly increased their courtship rate and mate guarding.Unexpectedly, they also increased their food provisioning tothe nestlings, even though loaded females did not reduce theirnestling-feeding rate. The increase in male feeding rate maybe explained as a way for the male to reduce the female's propensityto switch mate and desert or to increase her propensity to copulatewith the male to obtain paternity in her next brood. Altogether,our results demonstrate that the perception of the risk of beingdeserted by the female does not necessarily induce males todesert first, contrary to what is generally assumed by theoreticalmodels.     

DNA methylation in imprinted genes IGF2 and GNASXL is associated with prenatal maternal stress

Epigenetic regulation of imprinted genes during embryonic development is influenced by the prenatal environment. Our aim was to examine the effect of maternal emotional stress and cortisol levels during pregnancy on methylation of imprinted genes, insulin‐like growth factor 2 (IGF2) and guanine nucleotide‐binding protein, alpha stimulating extra‐large (GNASXL), using umbilical cord blood DNA. Maternal depressed mood (Edinburgh Depression Scale; EDS), pregnancy‐related anxiety questionnaire (PRAQ) and cortisol day profiles were assessed throughout pregnancy. At birth, a cord blood sample (n = 80) was taken to study DNA methylation of IGF2 DMR0 (differentially methylated region), IGF2 anti‐sense (IGF2AS) and GNASXL using Sequenom Epi TYPER. Linear mixed models were used to examine the relationship between DNA methylation and maternal stress, while correcting for confounders. We also studied the association of DNA methylation with the child ponderal index at birth. We found a cytosine–guanine dinucleotide (CpG)‐specific association of PRAQ subscales with IGF2 DMR0 (CpG5, P < 0.0001) and GNASXL (CpG11, P = 0.0003), while IGF2AS was associated with maternal EDS scores (CpG33, P = 0.0003) and cortisol levels (CpG33, P = 0.0006; CpG37‐38, P = 0.0005). However, there was no association of methylation with ponderal index at birth. In conclusion, maternal stress during pregnancy, as defined by cortisol measurements, EDS and PRAQ scores, is associated with DNA methylation of imprinted genes IGF2 and GNASXL. Our results provide further evidence that prenatal adversity can influence imprinted gene methylation, although future studies are needed to unravel the exact mechanisms.     

DNA methylation at differentially methylated regions of imprinted genes is resistant to developmental programming by maternal nutrition

The nutritional environment in which the mammalian fetus or infant develop is recognized as influencing the risk of chronic diseases, such as type 2 diabetes and hypertension, in a phenomenon that has become known as developmental programming. The late onset of such diseases in response to earlier transient experiences has led to the suggestion that developmental programming may have an epigenetic component, because epigenetic marks such as DNA methylation or histone tail modifications could provide a persistent memory of earlier nutritional states. One class of genes that has been considered a potential target or mediator of programming events is imprinted genes, because these genes critically depend upon epigenetic modifications for correct expression and because many imprinted genes have roles in controlling fetal growth as well as neonatal and adult metabolism. In this study, we have used an established model of developmental programming—isocaloric protein restriction to female mice during gestation or lactation—to examine whether there are effects on expression and DNA methylation of imprinted genes in the offspring. We find that although expression of some imprinted genes in liver of offspring is robustly and sustainably changed, methylation of the differentially methylated regions (DMRs) that control their monoallelic expression remains largely unaltered. We conclude that deregulation of imprinting through a general effect on DMR methylation is unlikely to be a common factor in developmental programming.     

DNA methylation at differentially methylated regions of imprinted genes is resistant to developmental programming by maternal nutrition

The nutritional environment in which the mammalian fetus or infant develop is recognized as influencing the risk of chronic diseases, such as type 2 diabetes and hypertension, in a phenomenon that has become known as developmental programming. The late onset of such diseases in response to earlier transient experiences has led to the suggestion that developmental programming may have an epigenetic component, because epigenetic marks such as DNA methylation or histone tail modifications could provide a persistent memory of earlier nutritional states. One class of genes that has been considered a potential target or mediator of programming events is imprinted genes, because these genes critically depend upon epigenetic modifications for correct expression and because many imprinted genes have roles in controlling fetal growth as well as neonatal and adult metabolism. In this study, we have used an established model of developmental programming—isocaloric protein restriction to female mice during gestation or lactation—to examine whether there are effects on expression and DNA methylation of imprinted genes in the offspring. We find that although expression of some imprinted genes in liver of offspring is robustly and sustainably changed, methylation of the differentially methylated regions (DMRs) that control their monoallelic expression remains largely unaltered. We conclude that deregulation of imprinting through a general effect on DMR methylation is unlikely to be a common factor in developmental programming.     

Should Y stay or should Y go: The evolution of non‐recombining sex chromosomes

Gradual degradation seems inevitable for non‐recombining sex chromosomes. This has been supported by the observation of degenerated non‐recombining sex chromosomes in a variety of species. The human Y chromosome has also degenerated significantly during its evolution, and theories have been advanced that the Y chromosome could disappear within the next ～5 million years, if the degeneration rate it has experienced continues. However, recent studies suggest that this is unlikely. Conservative evolutionary forces such as strong purifying selection and intrachromosomal repair through gene conversion balance the degeneration tendency of the Y chromosome and maintain its integrity after an initial period of faster degeneration. We discuss the evidence both for and against the extinction of the Y chromosome. We also discuss potential insights gained on the evolution of sex‐determining chromosomes by studying simpler sex‐determining chromosomal regions of unicellular and multicellular microorganisms.     

Disruption of imprinted gene expression and DNA methylation status in porcine parthenogenetic fetuses and placentas

Parthenogenetically activated oocytes cannot develop to term in mammals due to the lack of paternal gene expression and failed X chromosome inactivation (XCI). To further characterize porcine parthenogenesis, the expression of 18 imprinted genes was compared between parthenogenetic (PA) and normally fertilized embryos (Con) using quantitative real-time PCR (qRT-PCR). The results revealed that maternally expressed genes were over-expressed, whereas paternally expressed genes were significantly reduced in PA fetuses and placentas. The results of bisulfite sequencing PCR (BSP) demonstrated that PRE-1 and Satellite were hypermethylated in both Con and PA fetuses and placentas, while XIST DMRs were hypomethylated only in PA samples. Taken together, these results suggest that the aberrant methylation profile of XIST DMRs and abnormal imprinted gene expression may be responsible for developmental failure and impaired growth in porcine parthenogenesis.     

Integrated analysis of DNA methylation and mRNA expression profiling reveals candidate genes associated with cisplatin resistance in non-small cell lung cancer

DNA methylation plays a critical role during the development of acquired chemoresistance. The aim of this study was to identify candidate DNA methylation drivers of cisplatin (DDP) resistance in non-small cell lung cancer (NSCLC). The A549/DDP cell line was established by continuous exposure of A549 cells to increasing concentrations of DDP. Gene expression and methylation profiling were determined by high-throughput microarrays. Relationship of methylation status and DDP response was validated in primary tumor cell culture and the Cancer Genome Atlas (TCGA) samples. Cell proliferation, apoptosis, cell cycle, and response to DDP were determined in vitro and in vivo. A total of 372 genes showed hypermethylation and downregulation in A549/DDP cells, and these genes were involved in most fundamental biological processes. Ten candidate genes (S100P, GDA, WISP2, LOXL1, TIMP4, ICAM1, CLMP, HSP8, GAS1, BMP2) were selected, and exhibited varying degrees of association with DDP resistance. Low dose combination of 5-aza-2′-deoxycytidine (5-Aza-dC) and trichostatin A (TSA) reversed drug resistance of A549/DDP cells in vitro and in vivo, along with demethylation and restoration of expression of candidate genes (GAS1, TIMP4, ICAM1 and WISP2). Forced expression of GAS1 in A549/DDP cells by gene transfection contributed to increased sensitivity to DDP, proliferation inhibition, cell cycle arrest, apoptosis enhancement, and in vivo growth retardation. Together, our study demonstrated that a panel of candidate genes downregulated by DNA methylation induced DDP resistance in NSCLC, and showed that epigenetic therapy resensitized cells to DDP.     

A cross-talk between DNA methylation and H3 lysine 9 dimethylation at the KvDMR1 region controls the induction of Cdkn1c in muscle cells

The cdk inhibitor p57^kip2, encoded by the Cdkn1c gene, plays a critical role in mammalian development and in the differentiation of several tissues. Cdkn1c protein levels are carefully regulated via imprinting and other epigenetic mechanisms affecting both the promoter and distant regulatory elements, which restrict its expression to particular developmental phases or specific cell types. Inappropriate activation of these regulatory mechanisms leads to Cdkn1c silencing, causing growth disorders and cancer. We have previously reported that, in skeletal muscle cells, induction of Cdkn1c expression requires the binding of the bHLH myogenic factor MyoD to a long-distance regulatory element within the imprinting control region KvDMR1. Interestingly, MyoD binding to KvDMR1 is prevented in myogenic cell types refractory to the induction of Cdkn1c. In the present work, we took advantage of this model system to investigate the epigenetic determinants of the differential interaction of MyoD with KvDMR1. We show that treatment with the DNA demethylating agent 5-azacytidine restores the binding of MyoD to KvDMR1 in cells unresponsive to Cdkn1c induction. This, in turn, promotes the release of a repressive chromatin loop between KvDMR1 and Cdkn1c promoter and, thus, the upregulation of the gene. Analysis of the chromatin status of Cdkn1c promoter and KvDMR1 in unresponsive compared to responsive cell types showed that their differential responsiveness to the MyoD-dependent induction of the gene does not involve just their methylation status but, rather, the differential H3 lysine 9 dimethylation at KvDMR1. Finally, we report that the same histone modification also marks the KvDMR1 region of human cancer cells in which Cdkn1c is silenced. On the basis of these results, we suggest that the epigenetic status of KvDMR1 represents a critical determinant of the cell type-restricted expression of Cdkn1c and, possibly, of its aberrant silencing in some pathological conditions.     

Integrated analysis of DNA methylation and mRNA expression profiling reveals candidate genes associated with cisplatin resistance in non-small cell lung cancer

DNA methylation plays a critical role during the development of acquired chemoresistance. The aim of this study was to identify candidate DNA methylation drivers of cisplatin (DDP) resistance in non-small cell lung cancer (NSCLC). The A549/DDP cell line was established by continuous exposure of A549 cells to increasing concentrations of DDP. Gene expression and methylation profiling were determined by high-throughput microarrays. Relationship of methylation status and DDP response was validated in primary tumor cell culture and the Cancer Genome Atlas (TCGA) samples. Cell proliferation, apoptosis, cell cycle, and response to DDP were determined in vitro and in vivo. A total of 372 genes showed hypermethylation and downregulation in A549/DDP cells, and these genes were involved in most fundamental biological processes. Ten candidate genes (S100P, GDA, WISP2, LOXL1, TIMP4, ICAM1, CLMP, HSP8, GAS1, BMP2) were selected, and exhibited varying degrees of association with DDP resistance. Low dose combination of 5-aza-2′-deoxycytidine (5-Aza-dC) and trichostatin A (TSA) reversed drug resistance of A549/DDP cells in vitro and in vivo, along with demethylation and restoration of expression of candidate genes (GAS1, TIMP4, ICAM1 and WISP2). Forced expression of GAS1 in A549/DDP cells by gene transfection contributed to increased sensitivity to DDP, proliferation inhibition, cell cycle arrest, apoptosis enhancement, and in vivo growth retardation. Together, our study demonstrated that a panel of candidate genes downregulated by DNA methylation induced DDP resistance in NSCLC, and showed that epigenetic therapy resensitized cells to DDP.     

Gene expression and DNA methylation changes in the hypothalamus and hippocampus of adult rats developmentally exposed to bisphenol A or ethinyl estradiol: a CLARITY-BPA consortium study

Bisphenol A (BPA), an endocrine disrupting chemical (EDC), is a ubiquitous pollutant. As part of the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA), we sought to determine whether exposure of Sprague-Dawley rats to 2,500 μg/kg/day BPA (BPA) or 0.5 μg/kg/day ethinyl estradiol (EE) from gestational day 6 through postnatal day 21 induces behavior-relevant gene expression and DNA methylation changes in hippocampus and hypothalamus at adulthood. RNA and DNA were isolated from both regions. Expression of ten genes (Dnmt1, Dnmt3a, Dnmt3b, Esr1, Esr2, Avp, Ar, Oxt, Otr, and Bdnf) presumably altered by early-life BPA/EE exposure was examined. Three genes (Bdnf, Dnmt3b, and Esr1) were studied for DNA methylation changes in their putative 5? promoter regions. Molecular changes in hippocampus were correlated to prior Barnes maze performance, including sniffing correct holes, distance traveled, and velocity. Exposure to BPA and/or EE disrupted patterns of sexually dimorphic gene expression/promoter DNA methylation observed in hippocampus and hypothalamus of controls. In the hippocampus of female offspring, BPA exposure resulted in hypermethylation of the putative 5? promoter region of Bdnf, while EE exposure induced hypomethylation. Bdnf methylation was weakly associated with Bdnf expression in hippocampi of female rats. Hippocampal Bdnf expression in females showed a weak negative association with sniffing correct hole in Barnes maze. Hippocampal expression of Avp, Esr2, Oxt, and Otr was strongly associated with velocity of control rats in Barnes maze. Findings suggest BPA exposure induced non-EE-like gene expression and epigenetic changes in adult rat hippocampi, a region involved in spatial navigation.     

A DNA repair process in Escherichia coli corrects U:G and T:G mismatches to C:G at sites of cytosine methylation

Escherichia coli contains a base mismatch correction system called VSP repair that is known to correct T:G mismatches to C:G when they occur in certain sequence contexts. The preferred sequence context for this process is the site for methylation by the E. coli DNA cytosine methylase (Dcm). For this reason, VSP repair is thought to counteract potential mutagenic effects of deamination of 5-methylcytosine to thymine. We have developed a genetic reversion assay that quantitates the frequency of C to T mutations at Dcm sites and the removal of such mutations by DNA repair processes. Using this assay, we have studied the repair of U: G mismatches in DNA to C: G and have found that VSP repair is capable of correcting these mismatches. Although VSP repair substantially affects the reversion frequency, it may not be as efficient at correcting U: G mismatches as the uracil DNA glycosylase-mediated repair process.     

Comparison of sister-chromatid exchange induction caused by nitrosoureas that alkylate or alkylate and crosslink DNA

We have investigated the induction of sister-chromatid exchanges (SCEs) in 9L rat brain tumor cells treated with the alkylating agent 1-ethyl-1-nitrosourea (ENU) and 3-(4-amino-2-methyl-5-pyrimidinyl)methyl-1-(2-chloroethyl)-1-nitrosourea (ACNU), an agent that both alkylates and crosslinks DNA. Induction of SCEs by ACNU was found to be 143-fold greater than for ENU. However, on an equimolar basis, the alkylation of DNA by 14C-ACNU was approximately 3.2-fold higher than for 14C-ENU. After correction for this difference was made, the induction of SCEs by ACNU was calculated to be 45-fold greater than for ENU. While DNA alkylation products formed by ACNU and ENU are similar, the chloroethyl alkylation product(s) of ACNU can form DNA-interstrand crosslinks; the ethyl alkylation product(s) of ENU cannot. Based on these findings, we propose that the increased induction of SCEs caused by ACNU is a result of the formation of DNA interstrand crosslinks.     

Novel mutations of Escherichia coli that produce recombinogenic lesions in DNA: V. Recombinogenic plasmids from arl mutants of Escherichia coli are unusually sensitive to nuclease S1 and partially deficient in cytosine methylation at C-C-(A/T)-G-G sequences

Two novel phenotypes previously associated with arl mutations of Escherichia coli, increased frequencies of genetic recombination and unusual sensitivity of DNA to the single-strand-specific nuclease S₁, have been defined most completely by the properties of λ bacteriophages grown on arl bacteria (Arl^? phages). We now find that plasmids maintained in arl mutants (Arl^? plasmids) exhibit elevated recombination frequencies, unusual sensitivity to nuclease S₁ (in a limited number of regions) and a new Arl phenotype, partially deficient methylation of the inner cytosine at C-C-(A/T)-G-G sequences.A variety of Arl^? plasmids (all pBR322 derivatives) show elevated recombination (4 to 10-fold) by three different assays (frequencies of homomultimers and of heteromultimers, efficiency of intramolecular recombination). Plasmids from arl bacteria (after conversion to linear form) are nicked by nuclease S₁ about 0.7 times per duplex; Arl⁺ plasmids are nuclease S₁-resistant. Restriction endonuclease EcoRII (recognition sequence, C-C-(A/T)-G-G) cuts Arl^? plasmid DNA more readily than Arl⁺ DNA, but Arl^? plasmids are still more EcoRII-resistant than Dcm^? plasmids (from E. coli dcm mutants, which lack the chromosomal cytosine methylase; recognition sequence, also C-C-(A/T)-G-G). By chromatographic analyses, Arl^? plasmid DNA contains less 5-methylcytosine than Arl⁺ (0.07% versus 0.15%). although the 6-methyladenine content is the same (0.5mol%).     

Epigenetic silencing of genes enhanced by collective role of reactive oxygen species and MAPK signaling downstream ERK/Snail axis: Ectopic application of hydrogen peroxide repress CDH1 gene by enhanced DNA methyltransferase activity in human breast cancer

Loss of E-cadherin and epithelial to mesenchymal transition (EMT) are key steps in cancer progression. Reactive oxygen species (ROS) play significant roles in cellular physiology and homeostasis. Roles of E-cadherin (CDH1), EMT and ROS are intriguingly illustrated in many cancers without focusing their collective concert during cancer progression. We report that hydrogen peroxide (H₂O₂) treatment modulate CDH1 gene expression by epigenetic modification(s). Sublethal dosage of H₂O₂ treatment decrease E-cadherin, increase DNMT1, HDAC1, Snail, Slug and enrich H3K9me3 and H3K27me3 in the CDH1 promoter. The effect of H₂O₂ was attenuated by ROS scavengers; NAC, lupeol and beta-sitosterol. DNMT inhibitor, AZA prevented the H₂O₂ induced promoter-CpG-island methylation of CDH1. Treatment of cells with U0126 (inhibitor of ERK) reduced the expression of DNMT1, Snail and Slug, increased CDH1. This implicates that CDH1 is synergistically repressed by histone methylation, DNA methylation and histone deacetylation mediated chromatin remodelling and activation of Snail and Slug through ERK pathway. Increased ROS leads to activation of epigenetic machineries and EMT activators Snail/Slug which in their course of action inactivates CDH1 gene and lack of E-cadherin protein promotes EMT in breast cancer cells. ROS and ERK signaling facilitate epigenetic silencing and support the fact that subtle increase of ROS above basal level act as key cell signaling molecules. Free radical scavengers, lupeol and beta-sitosterol may be tested for therapeutic intervention of breast cancer. This work broadens the amplitude of epigenome and open avenues for investigations on conjoint effects of canonical and intrinsic metabolite signaling and epigenetic modulations in cancer.