Article

DNA (cytosine-5)-methyltransferase in mouse cells and tissue. Studies with a mechanism-based probe

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

The mechanisms that establish and maintain methylation patterns in the mammalian genome are very poorly understood, even though perturbations of methylation patterns lead to a loss of genomic imprinting, ectopic X chromosome inactivation, and death of mammalian embryos. A family of sequence-specific DNA methyltransferases has been proposed to be responsible for the wave of de novo methylation that occurs in the early embryo, although no such enzyme has been identified. A universal mechanism-based probe for DNA (cytosine-5)-methyltransferases was used to screen tissues and cell types known to be active in de novo methylation for new species of DNA methyltransferase. All identifiable de novo methyltransferase activity was found to reside in Dnmt1. As this enzyme is the predominant de novo methyltransferase at all developmental stages inspected, it does not fit the definition of maintenance methyltransferase or hemimethylase. Recent genetic data indicate that de novo methylation of retroviral DNA in embryonic stem cells is likely to involve one or more additional DNA methyltransferases. Such enzymes were not detected and are either present in very small amounts or are very different from Dnmt1. A new method was developed and used to determine the sequence specificity of intact Dnmt1 in whole-cell lysates. Specificity was found to be confined to the sequence 5′-CpG-3′; there was little dependence on sequence context or density of CpG dinucleotides. These data suggest that any sequence-specific de novo methylation mediated by Dnmt1 is either under the control of regulatory factors that interact with Dnmt1, or is cued by alternative secondary structures in DNA.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... Dnmt3 is involved in de novo methylation and establishes new methylation patterns during gametogenesis; it consists of three genes in vertebrates: Dnmt3a, Dnmt3b and Dnmt3L 18,19 . Dnmt1 has a 5-to 30-fold preference for hemimethylated DNA substrates over unmethylated substrates and has been implicated in the maintenance of previously established methylation patterns across cell generations 20,21 . Dnmt1 contains three isoforms, Dnmt1o, Dnmt1s and Dnmt1p 22,23 . ...
Article
Full-text available
The Bemisia tabaci Mediterranean (MED) cryptic species has been rapidly invading most parts of the world owing to its strong ecological adaptability, particularly its strong resistance to temperature stress. Epigenetic mechanisms play important roles in mediating ecological plasticity. In particular, DNA methylation has been the focus of attempts to understand the mechanism of phenotypic plasticity. The relationship between temperature and DNA methylation and how it affects the adaptability of invasive insects remain unknown. To investigate the temperature resistance role of DNA methyltransferase 1 (Dnmt1) in MED, we cloned and sequenced BtDnmt1 homology and identified its functions under various temperature conditions. The full-length cDNA of MED BtDnmt1 homology was 5,958 bp and has a 4,287 bp open reading frame that encodes a 1,428-amino-acid protein. BtDnmt1 mRNA expression levels were significantly down-regulated after feeding with dsRNA. Furthermore, after feeding with dsBtDnmt1, the MED adults exhibited significantly higher mortality under temperature stress conditions than the controls, suggesting that MED BtDnmt1 homology plays an essential role in the temperature tolerance capacity of MED. Our data improve our understanding of the temperature resistance and temperature adaptability mechanisms that have allowed the successful invasion and colonization of various environments by this alien species.
... 22 DNA methyltransferase 1 (Dnmt1) preferentially binds to hemimethylated DNA and is crucial for restoring DNA methylation patterns at replication foci, therefore accounting for maintenance of DNA methylation during S-phase. [23][24][25] In contrast, Dnmt3 family members Dnmt3a and Dnmt3b are not only essential for de novo DNA methylation at specific sites in early postimplantation embryos, but also for subsequent embryonic development and tissue differentiation. 26 In this study, we investigate the link between environment, epigenetics, and nephron formation. ...
Article
Background: Nephron number is a major determinant of long-term renal function and cardiovascular risk. Observational studies suggest that maternal nutritional and metabolic factors during gestation contribute to the high variability of nephron endowment. However, the underlying molecular mechanisms have been unclear. Methods: We used mouse models, including DNA methyltransferase (Dnmt1, Dnmt3a, and Dnmt3b) knockout mice, optical projection tomography, three-dimensional reconstructions of the nephrogenic niche, and transcriptome and DNA methylation analysis to characterize the role of DNA methylation for kidney development. Results: We demonstrate that DNA hypomethylation is a key feature of nutritional kidney growth restriction in vitro and in vivo, and that DNA methyltransferases Dnmt1 and Dnmt3a are highly enriched in the nephrogenic zone of the developing kidneys. Deletion of Dnmt1 in nephron progenitor cells (in contrast to deletion of Dnmt3a or Dnm3b) mimics nutritional models of kidney growth restriction and results in a substantial reduction of nephron number as well as renal hypoplasia at birth. In Dnmt1-deficient mice, optical projection tomography and three-dimensional reconstructions uncovered a significant reduction of stem cell niches and progenitor cells. RNA sequencing analysis revealed that global DNA hypomethylation interferes in the progenitor cell regulatory network, leading to downregulation of genes crucial for initiation of nephrogenesis, Wt1 and its target Wnt4. Derepression of germline genes, protocadherins, Rhox genes, and endogenous retroviral elements resulted in the upregulation of IFN targets and inhibitors of cell cycle progression. Conclusions: These findings establish DNA methylation as a key regulatory event of prenatal renal programming, which possibly represents a fundamental link between maternal nutritional factors during gestation and reduced nephron number.
... In 2008, by utilising a unique approach using Multi-PK antibodies (which can recognise multiple protein kinases) [47,48], we discovered that the catalytic fragment of CDKL5 phosphorylates DNA methyltransferase 1 (Dnmt1) in vitro [49]. Since Dnmt1 preferentially methylates hemimethylated DNA, it might act to maintain genome methylation patterns across the replication cycle [50]. CDKL5 phosphorylation sites are located at the N-terminus of Dnmt1 (residues 1-290), but the specific location of these sites remains unknown. ...
Article
Full-text available
Cyclin-dependent kinase-like 5 (CDKL5, also known as STK9) is a serine/threonine protein kinase originally identified in 1998 during a transcriptional mapping project of the human X chromosome. Thereafter, a mutation in CDKL5 was reported in individuals with the atypical Rett syndrome, a neurodevelopmental disorder, suggesting that CDKL5 plays an important regulatory role in neuronal function. The disease associated with CDKL5 mutation has recently been recognised as CDKL5 deficiency disorder (CDD) and has been distinguished from the Rett syndrome owing to its symptomatic manifestation. Because CDKL5 mutations identified in patients with CDD cause enzymatic loss of function, CDKL5 catalytic activity is likely strongly associated with the disease. Consequently, the exploration of CDKL5 substrate characteristics and regulatory mechanisms of its catalytic activity are important for identifying therapeutic target molecules and developing new treatment. In this review, we summarise recent findings on the phosphorylation of CDKL5 substrates and the mechanisms of CDKL5 phosphorylation and dephosphorylation. We also discuss the relationship between changes in the phosphorylation signalling pathways and the Cdkl5 knockout mouse phenotype and consider future prospects for the treatment of mental and neurological disease associated with CDKL5 mutations.
... This module showed a strong GO hit for chromatin modification (GO:0016568). Genes in the turquoise module within this category include many wellknown chromatin modification genes such as Dnmt1, Dnmt3b, Hdac8, Bcor, Crebbp, Ctcf, Bptf, Smarca5, and Smarcc1 [40][41][42][43][44][45] (Fig 7b). The grey60 module also showed a significant GO hit for chromatin (GO:0000785). ...
Article
Full-text available
Progressive increases in ethanol consumption is a hallmark of alcohol use disorder (AUD). Persistent changes in brain gene expression are hypothesized to underlie the altered neural signaling producing abusive consumption in AUD. To identify brain regional gene expression networks contributing to progressive ethanol consumption, we performed microarray and scale-free network analysis of expression responses in a C57BL/6J mouse model utilizing chronic intermittent ethanol by vapor chamber (CIE) in combination with limited access oral ethanol consumption. This model has previously been shown to produce long-lasting increased ethanol consumption, particularly when combining oral ethanol access with repeated cycles of intermittent vapor exposure. The interaction of CIE and oral consumption was studied by expression profiling and network analysis in medial prefrontal cortex, nucleus accumbens, hippocampus, bed nucleus of the stria terminalis, and central nucleus of the amygdala. Brain region expression networks were analyzed for ethanol-responsive gene expression, correlation with ethanol consumption and functional content using extensive bioinformatics studies. In all brain-regions studied the largest number of changes in gene expression were seen when comparing ethanol naïve mice to those exposed to CIE and drinking. In the prefrontal cortex, however, unique patterns of gene expression were seen compared to other brain-regions. Network analysis identified modules of co-expressed genes in all brain regions. The prefrontal cortex and nucleus accumbens showed the greatest number of modules with significant correlation to drinking behavior. Across brain-regions, however, many modules with strong correlations to drinking, both baseline intake and amount consumed after CIE, showed functional enrichment for synaptic transmission and synaptic plasticity.
... We have collected some specifi c data from that review and accumulated here. Based on such DNMTi sources/origin and type/class, those DNMTi include fl avonoids, genistein (124) from soybean Genista tinctoria, quercetin (125) from fruits, vegetables and beverages [104][105][106][107][108], luteolin (130) from Terminalia chebula [104,114,115], silibinin (134) from Silybum marianum, and kazinol Q (135) from Broussonetia kazinoki [104,[119][120][121][122]. Including quinones, nanaomycin A (136) from Streptomyces, laccaic acid (137) from Kerria lacca, hypericin (138) from Hypericum [104,[124][125][126][127][128]. ...
Article
Full-text available
Natural product search is an enduring revitalization upon the exploration of a huge already exotic potential for Secondary Metabolite (SM) production obscure in microbial genomes. Filamentous fungi genomes have an immense number of “orphan” SM gene clusters. Current evaluation indicates that only 5% of extant fungal species have been explored, thus the apparent for the disclosure of novel metabolites in fungi is extensive. In this situation, fungi burgeoning in severe environments are of special interest since they are distinguished producers of astonishing chemical structures. Genome mining strategies, more specifically epigenetic strategies are playing an important role in natural product discovery. This review has been organized and written to focus on available epigenetic approaches, targeting on DNA methyltransferase and histone deacetylase inhibitors along with reported novel secondary metabolites. To the best of our knowledge, this review article is the first attempt to incorporate the facts regarding DNA methyltransferase inhibitors and histone deacetylase inhibitors along with reported novel secondary metabolites with their recorded bioactivities.
... In mammals, CpG methylation is established and maintained by DNA methyltransferases (DNMTs) (6). DNMT1 largely functions as a maintenance methyltransferase by copying the methylation pattern from parent to daughter strand during DNA replication (7,8). The DNMT3 family includes two active homologs, DNMT3A and DNMT3B, and an inactive homolog DNMT3L (9,10). ...
Article
Full-text available
We have previously shown that the highly prevalent acute myeloid leukemia (AML) mutation, Arg882His, in DNMT3A disrupts its cooperative mechanism and leads to reduced enzymatic activity, thus explaining the genomic hypomethylation in AML cells. However, the underlying cause of the oncogenic effect of Arg882His in DNMT3A is not fully understood. Here, we discovered that DNMT3A WT enzyme under conditions that favor non-cooperative kinetic mechanism as well as DNMT3A Arg882His variant acquire CpG flanking sequence preference akin to that of DNMT3B, which is non-cooperative. We tested if DNMT3A Arg882His could preferably methylate DNMT3B-specific target sites in vivo. Rescue experiments in Dnmt3a/3b double knockout mouse embryonic stem cells show that the corresponding Arg878His mutation in mouse DNMT3A severely impairs its ability to methylate major satellite DNA, a DNMT3A-preferred target, but has no overt effect on the ability to methylate minor satellite DNA, a DNMT3B-preferred target. We also observed a previously unappreciated CpG flanking sequence bias in major and minor satellite repeats that is consistent with DNMT3A and DNMT3B specificity suggesting that DNA methylation patterns are guided by the sequence preference of these enzymes. We speculate that aberrant methylation of DNMT3B target sites could contribute to the oncogenic potential of DNMT3A AML variant.
... The difference between the expression levels was not statistically significant (P = .775) F I G U R E 4 Schematic diagram representing the possible role of hsa_circ_0060927 in sponging miR-1264 that results in the stability of DNMT1 mRNA and therefore methylation of target genes such as SOCS3 responsible for the accuracy of DNA methylation pattern during DNA replication and de novo methylation.31,32 Moreover, aberrant methylation of SOCS3 promoter region has been reported in many human cancers.33 ...
Article
Full-text available
Background: As a novel class of non-coding RNAs, the role of circular RNAs (circRNAs) in tumor biogenesis and progression has been proved in a number of human tumors; however, up to now, the relation between circRNAs and uterine leiomyomas (ULM) remains unclear. Methods: In this study, we have estimated the expression level of CYP24A1 hsa_circ_0060927 in uterine leiomyoma and adjacent tissues considering the mediator complex subunit 12 gene (MED12) mutation profile by quantitative real-time polymerase chain reaction (qRT-PCRs). Results: Using Sanger sequencing method, somatic mutations in the MED12 exon 2 were detected in 14 (35.90%) ULM samples, including 10 (71.43%) missense mutations and 4 (28.57%) in-frame deletions. Our results revealed that hsa_circ_0060927 was ectopically expressed in 33.33% of ULM tissues; although, this expression was independent of the MED12 mutation profile in the ULM samples. Conclusions: Present results provide primary evidence for the role of circular RNAs in the leiomyoma development; however, further studies are essential to confirm the importance of these molecules as potential biomarkers for diagnosis and/or prognosis in ULM.
... However, MEFs lacking all three Tet genes fail to initiate MET during reprogramming 13 , suggesting that the relationship between DNA demethylation and reprogramming is highly complex. DNMT1 has been suggested to have a higher ability to methylate hemi-methylated CpG sites than to methylate un-methylated CpG sites 14,15 . If TET1 has different abilities in demethylating hemi-methylated and fullmethylated CpG sites, the relationship between the two types of DNA demethylation should be further explored. ...
Article
Full-text available
The relationship between active DNA demethylation induced by overexpressing Tet1 and passive DNA demethylation induced by suppressing Dnmt1 remains unclear. Here, we found that DNMT1 preferentially methylated, but TET1 preferentially demethylated, hemi-methylated CpG sites. These phenomena resulted in a significant overlap in the targets of these two types of DNA demethylation and the counteractions of Dnmt1 and Tet1 during somatic cell reprogramming. Since the hemi-methylated CpG sites generated during cell proliferation were enriched at core pluripotency loci, DNA demethylation induced by Tet1 or sh-RNA against Dnmt1 (sh-Dnmt1) was enriched in these loci, which, in combination with Yamanaka factors, led to the up-regulation of these genes and promoted somatic cell reprogramming. In addition, since sh-Dnmt1 induces DNA demethylation by impairing the further methylation of hemi-methylated CpG sites generated during cell proliferation, while Tet1 induced DNA demethylation by demethylating these hemi-methylated CpG sites, Tet1-induced DNA demethylation, compared with sh-Dnmt1-induced DNA demethylation, exhibited a higher ability to open the chromatin structure and up-regulate gene expression. Thus, Tet1-induced but not sh-Dnmt1-induced DNA demethylation led to the up-regulation of an additional set of genes that can promote the epithelial-mesenchymal transition and impair reprogramming. When vitamin C was used to further increase the demethylation ability of TET1 during reprogramming, Tet1 induced a larger up-regulation of these genes and significantly impaired reprogramming. Therefore, the current studies provide additional information regarding DNA demethylation during somatic cell reprogramming.
... It uses hemimethylated templates to recruit the actively replicating DNA through an association with proliferating cell nuclear antigen (PCNA-replication fork clamp) ( Figure 1) [16]. After that Dnmt1 transmit the methylation state of the template strand to the nascent strand [60]. The isoforms (Dnmt1s, Dnmt1o and Dnmt1p) of Dnmt1 transcripts are produced by usage of multiple first exon. ...
Chapter
Full-text available
Embryonic stem cells (ESCs) are pluripotent stem cells, which differentiate into all somatic cell type and maintain the pluripotency state by initiating the transcription profiles. Several studies have revealed the mechanisms by which gene expression is regulated in ESCs and it is crucial for pluripotent nature of ESCs. Epigenetic mechanisms are the covalent modification of histone, DNA methylation and chromatin remodeling.The epigenetic nature of the ESCs is unique and it is associated to the gene expressionand pluripotency. DNA methylation mechanism plays a major role in gene regulation.The mechanism is clearly seen in the case of imprinted genes where methylation of promoter regions directs the repression of gene expression.
... Recent structural and biochemical evidence has revealed that both the RFTS and CXXC domains regulate the activity of DNMT1 through autoinhibitory mechanisms: the RFTS domain directly interacts with the MTase domain to inhibit DNA binding (10)(11)(12)(13)(14), whereas the CXXC domain specifically recognizes unmethylated CpG nucleotides, which in turn blocks the de novo methylation activity of DNMT1 (8,15). These N-terminal domain-mediated allosteric regulations, together with an inherent enzymatic preference of the MTase domain for hemimethylated CpG sites (9,16), shape the enzymatic specificity of DNMT1 in maintaining DNA methylation. ...
Article
Significance DNA methylation and histone modifications are two key epigenetic mechanisms in regulating gene expression, heterochromatin assembly, and genome stability. In mammals, maintenance of DNA methylation is mainly mediated by DNA methyltransferase 1 (DNMT1) in a replication-dependent manner. The spatiotemporal regulation of DNMT1 is essential for faithful propagation of DNA methylation patterns between cell generations. Here, we report the direct readout of the heterochromatic mark H3K9me3 by the RFTS domain of DNMT1, which serves to enhance the enzymatic stimulation of DNMT1 by previously characterized H3 ubiquitylation and mediates the cellular colocalization of DNMT1 and H3K9me3. This study uncovers a direct link between the repressive histone modification and DNMT1-mediated DNA methylation.
... DNMT1 was the first cloned DNA methyltransferase [13] and is highly conserved between mouse and human [14]. DNMT1 contains 1620 amino acids, has a strong preference for hemimethylated DNA [15], and functions in maintaining the pre-existing DNA methylation patterns during DNA replication (Figure 1a). In contrast, DNMT3a and DNMT3b function mostly in establishing new DNA methylation patterns by catalyzing the methylation of unmethylated DNA elements in early embryonic development [6,16] (Figure 1b). ...
Article
Full-text available
Ageing, a leading cause of the decline/deficits in human learning, memory, and cognitive abilities, is a major risk factor for age-associated neurodegenerative disorders such as Alzheimer’s disease. Emerging evidence suggests that epigenetics, an inheritable but reversible biochemical process, plays a crucial role in the pathogenesis of age-related neurological disorders. DNA methylation, the best-known epigenetic mark, has attracted most attention in this regard. DNA methyltransferases (DNMTs) are key enzymes in mediating the DNA methylation process, by which a methyl group is transferred, faithfully or anew, to genomic DNA sequences. Biologically, DNMTs are important for gene imprinting. Accumulating evidence suggests that DNMTs not only play critical roles, including gene imprinting and transcription regulation, in early development stages of the central nervous system (CNS), but also are indispensable in adult learning, memory, and cognition. Therefore, the impact of DNMTs and DNA methylation on age-associated cognitive functions and neurodegenerative diseases has emerged as a pivotal topic in the field. In this review, the effects of each DNMT on CNS development and healthy and pathological ageing are discussed.
... On retrouve la quasi-totalité de ces motifs dans toutes les DNMTs depuis la bactérie jusqu'à 1'homme en passant par les champignons et les plantes (Colot et al. 1999 Yoder et al. (Yoder et al. 1997) ont montré que chez la souris, la protéine DNMTl avait une préférence 5 à 30 fois supérieure pour l'ADN hémiméthylé par rapport à de l'ADN non méthylé, d'où la conclusion qu'elle serait responsable de la méthylation dite de maintenance et agirait notamment après le passage de la fourche de réplication. Récemment, Fuks et al. (Fuks et al. 2000) 1998). ...
Thesis
La méthylation des cytosines et l'acétylationJdésacétylation des histones constituent deux modifications épigénétiques importantes du génome eucaryote. Elle sont, toutes les deux, impliquées dans la répression transcriptionnelle. La découverte de la famille protéique MeCP (Methyl-CpG-binding protein) et notamment de la protéine MeCP2, archétype de cettefamille, a permis d'établir un lien entre ces deux modifications épigénétiques. En effet, la protéine MeCP2, une fois liée aux cytosines méthylées des dinucléotides 5'CpG3' réprime indirectement la transcription par l'intermédiaire d'un complexe multiprotéique comprenant en plus du co-répresseur Sin-3A, les histones désacétylases 1 et 2 (HDACI et 2). Les HDACI et 2, en désacétylant les histones du nucléosome entraînent la condensation de la chromatine.Récemment, le gène MECP2 codant la protéine MeCP2 a été impliqué dans un retard mental syndromique lié au chromosome X : le syndrome de Rett. Le syndrome de Rett, pathologie rare (1 fille sur 10 000 à 15 000) se caractérise par une période périnatale normale (jusqu'à 618 mois) avant l'apparition des premiers symptômes correspondant à une régression des acquisitions psychomotrices.Nous avons recherché les mutations dans le gène MECP2 chez 255 patientes atteintes d'un syndrôme de Rett et chez 15 garçons retardés mentaux. Nous avons mis en évidence un spectre mutationnel hétérogène du gène MECP2 chez les patientes atteintes d'un syndrome de Rett. En effet, en plus de mutations ponctuelles, le gène MECP2 peut être remanié sur plusieurs centaines de paires de bases. D'autre part, nous rapportons, pour la première fois chez des filles, l'existence de mutations en mosaïque somatique.
... Realization of this process imposes a biological prohibition on the occurrence of par thenogenesis and androgenesis in mammals (McGrath, Solter, 1984;Surani et al., 1984). DNA methylation is normally carried out with the help of methyltransferase, while total demethylation, with the help of nonspecific DNA demethylase (Carlson et al., 1992;Kafri et al., 1993;Yoder et al., 1997). On the other hand, during site specific methylation or demethylation, transcription factors which do not code for RNA, as well as nuclear receptors for hormones (steroids, thyroids), retinoic acid, and others, may be involved in the action (Brandeis et al., 1993;Mark et al., 2006). ...
Article
Full-text available
The action of two types of substances has been studied: 5�azadeoxycytidine and retinoic acid, which have a demethylation effect on DNA in the development process of diploid parthenogenetic mouse embryos. The effect of 5�azadeoxycytidine on hybrid mice (CBA × C57BL/6)F1 in vitro for 6 h, in the presence of single cell parthenogenetic embryos during the S�phase of the cell cycle has been studied. After developing to the blastocyst stage in vitro, parthenogenetic embryos were transplanted into the uterus of false pregnant females. It has been determined that a concentration of 0.1 μM 5�azadeoxycytidine activates embryonic development in the preimplantation period until the blastocyst stage (69% in experiment; 61% in the control) and during postimplantation, it increases the number of available space in the uterus for implantation (76% in experiment; 63% in the control). The effect of retinoic acid on parthenogenetic embryos from inbred C57BL/6 or CBA mice lines was studied by adding it to single cell embryos in a medium in vitro for 96 h. Treating parthenogenetic embryos C57BL/6 with retinoic acid concentrations 0.1 μM or 0.5 μM significantly increased the number of spaces for embryo implantation, 76% and 78% respectively, as against 57% for untreated embryos. Addition of similar doses of retinoic acid to the nutrient medium containing CBA parthenogenetic mouse embryos does not improve implantation (as with embryos C57BL/6), and a concentration of 2.0 μM is toxic to the embryos. During the period of postimplantation, parthenogenetic embryos of mouse lines C57BL/6 treated with retinoic acid just as the controls, did not develop to the somite stage. Mouse lines CBA had 45% of their embryos which were used as controls, developing to the advanced somite stages. However, the number of embryos treated with retinoic acid does not increase. Thus the treatment of two parthenogenetic embryos from inbred mice lines and their hybrids with compounds which demethylate DNA (5azadeoxycytidine and retinoic acid) creates an opportunity for partial modulation of genomic imprinting and an increase in the survival rate of such embryos.
... The observed spreading of H3K4me3 into gene bodies in the absence of de novo DNMTs shows that DNA methylation actively protects against aberrant accumulation of H3K4me3 in vivo. Whereas promoter DNA methylation has been negatively associated with gene expression in many cell types, gene-body methylation is positively correlated with gene expression and has been proposed to be a mechanism suppressing cryptic promoters in transcribed regions [41][42][43] . Our results further support this notion, showing that DNA methylation in gene bodies prevents accumulation of active histone marks, such as H3K4me3, at CpG-rich domains. ...
Article
Full-text available
Histone 3 K4 trimethylation (depositing H3K4me3 marks) is typically associated with active promoters yet paradoxically occurs at untranscribed domains. Research to delineate the mechanisms of targeting H3K4 methyltransferases is ongoing. The oocyte provides an attractive system to investigate these mechanisms, because extensive H3K4me3 acquisition occurs in nondividing cells. We developed low-input chromatin immunoprecipitation to interrogate H3K4me3, H3K27ac and H3K27me3 marks throughout oogenesis. In nongrowing oocytes, H3K4me3 was restricted to active promoters, but as oogenesis progressed, H3K4me3 accumulated in a transcription-independent manner and was targeted to intergenic regions, putative enhancers and silent H3K27me3-marked promoters. Ablation of the H3K4 methyltransferase gene Mll2 resulted in loss of transcription-independent H3K4 trimethylation but had limited effects on transcription-coupled H3K4 trimethylation or gene expression. Deletion of Dnmt3a and Dnmt3b showed that DNA methylation protects regions from acquiring H3K4me3. Our findings reveal two independent mechanisms of targeting H3K4me3 to genomic elements, with MLL2 recruited to unmethylated CpG-rich regions independently of transcription.
... In mammals, cytosine methylation is catalysed and maintained by a conserved family of enzymes known as DNA methyltransferases (Dnmts), which are subdivided into three families: Dnmt1, Dnmt2 and Dnmt3. Dnmt1 shows a five-to a 30-fold preference for hemimethylated DNA substrates over unmethylated substrates and is implicated in the maintenance of previously established methylation patterns across cell generations (Yoder et al., 1997;Tatematsu et al., 2000). Dnmt2 was originally misclassified and is implicated in transfer RNA methylation (Goll et al., 2006;Tuorto et al., 2015). ...
Article
The Bemisia tabaci Mediterranean (MED) cryptic species is an invasive pest, distributed worldwide, with high ecological adaptability and thermotolerance. DNA methylation (a reversible chromatin modification) is one possible change that may occur within an organism subjected to environmental stress. To assess the effects of temperature stress on DNA methyltransferase 3 (Dnmt3) in MED, we cloned and sequenced BtDnmt3 and identified its functions in response to high and low temperatures. The full-length cDNA of BtDnmt3 was 3913 bp, with an open reading frame of 1962 bp, encoding a 73.89 kDa protein. In situ hybridization showed that BtDnmt3 was expressed mainly in the posterior region. BtDnmt3 messenger RNA expression levels were significantly down-regulated after exposure to heat shock and significantly up-regulated after exposure to cold shock. Furthermore, after feeding on double-stranded RNA specific for BtDnmt3, both heat resistance and cold resistance were significantly decreased, suggesting that BtDnmt3 is associated with thermal stress response and indicating a differential response to high- and low-temperature stress in MED. Together, these results highlight a potential role for DNA methylation in thermal resistance, which is a process important to successful invasion and colonization of an alien species in various environments.
... It is important to note that DNMT1 also participates in de novo methylation (Lorincz et al. 2002). Consistently, lack of Dnmt1 gene (Dnmt1 À/À ) causes embryonic lethality, loss of imprinting, and global demethylation of the genome (Yoder et al. 1997;Kurihara et al. 2008;Okano et al. 1998a). To date, three functional DNMT1 isoforms have been identified: the pachytene spermatocyte form of DNMT1 (DNMT1p), the oocyte-specific form of DNMT1 (DNMT1o), and the somatic form of DNMT1 (DNMT1s) (Ko et al. 2005). ...
Epigenetic mechanisms play important roles in properly occurring mammalian oogenesis. One of these mechanisms is DNA methylation adding a methyl group to the fifth carbon atom of the cytosine residues using S-adenosyl-l-methionine as a methyl donor. DNA methylation generally takes place at cytosine-phosphate-guanine (CpG) dinucleotide sites and rarely occurs at cytosine-phosphate-thymine (CpT), cytosine-phosphate-adenine (CpA), or cytosine-phosphate-cytosine sites, known as non-CpG sites. Basically, two different DNA methylation processes are identified: de novo methylation and maintenance methylation. While the de novo methylation functions in methylation of unmethylated DNA strands, maintenance methylation is capable of methylating hemi-methylated DNA strands following DNA replication. Both DNA methylation processes are catalyzed by special DNA methyltransferase (DNMT) enzymes. To date, five different DNMTs have been identified: DNMT1, DNMT3A, DNMT3B, DNMT3L, and DNMT2. In this chapter, we focus particularly on temporal and spatial expression of DNMTs in mammalian oocytes and granulosa cells.
... Nutrição em foco: uma abordagem holística intermediário S-adenosil-L-homocisteína. A inserção de novas marcas de metilação no DNA é catalizado pelas enzimas da família DNMT3 (DNMT3A e DNMT3B), também chamadas de "DNMTs de novo"(OKANO et al., 1999;UYSAL;AKKOYUNLU;OZTURK, 2015), já a manutenção desses padrões de metilação são mantidos pela DNMT1, durante a replicação celular(BOSTICK et al., 2007;HERMANN;GOYAL;JELTSCH, 2004;YODER et al., 1997). A hipermetilação de ilhas CpGs -regiões do DNA commais de 200 pares de bases com alta densidade de citosina e guanina -localizados em regiões gênicas regulatórias, como regiões de promotores e de enhancers pode alterar a afinidade e especificidade da ligação de proteínas reguladoras a essa região, promovendo o silenciamento gênico (SHIMBO; WADE, 2016). ...
... Hypoxia and hypoxia-ischemia have extensive and far-reaching effects on gene expression and protein synthesis. Epigenetic alternation plays an important role in the changes of gene expression induced by ischemia and hypoxia [23]. At present, the research on epigenetic regulation mainly focuses on DNA CG island methylation modification, histone methylation and acetylation modification. ...
Preprint
Full-text available
Ischemic stroke is an important clinical problem with few effective treatments. Many studies have shown that exogenous tissue kallikrein (TK) can protect neurons against hypoxia/reoxygenation injury. In the present study, we explored the possible molecular mechanisms underlying the regulation and function of endogenous TK. Western blot, chromatin immunoprecipitation (ChIP) and real-time PCR (RT-PCR) revealed that cerebral ischemic preconditioning (IP) upregulated the expression of endogenous TK by regulating the acetylation of histone H3. Cresyl violet staining was used to assess the neuroprotective role of endogenous TK on rat hippocampal CA1 neurons against cerebral ischemia/reperfusion (I/R) injury. Western blot results showed that IP activated the expression of p-Raf, p-MEK1/2 and p-ERK1/2 by Western blot. Moreover, Western blotfurther determined that endogenous TK upregulated the expression of p-Bad, depressed the release of cytochrome c and Bax from mitochondria to the cytosol and inhibited caspase-3 activation. In conclusion, endogenous TK can play a neuroprotective role and its upregulation induced by IP treatment can activate the phosphorylation of Raf, MEK1/2 and p-ERK1/2, depress the release of cytochrome c and Bax from mitochondria to the cytosol and inhibit caspase-3 activation.
... Advances in technologies offered quantitative and baselevel ultra-resolution methylome maps. DNA methylation involves the addition of a methyl group to the carbon-5 position of cytosine in the context of cytosine followed by guanine (CpG dinucleotides), referred to as 5-methylcytosine (5mC), although to a lesser extent, DNA methylation also occurs at cytosine bases in a non-CpG context (143,144). ...
Article
Full-text available
Bull fertility is an important economic trait in sustainable cattle production, as infertile or subfertile bulls give rise to large economic losses. Current methods to assess bull fertility are tedious and not totally accurate. The massive collection of functional data analyses, including genomics, proteomics, metabolomics, transcriptomics, and epigenomics, helps researchers generate extensive knowledge to better understand the unraveling physiological mechanisms underlying subpar male fertility. This review focuses on the sperm phenomes of the functional genome and epigenome that are associated with bull fertility. Findings from multiple sources were integrated to generate new knowledge that is transferable to applied andrology. Diverse methods encompassing analyses of molecular and cellular dynamics in the fertility-associated molecules and conventional sperm parameters can be considered an effective approach to determine bull fertility for efficient and sustainable cattle production. In addition to gene expression information, we also provide methodological information, which is important for the rigor and reliability of the studies. Fertility is a complex trait influenced by several factors and has low heritability, although heritability of scrotal circumference is high and that it is a known fertility maker. There is a need for new knowledge on the expression levels and functions of sperm RNA, proteins, and metabolites. The new knowledge can shed light on additional fertility markers that can be used in combination with scrotal circumference to predict the fertility of breeding bulls. This review provides a comprehensive review of sperm functional characteristics or phenotypes associated with bull fertility.
... Advances in technologies offered quantitative and baselevel ultra-resolution methylome maps. DNA methylation involves the addition of a methyl group to the carbon-5 position of cytosine in the context of cytosine followed by guanine (CpG dinucleotides), referred to as 5-methylcytosine (5mC), although to a lesser extent, DNA methylation also occurs at cytosine bases in a non-CpG context (143,144). ...
Chapter
Bull fertility is an important economic trait in sustainable cattle production, as infertile or subfertile bulls give rise to large economic losses. Current methods to assess bull fertility are tedious and not totally accurate. The massive collection of functional data analyses, including genomics, proteomics, metabolomics, transcriptomics, and epigenomics, helps researchers generate extensive knowledge to better understand the unraveling physiological mechanisms underlying subpar male fertility. This review focuses on the sperm phenomes of the functional genome and epigenome that are associated with bull fertility. Findings from multiple sources were integrated to generate new knowledge that is transferable to applied andrology. Diverse methods encompassing analyses of molecular and cellular dynamics in the fertility-associated molecules and conventional sperm parameters can be considered an effective approach to determine bull fertility for efficient and sustainable cattle production. In addition to gene expression information, we also provide methodological information, which is important for the rigor and reliability of the studies. Fertility is a complex trait influenced by several factors and has low heritability, although heritability of scrotal circumference is high and that it is a known fertility maker. There is a need for new knowledge on the expression levels and functions of sperm RNA, proteins, and metabolites. The new knowledge can shed light on additional fertility markers that can be used in combination with scrotal circumference to predict the fertility of breeding bulls. This review provides a comprehensive review of sperm functional characteristics or phenotypes associated with bull fertility.
... The preferred substrate for this enzyme was demonstrated to be hemimethylated DNA, suggesting that it could facilitate the semi-conservative inheritance of methyl moieties during DNA replication [22]. Although DNMT1 is the only DNA methyltransferase that harbours this substrate specificity and has therefore been designated as the maintenance DNMT, it in fact also contains considerable de novo methylation activity both in vitro and in vivo [23,24]. Vice versa, in certain cell types the de novo methyltransferases DNMT3A and DNMT3B have been shown to contribute to maintenance methylation as well [25,26]. ...
Article
Full-text available
DNA methylation is an epigenetic modification that plays a central regulatory role in various biological processes. Methyl groups are coupled to cytosines by the family of DNA methyltransferases (DNMTs), where DNMT1 is the main maintenance enzyme and the DNMT3 branch of the family is mostly responsible for de novo methylation. The regulation and function of DNA methylation are dependent on the genomic and chromatin context, such as binding sites for transcription factors or the presence of histone marks. Yet how local context, especially chromatin marks, influences the recruitment of the different DNMTs to their genomic target sites remains to be completely revealed. Elucidating the crosstalk between different histone modifications and DNA methylation, and their combined effect on the genome-wide epigenetic landscape, is of particular interest. Aberrant distribution of chromatin marks that guide DNMT activity or DNMT mutations that influence their correct recruitment to the genome have a profound impact on the deposition of DNA methylation, with consequences for genome function and gene activity. In this review, we describe the current state of knowledge on this topic and provide an overview on how chromatin marks can guide DNMT recruitment in healthy and diseased cells.
... Advances in technologies offered quantitative and baselevel ultra-resolution methylome maps. DNA methylation involves the addition of a methyl group to the carbon-5 position of cytosine in the context of cytosine followed by guanine (CpG dinucleotides), referred to as 5-methylcytosine (5mC), although to a lesser extent, DNA methylation also occurs at cytosine bases in a non-CpG context (143,144). ...
... t la méthylation de l'ADN. Il s'agit d'une modification biochimique qui aboutit à l'addition d'un groupement méthyl sur le carbone numéro 5 du cycle pyrimidine d'un résidu cytosine de l'ADN. Cette méthylation se produit essentiellement au niveau des enchaînements 5'CG3', appelés îlots CpG et elle est catalysée par les DNA methyltransférases (DNMT).(Yoder et al., 1997). Dans le génome humain environ 80 % de des CpG sont méthylées(Cheung et al., 2005). Les enzymes qui catalysent cette réaction (DNMT) sont regroupées en trois familles dont chaque une d'elle possède des fonctions bien précises. ...
Thesis
Full-text available
Male infertility is a relatively common pathology; the genetic etiology may be a cause of this abnormality for these males presumed sterile. Thanks to new techniques of Medically Assisted Procreation that infertile men could become biological fathers. However, some questions about the genetic and epigenetic information transmitted to the offspring as well as the modalities of this transmission are raised. A genetic study was carried out over 101 infertile male patients candidates for the Medically Assisted Procreation, these subjects are registered with their spouses in a schedule of intra-cytoplasmic microinjection of the spermatozoon. This study was performed in a prospective setting using cytogenetic tools. Two approaches have been tried in order to assess the quality of the sperm genome. One by analyzing the quality of sperm DNA by measuring the rate of degradation and fragmentation of the latter, another by exploring the quality of spermatic chromatin by measuring its decondensation rate. The study of the association between the quality of the spermatic genome and the various classical spermiological factors shows that the alteration of spermatogenesis is related to a deterioration of the spermatic genome. Regarding the behavioral, toxic and environmental factors, our study showed that these factors did not act on the quality of the spermatic genome. Nevertheless, smoking intoxication seems to alter the chromatin structure of a specific category of patients (p = 0.03). The evaluation of the various parameters was related to the status of Medically Assisted Reproduction especially those of microinjection into cytoplasmic sperm. Our results showed that alterations in the quality of the spermatic genome do not influence either the fertilization process or preimplantation embryonic development (p > 0.05). Our findings indicate also that the quality of the spermatic genome is not related to pregnancy. Nonetheless, it may have an impact on abortions. Regarding births, the integrity of the spermatic nucleus may influence the birth rate (p = 0.064). Moreover, a differentiated sperm cell with an unaltered DNA can give birth even if its chromatin is poorly structured or even decondensed
... DNMT1 is a maintenance methyltransferase, responsible for maintaining the DNA methylation profile during the semiconservative DNA replication. This enzyme has a high affinity for hemimethylated DNA (5-30 fold preference) and it works restoring pre-existing methylation patterns in the parenteral ribbon to the newly synthesized ribbon after cell replication [45][46][47]. Ubiquitin-like with plant homeodomain and RING finger domain 1 protein (UHRF1) protein, also known as ICBP90 in humans and Np95 in mice, is also required for maintaining DNA methylation. During cell replication, UHRF1 protein recognizes and binds to hemimethylated CpG sequences through its SET and RINGassociated (SRA) domain, and acts by forming complexes with DNMT1, recruiting it to replication sites to restore methylation on the nascent strand [45,48,49]. ...
Article
Full-text available
Scientific advances in recent decades have revealed an incredible degree of plasticity in gene expression in response to various environmental, nutritional, physiological, pathological, and behavioral conditions. Epigenetics emerges in this sense, as the link between the internal (genetic) and external (environmental) factors underlying the expression of the phenotype. Methylation of DNA and histone post-translationa modifications are canonical epigenetic events. Additionally, noncoding RNAs molecules (microRNAs and lncRNAs) have also been proposed as another layer of epigenetic regulation. Together, these events are responsible for regulating gene expression throughout life, controlling cellular fate in both normal and pathological development. Despite being a relatively recent science, epigenetics has been arousing the interest of researchers from different segments of the life sciences and the general public. This review highlights the recent advances in the characterization of the epigenetic events and points promising use of these brands for the diagnosis, prognosis, and therapy of diseases. We also present several classes of epigenetic modifying compounds with therapeutic applications (so-call epidrugs) and their current status in clinical trials and approved by the FDA. In summary, hopefully, we provide the reader with theoretical bases for a better understanding of the epigenetic mechanisms and of the promising application of these marks and events in the medical clinic.
Chapter
Here, we discuss how epigenetic processes, including posttranslational modifications of histones and posttranscriptional modifications of RNA, differ in pluripotent embryonic stem cells (ESCs) in comparison with their differentiated counterparts. Additionally, we discuss the specificity of DNA repair epigenetics in ESCs, in which the OCT4 protein play an important role, not only in pluripotency maintenance but also in the DNA damage response. Additionally, in ESC biology, the N⁶-methyladenosine (m⁶A) modification of RNA was found to be functionally essential. It is known that m⁶A RNAs regulate the balance between the self-renewal capability and differentiation properties of ESCs. This posttranscriptional modification, regulating gene expression and the DNA damage response, appears to also be crucial, especially during embryonic development.
Chapter
Early developmental exposure to ethanol, a known teratogen, can result in a range of neurodevelopmental disorders, collectively referred to as Fetal Alcohol Spectrum Disorders (FASDs). Changes in the environment, including exposure to teratogens, can result in long term alterations to the epigenetic landscape of a cell, thereby altering gene expression. Noncoding RNAs (ncRNAs) can affect transcription and translation of networks of genes. ncRNAs are dynamically expressed during development and have been identified as a target of alcohol. ncRNAs therefore make for attractive targets for novel therapeutics to address the developmental deficits associated with FASDs.
Article
Full-text available
Once perceived as a disorder treated by vasodilation, pulmonary artery hypertension (PAH) has emerged as a pulmonary vascular disease with severe endothelial cell dysfunction. In the absence of a cure, many studies seek to understand the detailed mechanisms of EC regulation to potentially create more therapeutic options for PAH. Endothelial dysfunction is characterized by complex phenotypic changes including unchecked proliferation, apoptosis-resistance, enhanced inflammatory signaling and metabolic reprogramming. Recent studies have highlighted the role of epigenetic modifications leading to pro-inflammatory response pathways, endothelial dysfunction, and the progression of PAH. This review summarizes the existing literature on epigenetic mechanisms such as DNA methylation, histone modifications, and non-coding RNAs, which can lead to aberrant endothelial function. Our goal is to develop a conceptual framework for immune dysregulation and epigenetic changes in endothelial cells in the context of PAH. These studies as well as others may lead to advances in therapeutics to treat this devastating disease.
Thesis
The first cell fate decision in development occurs at the blastocyst stage with the emergence of the trophectoderm (TE) and the inner cell mass (ICM). The TE is the precursor population of all major placental cell types. Reflecting this developmental plasticity, trophoblast stem cells (TSCs) can be derived from the TE of mouse blastocysts. TSCs have proven an invaluable research tool to study processes of early placentation in vitro. Despite the placenta’s central role in reproduction, our understanding of the regulatory networks that orchestrate TSC self-renewal and differentiation remains incomplete. In this project, I characterised an epigenetic modifier, KDM1B, for its role in TSC self-renewal and differentiation. I identified this factor as a putative novel regulator of trophoblast stem cell fate and in vitro differentiation from transcriptomics data as its expression is markedly induced at the onset of differentiation. Furthermore, Kdm1b had been implicated in mouse development and placentation, via directing DNA methylation of maternal imprints in the oocyte. KDM1B is a histone lysine demethylase whose activity is directed to H3K4me1 and H3K4me2, particularly within the gene body of actively transcribed genes. By generating CRISPR-Cas9-mediated knockout TSCs ablated for Kdm1b, I show that Esrrb is consistently down-regulated but Gcm1 is up-regulated in Kdm1b-/- TSC clones as measured by RT-qPCR, indicative of precocious differentiation into the syncytiotrophoblast lineage. By performing a large cohort of integrated genome-wide analyses, notably RNA-seq and chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) for the relevant histone modifications H3K4me1, H3K4me2, H3K4me3 and H3K36me3, I show that KDM1B regulates intragenic H3K4me1-marked enhancers, while not impacting H3K4me3. Remarkably, KDM1B null trophoblast cells also show an increased instability of chromosome 13, the same chromosome on which Kdm1b itself is located. This chromosome hosts several gene families that arose from gene duplication events, with vital roles in trophoblast development. The instability of chromosome 13 that gave rise to these gene families is apparently exacerbated by loss of KDM1B, or by CRISPR Cas9-induced cutting of the chromosome. In addition to interrogating the role of KDM1B during differentiation, I utilised the transcriptomic and ChIP-seq data to identify several trophoblast-specific transcripts via location of distal H3K4me3 peaks. Using previously published ChIP-seq data in combination with my own, I link enhancer activity in stem cells to gene expression throughout differentiation. Finally, I identify a striking and novel redistribution of H3K36me3 in 5 day differentiated trophoblast cells, to the promoter regions of expressed genes. In conclusion, this work presents an in-depth analysis of the transcriptional and epigenomic rearrangements that occur both in WT trophoblast and as a consequence of Kdm1b deletion. These data lend important insights into the functions of this epigenetic modifier in the fine-tuning of the transcriptional networks that direct TSC self-renewal and differentiation.
Thesis
Aktuelle Studien berichteten über veränderte DNA-Methylierung in insuffizienten menschlichen Herzen. Diese Ergebnisse lassen auf eine de novo DNA-Methylierung bei der Entwicklung einer chronischen Herzinsuffizienz schließen. In der vorliegenden Arbeit wurde untersucht, ob der Kardiomyozyten-spezifische Verlust der de novo DNA-Methyltransferasen Dnmt3a und Dnmt3b die kardiale Funktion und das Remodeling nach chronischer, linksventrikulärer Druckbelastung verändert. Um Mäuse zu generieren, welche spezifisch in Kardiomyozyten Dnmt3a und Dnmt3b nicht exprimierten, wurden Mäuse mit homozygot gefloxten Dnmt3afl- und Dnmt3bfl- Allelen mit Mäusen gekreuzt, welche die Cre-Rekombinase unter Kontrolle des Promotors des atrialen Myosin-Leichtketten-Gens exprimierten. Die Effizienz der kombinierten Dnmt3a/3b-Deletion wurde sowohl auf DNA- als auch auf mRNAEbene überprüft. Anschließend wurden die Mäuse ohne („basal“) oder nach linksventrikulärer Druckbelastung mittels transversaler Aortenkonstriktion (TAC) auf den kardialen Phänotyp hin untersucht. Unter gleichen Bedingungen wurden Genomweite Transkriptionsprofile erstellt und die DNA-Methylierung von Promotoren unterschiedlich regulierter Gene mittels Pyrosequenzierung bestimmt. In den Kardiomyozyten der Doppelknockout-Mäuse (DKO) konnte die Abwesenheit der Dnmt3a- und Dnmt3b-Transkripte nachgewiesen werden. Die kardiale Phänotypisierung zeigte keine signifikanten Unterschiede zwischen DKO- und Kontrollmäusen, weder basal noch nach TAC. In der Transkriptomanalyse wurde eine Hochregulierung von 44 und eine Herunterregulierung von 9 Genen in den DKO-Mäusen nachgewiesen. Beim Vergleich der Genotypen nach TAC zeigten sich ähnliche Veränderungen mit substanzieller Überlappung der auch basal regulierten Gene. Promotoren einiger hochregulierter Gene waren in der DKO-Gruppe größtenteils unmethyliert verglichen mit der Kontrollgruppe. Die Abwesenheit einer kardialen Pathologie trotz Nachweis des molekularen Phänotyps lässt darauf schließen, dass die de novo DNA-Methylierung in Herzmuskelzellen keinen Einfluss auf adaptive Mechanismen nach chronischer Druckbelastung des Herzens hat.
Article
Arabidopsis ASYMMETRIC LEAVES2 (AS2) plays a critical role in leaf adaxial-abaxial partitioning by repressing expression of the abaxial-determining gene ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3). We previously reported that six CpG dinucleotides in its exon 6 are thoroughly methylated by METHYLTRASFERASE1, that CpG methylation levels are inversely correlated with ETT/ARF3 transcript levels, and that methylation levels at three out of six are decreased in as2-1, All these imply that AS2 is involved in epigenetic repression of ETT/ARF3 by gene body methylation. The mechanism of the epigenetic repression by AS2, however, is unknown. Here, we tested mutations of NUCLEOLIN1 (NUC1) and RNA HELICASE10 (RH10) encoding nucleolus-localized proteins for the methylation in exon 6 as these mutations enhance the level of ETT/ARF3 transcripts in as2. Methylation levels at three specific CpGs were decreased in rh10-1, and two of those three overlapped with those in as2-1. Methylation levels at two specific CpGs were decreased in nuc1-1, and one of those three overlapped with that in as2-1. No site was affected commonly by both rh10-1 and nuc1-1. One specific CpG was unaffected by these mutations. These results imply that RH10, NUC1 and AS2 are involved in maintaining methylation at five CpGs in exon 6 might be through at least several independent pathways, which might interact with each other. Furthermore, we found that AS2 binds specifically the sequence containing CpGs in exon 1 of ETT/ARF3, and that the binding requires the zinc-finger-like motif in AS2 that is structurally similar to the zinc finger-CxxC domain in vertebrate DNA methyltransferase1.
Article
DNA interacting enzymes recognize their target sequences embedded in variable flanking sequence context. The influence of flanking sequences on enzymatic activities of DNA methyltransferases (DNMTs) can be systematically studied with "deep enzymology" approaches using pools of double-stranded DNA substrates, which contain target sites in random flanking sequence context. After incubation with DNMTs and bisulfite conversion, the methylation states and flanking sequences of individual DNA molecules are determined by NGS. Deep enzymology studies with different human and mouse DNMTs revealed strong influences of flanking sequences on the CpG and non-CpG methylation activity and structure of DNMT-DNA complexes. Differences in flanking sequence preferences of DNMT3A and DNMT3B were shown to be related to the prominent role of DNMT3B in the methylation of human SATII repeat elements. Mutational studies in DNMT3B discovered alternative interaction networks between the enzyme and the DNA leading to a partial equalization of the effects of different flanking sequences. Structural studies in DNMT1 revealed striking correlations between enzymatic activities and flanking sequence dependent conformational changes upon DNA binding. Correlation of the biochemical data with cellular methylation patterns demonstrated that flanking sequence preferences are an important parameter that influences genomic DNA methylation patterns together with other mechanisms targeting DNMTs to genomic sites.
Thesis
1. Zusammenfassung 1.1 Hintergrund und Ziele Obwohl Alkoholabhängigkeit ein weit verbreitetes Problem darstellt, sind die molekularen Pathomechanismen dieser Erkrankung noch weitgehend ungeklärt. In jüngster Vergangenheit konnten in einigen Studien Zusammenhänge zwischen verschiedenen suchtspezifischen Genen, Genen, welche die Methylierung beeinflussen, und Alkoholkonsum beobachtet werden. Im Rahmen der hier vorliegenden Studie wurden die Expressionslevel mehrerer dieser Gene während des frühen Alkoholentzugs untersucht. Dabei handelt es sich um DNA-Methyltransferasen (DNMT-1, DNMT-3a und DNMT-3b), MBD2 (Methyl-CpG binding domain protein 2), CREB (cAMP response element binding protein), HERP (Homocysteine-induced endoplasmic reticulum stress protein) und Alpha-Synuclein. 1.2 Patienten und Methoden In die Studie wurden 30 männliche Patienten sofort nach stationärer Aufnahme zum Alkoholentzug eingeschlossen. Am Aufnahmetag auf Station, an Tag 1, Tag 3 und Tag 7 des Entzugs wurde jeweils venöses Blut in EDTA-Monovetten abgenommen, das bei -80 °C schockgefroren wurde. Mittels eines modifizierten Quiagen-Protokolls wurde RNA isoliert. Diese wurde durch reverse Transkription in eine komplementäre cDNA umgeschrieben, die wiederum die Grundlage für die quantitative Polymeraseketten-reaktion darstellte. Mit Hilfe von Fluoreszenzmessungen während der PCR-Zyklen wurde die relative DNA-Menge quantifiziert. Dabei wurde SYBR Green I verwendet, ein doppelstrang-spezifischer, sequenzunabhängiger Fluoreszenzfarbstoff. Als interner Standard lief bei allen Schritten Beta-Aktin mit. 1.3 Ergebnisse Während des frühen Alkoholentzugs zeigten sich in der vorliegenden Studie bei DNMT-1 nahezu gleichbleibende mRNA-Level. Bei DNMT-3a wurde ein Anstieg der mRNA-Level verzeichnet. Die mRNA-Level von DNMT-3b zeigten während des Beobachtungszeitraumes einen geringen Anstieg. Bei MBD2 fiel der mRNA-Level zunächst ab, stieg im Verlauf an und fiel dann wieder ab. Bei CREB wurde ein Anstieg des mRNA-Level mit Maximum am 3. Tag verzeichet. Die mRNA-Level von HERP und Alpha-Synuclein zeigten jeweils zu Beginn der Studie einen Anstieg, im weiteren Verlauf persistierten die mRNA-Level beider Proteine auf erhöhtem Niveau. Der Anstieg bei HERP war größer als der bei Alpha-Synuclein. Unter diesen Beobachtungen war die Veränderung der mRNA-Level bei HERP die einzige, die statistische Signifikanz erreichte. 1.4 Schlussfolgerungen In dieser Studie wurden explorativ die Expressionen von DNMT-1, DNMT-3a, DNMT-3b, MBD2, CREB, HERP und Alpha-Synuclein bei Patienten im frühen Alkoholentzug untersucht. Die Ergebnisse könnten als Grundlage zur Entwicklung neuer Therapieansätze dienen. Vorstellbar wäre auch die Verwendung von DNMT-3a oder HERP als Biomarker für den Verlauf des Alkoholentzugs.
Chapter
When reflecting about cell fate commitment we think of differentiation. Be it during embryonic development or in an adult stem cell niche, where cells of a higher potency specialize and cell fate decisions are taken. Under normal circumstances this process is definitive and irreversible. Cell fate commitment is achieved by the establishment of cell-type-specific transcriptional programmes, which in turn are guided, reinforced, and ultimately locked-in by epigenetic mechanisms. Yet, this plunging drift in cellular potency linked to epigenetically restricted access to genomic information is problematic for reproduction. Particularly in mammals where germ cells are not set aside early on like in other species. Instead they are rederived from the embryonic ectoderm, a differentiating embryonic tissue with somatic epigenetic features. The epigenomes of germ cell precursors are efficiently reprogrammed against the differentiation trend, only to specialize once more into highly differentiated, sex-specific gametes: oocyte and sperm. Their differentiation state is reflected in their specialized epigenomes, and erasure of these features is required to enable the acquisition of the totipotent cell fate to kick start embryonic development of the next generation. Recent technological advances have enabled unprecedented insights into the epigenetic dynamics, first of DNA methylation and then of histone modifications, greatly expanding the historically technically limited understanding of this processes. In this chapter we will focus on the details of embryonic epigenetic reprogramming, a cell fate determination process against the tide to a higher potency.
Article
Full-text available
Infertility is an often devastating diagnosis encountered by around one in six couples who are trying to conceive. Moving away from the long held belief that infertility is primarily a female issue, it is now recognised that half, if not more, of these cases may be due to male factors. Recent evidence has suggested that epigenetic abnormalities in chromatin dynamics, DNA methylation or sperm-borne RNAs may contribute to male infertility. In light of advances in deep sequencing technologies, researchers have been able to increase the coverage and depth of sequencing results which in turn has allowed more comprehensive analyses of spermatozoa chromatin dynamics and methylomes, and enabled the discovery of new subsets of sperm RNAs. This review examines the most current literature related to epigenetic processes in the male germline and the associations of aberrant modifications with fertility and development.
Article
Full-text available
Neurological disorders (NDs) comprise a heterogeneous group of conditions that affect the function of the nervous system. Often incurable, NDs have profound and detrimental consequences on the affected individuals’ lives. NDs have complex etiologies but commonly feature altered gene expression and dysfunctions of the essential chromatin-modifying factors. Hence, compounds that target DNA and histone modification pathways, the so-called epidrugs, constitute promising tools to treat NDs. Yet, targeting the entire epigenome might reveal insufficient to modify a chosen gene expression or even unnecessary and detrimental to the patients’ health. New technologies hold a promise to expand the clinical toolkit in the fight against NDs. (Epi)genome engineering using designer nucleases, including CRISPR-Cas9 and TALENs, can potentially help restore the correct gene expression patterns by targeting a defined gene or pathway, both genetically and epigenetically, with minimal off-target activity. Here, we review the implication of epigenetic machinery in NDs. We outline syndromes caused by mutations in chromatin-modifying enzymes and discuss the functional consequences of mutations in regulatory DNA in NDs. We review the approaches that allow modifying the (epi)genome, including tools based on TALENs and CRISPR-Cas9 technologies, and we highlight how these new strategies could potentially change clinical practices in the treatment of NDs.
Article
Full-text available
DNA methyltransferase 1 (DNMT1) is a multidomain protein believed to be involved only in the passive transmission of genomic methylation patterns via maintenance methylation. The mechanisms that regulate DNMT1 activity and targeting are complex and poorly understood. We used ES cells to investigate the function of the uncharacterized bromo-adjacent homology (BAH) domains and the glycine-lysine (GK) repeats that join the regulatory and catalytic domains of DNMT1. We removed the BAH domains by means of a CRISPR/Cas9-mediated deletion within the endogenous Dnmt1 locus. The internally deleted protein failed to associate with replication foci during S phase in vivo and lost the ability to mediate maintenance methylation. The data indicate that ablation of the BAH domains causes DNMT1 to be excluded from replication foci even in the presence of the replication foci targeting sequence (RFTS). The (GK) repeats resemble the N-terminal tails of histones H2A and H4 and are normally acetylated. Substitution of lysines within the (GK) repeats with arginines to prevent acetylation did not alter the maintenance activity of DNMT1 but unexpectedly activated de novo methylation of paternal imprinting control regions (ICRs) in mouse ES cells; maternal ICRs remained unmethylated. We propose a model under which DNMT1 deposits paternal imprints in male germ cells in an acetylation-dependent manner. These data reveal that DNMT1 responds to multiple regulatory inputs that control its localization as well as its activity, and is not purely a maintenance methyltransferase but can participate in the de novo methylation of a small but essential compartment of the genome.
Article
SETDB1 is a histone methyltransferase that converts H3K9me2 to H3K9me3. SETDB1 activity and H3K9me3 are crucial for the formation of obligately silenced heterochromatin such as that of centromeres. Here we show that a microRNA, miR-152-3p, is involved in the regulation of SETDB1 protein levels, but surprisingly, miR-152-3p plays a positive regulatory role for SETDB1 expression. Inhibition of miR-152-3p by anti-miR treatment resulted in a robust reduction in SETDB1 protein levels, though SETDB1 mRNA levels were unaffected. This was also accompanied by a blockade of the biochemical pathway proceeding from H3K9me2 to H3K9me3 as evidenced by quantitative nucleosome ELISA assays that showed that H3K9me2 accumulates in cells treated with an anti-miR that targets miR-152-3p. In addition, the action of a miR-152-3p mimic increased flux of the reaction leading to H3K9me3. We also performed site-directed mutagenesis of three predicted miR-152-3p target recognition sequences to yield three precise deletions. Deletion of one of the three sites recapitulated the positive regulatory aspect of the action of miR-152-3p upon SETDB1 expression in a luciferase reporter assay. Previous studies have shown that miR-152-3p negatively regulates DNMT1, the sole maintenance DNA methyltransferase which is required for levels of 5-methylcytosine levels within DNA. Our results shown that miR-152-3p positively regulates the production of H3K9me3 by regulating the production of SETDB1. Therefore, our findings provide strong evidence that miR-152-3p can serve as a toggle switch that regulates the balance between DNA methylation and H3K9 histone methylation in constitutive heterochromatin.
Thesis
Abnormal DNA methylation is one of the major hallmarks of cancer. The dynamic and reversible nature of this epigenetic modification has made it a potential target for cancer treatment. UHRF1, a pivotal DNA methylation maintenance protein, is also strongly involved in tumorogenesis. It isoverexpressed in a wide array of cancers and leads to silencing of TSGs and tumor growth. In this context, the aim of the thesis is to develop potential UHRF1 inhibitors that may be clinically effective for anti-cancer therapy. To reach this objective, a diverse approach was adopted including virtual screening, biophysical and biological techniques that helped to characterize the inhibitory activity of active molecules and understand their mechanism of action. The tests revealed one positive compound from the anthraquinone family that inhibited UHRF1 by binding to its SRA domain and impairing its interaction with DNMT1, the enzyme responsible for DNA methylation maintenance. This compound showed an anti-proliferative activity in various cancer cells.
Article
Epigenetics includes the study of external factors that can influence the expression of genes by altering the accessibility of DNA through methylation. To investigate the epigenetic influence of season, sperm head shape, and semen storage on placental and fetal tissues, pregnancies were generated in the summer or winter using boar semen from either least or most sperm head shape change, collected during cool or warm seasons, and stored as cooled‐extended or cryopreserved. The lowest (p < 0.05) ratios of 5‐methylcytosine to 5‐hydroxymethylcytosine activity (5mC:5hmC) in fetal liver were from summer breedings and in placental tissues from winter breedings. The relative expression of placental CDH1 tended ( p < 0.10) to be greater in placenta generated from cryopreserved semen or semen collected during cool periods. The relative expression of placental GNAS was affected ( p < 0.05) by the interaction of breeding and semen collection seasons. Cryopreserved semen increased ( p < 0.05) the placental relative expression of GNAS. Placental MEST and RHOBTB3 tended ( p < 0.10) to have a greater relative expression from pregnancies generated using semen collected during cool periods used during winter breedings. Within fetal liver, the relative expression of GNAS and HGF was greater ( p < 0.05) from winter breedings. Interaction of winter breedings and least sperm head shape change tended ( p < 0.10) to have the greatest fetal liver expression of CDH1. Seasonality of semen collection, breeding, and the effect on sperm head shape change had an influence on the expression of genes with known differentially methylated regions or response to methylation activity from embryonic and extraembryonic tissues. Highlight Methylation activity is dynamic throughout embryonic and extraembryonic development. Influence of season putatively alters the methylation profile and gene expression within placental and fetal livers of swine.
Article
Full-text available
DNA methylation is generally known to inactivate gene expression. The DNA methyltransferases (DNMTs), DNMT3A and DNMT3B, catalyze somatic cell lineage‐specific DNA methylation, while DNMT3A and DNMT3L catalyze germ cell lineage‐specific DNA methylation. How such lineage‐ and gene‐specific DNA methylation patterns are created remains to be elucidated. To better understand the regulatory mechanisms underlying DNA methylation, we generated transgenic mice that constitutively expressed DNMT3A and DNMT3L, and analyzed DNA methylation, gene expression, and their subsequent impact on ontogeny. All transgenic mice were born normally but died within 20 weeks accompanied with cardiac hypertrophy. Several genes were repressed in the hearts of transgenic mice compared with those in wild‐type mice. CpG islands of these downregulated genes were highly methylated in the transgenic mice. This abnormal methylation occurred in the perinatal stage. Conversely, monoallelic DNA methylation at imprinted loci was faithfully maintained in all transgenic mice, except H19. Thus, the loci preferred by DNMT3A and DNMT3L differ between somatic and germ cell lineages.
Article
DNA methylation is a class of epigenetic modification essential for coordinating gene expression timing and magnitude throughout normal brain development and for proper brain function following development. Aberrant methylation changes are associated with changes in chromatin architecture, transcriptional alterations and a host of neurological disorders and diseases. This review highlights recent advances in our understanding of the methylome's functionality and covers potential new roles for DNA methylation, their readers, writers, and erasers. Additionally, we examine novel insights into the relationship between the methylome, DNA-protein interactions, and their contribution to neurodegenerative diseases. Lastly, we outline the gaps in our knowledge that will likely be filled through the widespread use of newer technologies that provide greater resolution into how individual cell types are affected by disease and the contribution of each individual modification site to disease pathogenicity.
Preprint
Full-text available
The ciliary marginal zone (CMZ) of the zebrafish retina contains a population of actively proliferating resident stem cells, which generate retinal neurons throughout life. The maintenance methyltransferase, dnmt1 , is expressed within the CMZ. Loss of dnmt1 function results in gene misregulation and cell death in a variety of developmental contexts, however, its role in retinal stem cell (RSC) maintenance is currently unknown. Here, we demonstrate that zebrafish dnmt1 s872 mutants possess severe defects in RSC maintenance within the CMZ. Using a combination of immunohistochemistry, in situ hybridization, and a transgenic reporter assay, our results demonstrate a requirement for dnmt1 activity in the regulation of RSC proliferation, gene expression and in the repression of endogenous retroelements (REs). Ultimately, cell death is elevated in the dnmt1 -/- CMZ, but in a p53-independent manner. Using a transgenic reporter for RE transposition activity, we demonstrate increased transposition in the dnmt1 -/- CMZ. Taken together our data identify a critical role for dnmt1 function in RSC maintenance in the vertebrate eye.
Article
Oocyte cryopreservation has become an important component of assisted reproductive technology with increasing implication in female fertility preservation and animal reproduction. However, the possible adverse effects of oocyte cryopreservation on epigenetic status of the resulting embryos is still an open question. This study evaluated the effects of MII-oocyte vitrification on gene transcripts linked to epigenetic reprogramming in association with the developmental competence and epigenetic status of the resulting embryos at 2-cell and blastocyst stages in dromedary camel. The cleavage rate of vitrified oocytes following intracytoplasmic sperm injection was significantly increased compared with the control (98.2 ± 2 vs. 72.7 ± 4.1%, respectively), possibly due to the higher susceptibility of vitrified oocytes to spontaneous activation. Nonetheless, the competence of cleaved embryos derived from vitrified oocytes for development to the blastocyst and hatched blastocyst was significantly reduced compared with the control (7.7 ± 1.2 and 11.1 ± 11.1 compared with 28.1 ± 2.6 and 52.4 ± 9.9%, respectively). The relative transcript abundances of epigenetic reprogramming genes DNMT1 , DNMT3B , HDAC1 , and SUV39H1 were all significantly reduced in vitrified oocytes relative to the control. Evaluation of the epigenetic marks showed significant reductions in the levels of DNA methylation (6.1 ± 0.3 vs. 9.9 ± 0.5, respectively) and H3K9 acetylation (7.8 ± 0.2 vs. 10.7 ± 0.3, respectively) in 2-cell embryos in the vitrification group relative to the control. Development to the blastocyst stage partially adjusted the effects that oocyte vitrification had on the epigenetic status of embryos (DNA methylation: 4.9 ± 0.4 vs. 6.2 ± 0.6; H3K9 acetylation: 5.8 ± 0.3 vs. 8 ± 0.9, respectively). To conclude, oocyte vitrification may interfere with the critical stages of epigenetic reprogramming during preimplantation embryo development.
Chapter
Biological rhythms in neuroendocrine and immune systems are pervasive. Daily and seasonal changes in day lengths regulate multiple physiological and immunological parameters in a diverse range of animals. A series of studies have shown that epigenetic modifications exhibit naturally occurring rhythms across short- and long-term timescales. In this chapter, we describe daily, estrous and seasonal oscillations in epigenetic enzymes in neuroendocrine substrates, peripheral reproductive tissues and immune cells (e.g. leukocytes). The predominant focus of the chapter includes enzymes involved in DNA methylation and histone modifications, such as DNA methyltransferase and histone deacetylases. The findings presented herein highlight that epigenetic modifications can be permanent as well as transient with long-term consequences on the timing of physiological and behavioural processes. Moreover, the bidirectional interaction between the immune system and the neuroendocrine nucleus that controls biological rhythmicity, the suprachiasmatic nucleus, emphasizes the need to understand rhythmic changes in epigenetic enzymes and the consequences of disrupted daily and seasonal rhythms.
Article
Full-text available
Differential DNA methylation is characteristic of gene regulatory regions, such as enhancers, which mostly constitute low or intermediate CpG content in their DNA sequence. Consequently, quantification of changes in DNA methylation at these sites is challenging. Given that DNA methylation across most of the mammalian genome is maintained, the use of genome-wide bisulfite sequencing to measure fractional changes in DNA methylation at specific sites is an overexertion which is both expensive and cumbersome. Here, we developed a MethylRAD technique with an improved experimental plan and bioinformatic analysis tool to examine regional DNA methylation changes in embryonic stem cells (ESCs) during differentiation. The transcriptional silencing of pluripotency genes (PpGs) during ESC differentiation is accompanied by PpG enhancer (PpGe) silencing mediated by the demethylation of H3K4me1 by LSD1. Our MethylRAD data show that in the presence of LSD1 inhibitor, a significant fraction of LSD1-bound PpGe fails to gain DNA methylation. We further show that this effect is mostly observed in PpGes with low/intermediate CpG content. Underscoring the sensitivity and accuracy of MethylRAD sequencing, our study demonstrates that this method can detect small changes in DNA methylation in regulatory regions, including those with low/intermediate CpG content, thus asserting its use as a method of choice for diagnostic purposes.
Article
Full-text available
The ciliary marginal zone (CMZ) of the zebrafish retina contains a population of actively proliferating resident stem cells, which generate retinal neurons throughout life. The maintenance methyltransferase, dnmt1, is expressed within the CMZ. Loss of dnmt1 function results in gene misregulation and cell death in a variety of developmental contexts, however, its role in retinal stem cell (RSC) maintenance is currently unknown. Here, we demonstrate that zebrafish dnmt1s872 mutants possess severe defects in RSC maintenance within the CMZ. Using a combination of immunohistochemistry, in situ hybridization, and a transgenic reporter assay, our results demonstrate a requirement for dnmt1 activity in the regulation of RSC proliferation, gene expression and in the repression of endogenous retroelements (REs). Ultimately, cell death is elevated in the dnmt1−/− CMZ, but in a p53-independent manner. Using a transgenic reporter for RE transposition activity, we demonstrate increased transposition in the dnmt1−/− CMZ. Taken together our data identify a critical role for dnmt1 function in RSC maintenance in the vertebrate eye.
Article
DNA methylation is an important epigenetic mechanism of gene expression control. The present study aimed to evaluate the temporal effect of isocaloric high-sugar diet (HSD) intake on the development of nonalcoholic fatty liver disease (NAFLD) and the role of DNA methylation in this event. Newly weaned Wistar rats were divided into eight groups and fed a standard chow diet or an HSD ad libitum for 4 weeks, 8 weeks, 15 weeks, and 18 weeks. After the experimental periods, the animals were euthanized and their livers were removed for histological analysis, gene expression of maintenance methylase (Dnmt1), de novo methylases (Dnmt3a and Dnmt3b), demethylases (Tet2 and Tet3) of DNA, and global DNA methylation. HSD intake led to the gradual development of NAFLD. HSD intake for 18 weeks was associated with downregulation of Dnmt1 expression and global DNA hypomethylation; these results were negatively correlated with more severe steatosis scores observed in these animals. The HSD consumption for 18 weeks was also associated with a decrease in Dnmt3a and Tet2 expression. Interestingly, the expression of de novo methyltransferase Dnmt3b was reduced by HSD during all experimental periods. Together, these results indicate that the downregulation of de novo DNA methylation, Dnmt3b, induced by HSD is the primary factor in the development of NAFLD. On the other hand, disease progression is associated with downregulation of maintenance DNA methylation and global DNA hypomethylation. These results suggest a link between the dynamic changes in hepatic DNA methylation and the development of NAFLD induced by an HSD intake.
Article
Full-text available
We describe here the cloning, characterization and expression in E. coli of the gene coding for a DNA methylase from Spiropiasma sp. strain MQ1 (M Sssl). This enzyme methylates completely and exclusively CpG sequences (1). The Spiroplasma gene was transcribed In E. coli using its own promoter. Translation of the entire message required the use of an opal suppressor, suggesting that UGA triplets code for tryptophan in Spiroplasma. Sequence analysis of the gene revealed several UGA triplets, in a 1158 bp long open reading frame. The deduced amino acid sequence revealed in M⋅Sssl all common domains characteristic of bacterial cytosine DNA methylases. The putative sequence recognition domain of M⋅Sssl showed no obvious similarities with that of the mouse DNA methylase, in spite of their common sequence specificity. The cloned enzyme methylated exclusively CpG sequences both in vivo and in vitro. In contrast to the mammalian enzyme which is primarily a maintenance methylase, M⋅Sssl displayed de novo methylase activity, characteristic of prokaryotic cytosine DNA methylases.
Article
Full-text available
The sequence specificity of an extensively purified DNA methylase preparation from Krebs II mouse ascites cells has been examined. The enzyme appears to be highly sequence dependent. Moreover the sequence distribution of cytosine residues that are methylated, bears a very close resemblance to the sequence distribution of 57prime;-methyl cytosine found in vivo in a wide range of vertebrate cells and is consistent with methylation of cytosines in the sequence R-Yn-C-R.
Article
Full-text available
Methylated DNA in mammals is associated with transcriptional repression and nuclease resistant chromatin. In this review we discuss how these effects may be mediated by proteins that bind to methylated DNA.
Article
Full-text available
We have cloned a series of overlapping cDNA clones encoding a 5194 bp transcript for human DNA methyltransferase (DNA MTase). This sequence potentially codes for a protein of 1495 amino acids with a predicted molecular weight of 169 kDa. The human DNA MTase cDNA has eighty percent homology at the nucleotide level, and the predicted protein has seventy-four percent identity at the amino acid level, to the DNA MTase cDNA cloned from mouse cells. Like the murine DNA MTase, the amino terminal two-thirds of the human protein contains a cysteine-rich region suggestive of a metal-binding domain. The carboxy terminal one-third of the protein shows considerable similarity to prokaryotic (cytosine-5)-methyltransferases. The arrangement of multiple motifs conserved in the prokaryotic genes is preserved in the human DNA MTase, including the relative position of a proline-cysteine dipeptide thought to be an essential catalytic site in all (cytosine-5)-methyltransferases. A single 5.2 kb transcript was detected in all human tissues tested, with the highest levels of expression observed in RNA from placenta, brain, heart and lung. DNA MTase cDNA clones were used to screen a chromosome 19 genomic cosmid library. The DNA MTase-positive cosmids which are estimated to span a genomic distance of 93 kb have been localized to 19p13.2-p13.3 by fluorescence in situ hybridization. Isolation of the cDNA for human DNA MTase will allow further study of the regulation of DNA MTase expression, and of the role of this enzyme in establishing DNA methylation patterns in both normal and neoplastic cells.
Article
Full-text available
G.T mispairs, the sole mismatch type that can arise in "resting" mammalian DNA (through spontaneous hydrolytic deamination of 5-methylcytosine), are corrected in vivo with high efficiency and mostly to a G.C. We identified a protein factor, present in HeLa cell extracts, that binds selectively to DNA substrates containing this mismatch. The partially purified protein was shown by gel-filtration chromatography and UV cross-linking experiments to have an apparent molecular mass of 200 kDa. Its binding to G.T mispairs was not influenced by sequences flanking the mismatch, but methylation of guanines either within the mismatch itself or in its immediate vicinity abolished the formation of the protein-DNA complex. The protein appears to lack both endo- and exonuclease activities and requires neither magnesium nor zinc nor ATP for binding. We discuss the possible role of this protein in a repair pathway, which helps mammalian cells counter the mutagenic effect of the hydrolytic deamination of 5-methylcytosine.
Article
Full-text available
A DNA methyltransferase of Mr = 140,000 that is active on both unmethylated and hemimethylated DNA substrates has been purified from the murine plasma-cytoma cell line MPC 11. The maximal rate of methylation was obtained with maintenance methylation of hemimethylated Micrococcus luteus or M13 DNAs. At low enzyme concentrations, the highest rate of de novo methylation occurred with single-stranded DNA or relatively short duplex DNA containing single-stranded regions. Strong substrate inhibition was observed with hemimethylated but not unmethylated DNA substrates. Fully methylated single-stranded M13 phage DNA inhibited neither the de novo nor the maintenance reactions, but unmethylated single-stranded M13 DNA strongly inhibited the maintenance reaction. The kinetics observed with hemimethylated and single-stranded substrates could be explained if the enzyme were to bind irreversibly to a DNA molecule and to aggregate if present in molar excess. Such aggregates would be required for activity upon hemimethylated but not single-stranded DNA. For de novo methylation of duplex DNA, single-stranded regions or large amounts of methyltransferase appear to be required. The relative substrate preference for the enzyme is hemimethylated DNA greater than fully or partially single-stranded DNA greater than fully duplex DNA.
Article
Full-text available
This paper shows stage- and tissue-specific global demethylation and remethylation occurring during embryonic development. The egg genome is strikingly undermethylated and the sperm genome relatively methylated. Following a loss of genomic methylation during preimplantation development, embryonic and extraembryonic lineages are progressively and independently methylated to different final extents. Methylation continues postgastrulation and hence could be a mechanism initiating, or confirming, differential programming in the definitive germ layers. It is proposed that much of the methylation observed in somatic tissues acts to stabilize and reinforce prior events that regulate the activity of specific genes, chromosome domains or the X chromosome (in females). Fetal germ cell DNA is markedly undermethylated and we favour the idea that the germ lineage is set aside before the occurrence of extensive methylation of DNA in fetal precursor cells.
Article
Full-text available
We have investigated the block to expression of Moloney murine leukemia virus in murine embryonal carcinoma (EC) cells. Infected EC cells were found to contain up to 100 integrated proviral genomes. However, expression of virus as measured by XC plaque and virus-specific RNA synthesis did not occur at significant levels, in contrast to productively infected differentiated cells. Analysis of the DNA in the infected EC cells revealed that the proviral genomes were highly methylated, as shown by their resistance to cleavage by Sma I. Integrated proviral genomes in infected differentiated cells were readily cut by Sma I and thus were not methylated at these sites. Transfection of DNA from infected EC cells to cells permissive for virus expression failed to induce virus expression. The proviral genomes, however, were potentially infectious because they induced XC plaques when the recipient cells for transfection were treated with 5-azacytidine. This drug is believed to interfere with DNA methylation. We conclude that expression of proviral genomes introduced into EC cells is suppressed and that this inactivation can be correlated with the de novo methylation of the viral DNA. De novo methylation activity thus may be a characteristic of early embryonic cells.
Article
Full-text available
The expression of Forssman antigen on the surface of cells of post-implantation mouse embryos between 5 and 7 1/2 days old and of cells of the gonads from 10 1/2 days has been followed using the monoclonal antiserum M1/22.25. In the early post-implantation embryo a lineage-related distribution is found. The inner cell mass of the blastocyst was previously shown to be Forssman antigen positive and its derivative tissues the epiblast of the 5-day embryo and the primary embryonic endoderm are also positive. The endoderm cells remain positive both over the embryonic and extraembryonic portions of the embryo but the epiblast becomes Forssman antigen negative as it differentiates into embryonic ectoderm. The extraembryonic ectoderm which is derived from the Forssman negative trophectoderm remains negative throughout. The primordial germ cells are Forssman antigen positive from their first appearance in the germinal ridge until day 14 when they become negative but after that time it is other cells not related by direct lineage which become Forssman antigen positive. These are tentatively identified as Sertoli cells precursors as it is the Sertoli cells which are the antigen-positive population in the adult testis.
Article
Full-text available
In vertebrate DNA, 3% to 5% of cytosine residues are present as 5-methylcytosine, and it is generally accepted that essentially all of this methylation occurs at cytosines which are contained in the symmetrical dinucleotide CpG. In this report we demonstrate, using bisulphite genomic sequencing, that the methylation machinery of mammalian cells is capable of both maintenance and de novo methylation at CpNpG sites. The existence of inherited CpNpG methylation in mammalian cells has important implications in gene regulation and in the aetiology of disease.
Chapter
Drug effects on nucleic acids may entail the incorporation of drugs or their metabolites, thereby altering the sequence of the four major bases, or involve postsynthetic modifications, e.g., methylations of nucleic acid constituents. While in the past the incorporation of drugs such as purine or pyrimidine analogs into tRNA and other nucleic acids has been recognized as a major effect of these compounds, during the past 5 years our laboratory has presented evidence for pronounced, sometimes highly selective effects of drugs on modifications of nucleic acids. This will be the main subject of this chapter.
Chapter
The genomic DNAs of vertebrates and many invertebrates contain 5-methylcytosine (5-meCyt) as the only modified base (Table 10.1). Considerable interest in DNA methylation has been created by increasing evidence that links methylation patterns to patterns of gene expression in differentiation. An inverse correlation between methylation and transcriptional activity has been found for a number of developmentally regulated genes; it will be reviewed elsewhere (see Chapter 8).
Chapter
In 1975, two papers suggested a role for DNA methylation in X chromosome inactivation. In one paper (Riggs, 1975), I argued that: 1) DNA methylation should affect protein-DNA interactions; 2) methylation patterns and a maintenance methylase should exist; and 3) DNA methylation should be involved in mammalian cellular differentiative processes. Holliday and Pugh (1975) argued similarly, although less weight was given to X inactivation and more weight was given to the possibility that 5-methylcytosine (5-meCyt) might be deaminated to thymidine; thus a specific mutational change would be generated, as suggested by Scarano (1971). Recently, several studies of X chromosome inactivation have contributed to the emerging body of evidence supporting a role for DNA methylation in mammalian gene regulation; it is these studies that will be reviewed in this chapter. More comprehensive reviews of X chromosome inactivation have been published recently (Gartler and Riggs, 1983; Graves, 1983).
Article
Technological advances have made possible the development of high-resolution genetic linkage maps for the mouse. These maps in turn offer exciting prospects for understanding mammalian genome evolution through comparative mapping, for developing mouse models of human disease, and for identifying the function of all genes in the organism.
Article
The properties of the methyl-directed DNA (cytosine-5-)-methyltransferase (EC 2.1.1.37) suggest that it is the enzyme that maintains patterns of methylation in the human genome. Proposals for the enzyme's mechanism of action suggest that 5-methyldeoxycytidine is produced from deoxycytidine via a dihydrocytosine intermediate. We have used an oligodeoxynucleotide containing 5-fluorodeoxycytidine as a suicide substrate to capture the enzyme and the dihydrocytosine intermediate. Gel retardation experiments demonstrate the formation of the expected covalent complex between duplex DNA containing 5-fluorodeoxycytidine and the human enzyme. Formation of the complex was dependent upon the presence of the methyl donor S-adenosylmethionine, suggesting that it comprises an enzyme-linked 5-substituted dihydrocytosine moiety in DNA. Dihydrocytosine derivatives are extremely labile toward hydrolytic deamination in aqueous solution. Because C-to-T transition mutations are especially prevalent at CG sites in human DNA, we have used high-performance liquid chromatography to search for thymidine that might be generated by hydrolysis during the methyl transfer reaction. Despite the potential for deamination inherent in the formation of the intermediate, the methyltransferase did not produce detectable amounts of thymidine. The data suggest that the ability of the human methyltransferase to preserve genetic information when copying a methylation pattern (i.e., its fidelity) is comparable to the ability of a mammalian DNA polymerase to preserve genetic information when copying a DNA sequence. Thus the high frequency of C-to-T transitions at CG sites in human DNA does not appear to be due to the normal enzymatic maintenance of methylation patterns.
Article
A full-length cDNA, encoding a DNA (cytosine-5)-methyltransferase (DNA MTase), has been assembled from a series of overlapping cDNA clones isolated from P. lividus sea urchin embryo cDNA libraries. The cDNA contains 103 bp 5′-UTR, 4839 bp open reading frame corresponding to a 1612 amino acids (aa) protein and 2240 bp 3′-UTR including a terminal 18-bp poly(A) tail. Both the cDNA and the encoded protein are the longest so far reported for DNA MTases. The protein shows five distinct and sequential regions of identity with the other animal DNA MTases, with values of identity from zero to 80%. Northern blot analyses reveal a single RNA band of about 7.5 kb in length showing a highly regulated concentration pattern during development with peak value at the four blastomere stage.
Article
The large-scale chemical synthesis of an oligodeoxynucleotide containing 5-fluoro-2'-deoxycytidine (FdC) and its characterization are described. The FdC residue is introduced via the corresponding 4-O-(2,4,6-trimethylphenyl)-2'-deoxyuridine derivative, which undergoes clean conversion to FdC during removal of the oligonucleotide protecting groups with ammonia. A double-stranded oligodeoxynucleotide containing FdC inactivated the DNA methyltransferase enzyme M.Hae III by irreversible formation of a covalent protein-DNA complex.
Article
The overproduction, purification, and determination of the active-site catalytic nucleophile of the DNA (cytosine-5)-methyltransferase (DCMtase) enzyme M.HaeIII are reported. Incubation of purified M.HaeIII with an oligodeoxynucleotide specifically modified with the mechanism-based inhibitor 5-fluoro-2'-deoxycytidine [Osterman, D. G., et al. (1988) Biochemistry 27, 5204-5210], in the presence of the cofactor S-adenosyl-L-methionine (AdoMet), resulted in the formation of a covalent DNA-M.HaeIII complex, which was purified to homogeneity. Characterization of the intact complex showed it to consist of one molecule of the FdC-containing duplex oligonucleotide, one molecule of M.HaeIII, and one methyl group derived from AdoMet. Exhaustive proteolysis, reduction, and alkylation of the DNA-M.HaeIII complex led to the isolation of two DNA-bound peptides--one each from treatment with Pronase or trypsin--which were subjected to peptide sequencing in order to identify the DNA attachment site. Both peptides were derived from the region of M.HaeIII containing a Pro-Cys sequence that is conserved in all known DCMtases. At the position of this conserved Cys residue (Cys71), in the sequence of each peptide, was found an unidentified amino acid residue; all other amino acid residues were in accord with the known sequence. It is thus concluded that Cys71 of M.HaeIII forms a covalent bond to DNA during catalytic methyl transfer. This finding represents a direct experimental verification for the hypothesis that the conserved Cys residue of DCMtases is the catalytic nucleophile [Wu, J. C., & Santi, D. V. (1987) J. Biol. Chem. 262, 4778-4786].(ABSTRACT TRUNCATED AT 250 WORDS)
5-Fluoropyrimidines and 5-azapyrimidines were found in our laboratory to be specific inhibitors of modification reactions taking place at the 5 position of pyrimidines in nucleic acids. Thus, 5-fluorouracil and 5-fluorouridine specifically inhibit the formation of 5-methyluracil, pseudouridine, and 5,6-dihydrouracil in tRNA. 5-Fluorocytidine, which is partially biotransformed to 5-fluorouracil derivatives in mammalian cells, inhibits the formation of 5-methyluracil, pseudouridine, 5,6-dihydrouracil, and 5-methylcytosine, and 5-azacytidine is a specific inhibitor of the formation of 5-methylcytosine in tRNA and DNA. Inhibitory effects on tRNA modifications require RNA synthesis, as shown by the observation that various inhibitors of RNA synthesis block the drug effects. An inhibitory low-molecular-weight (4-7S) RNA, consisting mainly of tRNA and pre-tRNA, was isolated from livers of mice after treatment with 5-azacytidine. This RNA, when added to an in vitro tRNA methyltransferase assay, specifically interfered with the formation of 5-methylcytosine in substrate tRNA. Similarly, a DNA inhibiting the synthesis of 5-methylcytosine in an in vitro DNA methylation assay was isolated from L1210 leukemic cells treated with a high dose of 5-azacytidine for a short time. Our data are consistent with the hypothesis that incorporation of 5-azacytosine into positions that are normally occupied by C residues destined to become methylated is required for the inhibition to occur, and a similar situation probably applies to the 5-fluoropyrimidine analogs. Analog base moieties occupying such sites are likely to bind strongly, perhaps irreversibly, to the active sites of the particular modifying enzymes. All our observations with the 5-fluoro- and 5-azapyrimidines are in accord with this hypothesis. It was also observed that administration of 5-azacytidine to mice led to strong inhibition of tRNA cytosine-5-methyltransferase, while at the same time the activities and capacities of purine-specific tRNA methyltransferases became strongly elevated after an initial lag period. We speculate that such increases may represent a response of the cell to the methylation defect induced by the drug. Undermodified tRNAs present in neoplastic cells may also trigger an increased synthesis of modifying enzymes. A scheme has been presented which explains increased tRNA turnover and increased activities of modifying enzymes in neoplastic cells as a consequence of a primary defect in tRNA modification.
Article
A model based on DNA methylation is proposed to explain the initiation and maintenance of mammalian X inactivation and certain aspects of other permanent events in eukaryotic cell differentiation. A key feature of the model is the proposal of sequence-specific DNA methylases that methylate unmethylated sites with great difficulty but easily methylate half-methylated sites. Although such enzymes have not yet been detected in eukaryotes, they are known in bacteria. An argument is presented, based on recent data on DNA-binding proteins, that DNA methylation should affect the binding of regulatory proteins. In support of the model, short reviews are included covering both mammalian X inactivation and bacterial restriction and modification enzymes.
Article
The CpG islands found at the 5' ends of many mammalian genes are typically unmethylated despite being both exposed to diffusible protein factors in nuclei and rich in CpG, the target site for DNA methyltransferase. We show here that the CpG islands associated with the human Thy-1 and profilin genes are inherently resistant to de novo methylation by purified murine DNA methyltransferase, and that the higher than expected tendency of CpG sites in islands to be flanked on both sides by G-C base pairs is the likely reason for the resistance. Several lines of evidence indicate that DNA methyltransferase does not make base-specific contacts with residues that flank CpG sites, and it is likely that CpG sites within islands are resistant to de novo methylation because of local conformational features such as ease of strand separation, minor groove dimensions, and alternative secondary structures. A role for minor groove contacts is consistent with the presence within a putative regulatory domain of numerous modified beta turn structural elements that can make minor groove contacts.
Article
The properties of the methyl-directed DNA (cytosine-5-)-methyltransferase (EC 2.1.1.37) suggest that it is the enzyme that maintains patterns of methylation in the human genome. Proposals for the enzyme's mechanism of action suggest that 5-methyldeoxycytidine is produced from deoxycytidine via a dihydrocytosine intermediate. We have used an oligodeoxynucleotide containing 5-fluorodeoxycytidine as a suicide substrate to capture the enzyme and the dihydrocytosine intermediate. Gel retardation experiments demonstrate the formation of the expected covalent complex between duplex DNA containing 5-fluorodeoxycytidine and the human enzyme. Formation of the complex was dependent upon the presence of the methyl donor S-adenosylmethionine, suggesting that it comprises an enzyme-linked 5-substituted dihydrocytosine moiety in DNA. Dihydrocytosine derivatives are extremely labile toward hydrolytic deamination in aqueous solution. Because C-to-T transition mutations are especially prevalent at CG sites in human DNA, we have used high-performance liquid chromatography to search for thymidine that might be generated by hydrolysis during the methyl transfer reaction. Despite the potential for deamination inherent in the formation of the intermediate, the methyltransferase did not produce detectable amounts of thymidine. The data suggest that the ability of the human methyltransferase to preserve genetic information when copying a methylation pattern (i.e., its fidelity) is comparable to the ability of a mammalian DNA polymerase to preserve genetic information when copying a DNA sequence. Thus the high frequency of C-to-T transitions at CG sites in human DNA does not appear to be due to the normal enzymatic maintenance of methylation patterns.
Article
Gene targeting in embryonic stem (ES) cells has been used to mutate the murine DNA methyltransferase gene. ES cell lines homozygous for the mutation were generated by consecutive targeting of both wild-type alleles; the mutant cells were viable and showed no obvious abnormalities with respect to growth rate or morphology, and had only trace levels of DNA methyltransferase activity. A quantitative end-labeling assay showed that the level of m5C in the DNA of homozygous mutant cells was about one-third that of wild-type cells, and Southern blot analysis after cleavage of the DNA with a methylation-sensitive restriction endonuclease revealed substantial demethylation of endogenous retroviral DNA. The mutation was introduced into the germline of mice and found to cause a recessive lethal phenotype. Homozygous embryos were stunted, delayed in development, and did not survive past mid-gestation. The DNA of homozygous embryos showed a reduction of the level of m5C similar to that of homozygous ES cells. These results indicate that while a 3-fold reduction in levels of genomic m5C has no detectable effect on the viability or proliferation of ES cells in culture, a similar reduction of DNA methylation in embryos causes abnormal development and embryonic lethality.
Article
Mammalian DNA (cytosine-5) methyltransferase contains a C-terminal domain that is closely related to bacterial cytosine-5 restriction methyltransferase. This methyltransferase domain is linked to a large N-terminal domain. It is shown here that the N-terminal domain contains a Zn binding site and that the N- and C-terminal domains can be separated by cleavage with trypsin or Staphylococcus aureus protease V8; the protease V8 cleavage site was determined by Edman degradation to lie 10 residues C-terminal of the run of alternating lysyl and glycyl residues which joins the two domains and six residues N-terminal of the first sequence motif conserved between the mammalian and bacterial cytosine methyltransferases. While the intact enzyme had little activity on unmethylated DNA substrates, cleavage between the domains caused a large stimulation of the initial velocity of methylation of unmethylated DNA without substantial change in the rate of methylation of hemimethylated DNA. These findings indicate that the N-terminal domain of DNA methyltransferase ensures the clonal propagation of methylation patterns through inhibition of the de novo activity of the C-terminal domain. Mammalian DNA methyltransferase is likely to have arisen via fusion of a prokaryotic-like restriction methyltransferase and an unrelated DNA binding protein. Stimulation of the de novo activity of DNA methyltransferase by proteolytic cleavage in vivo may contribute to the process of ectopic methylation observed in the DNA of aging animals, tumors and in lines of cultured cells.
Article
DNA topoisomerases are essential nuclear enzymes that are involved in DNA replication. Clinically useful antitumor drugs such as doxorubicin, daunorubicin (anthracyclines), etoposide, teniposide (epipodophyllotoxins), and amsacrine (an aminoacridine) interfere with the function of topoisomerase II and camptothecin and its analogs inhibit topoisomerase I. Some mammalian tumor cells that express resistance to drugs that interfere with topoisomerase I or topoisomerase II have alterations in their respective topoisomerases. In this paper, we review the functions of the topoisomerases, discuss aspects of their cellular regulation, ask how interference with topoisomerase function can lead to tumor cell death, discuss the biochemical features of tumor cells that are resistant to these anti-topoisomerase drugs, and, in the context of drug resistance, we raise questions about how these drugs exert their cytotoxicity.
Article
The amino acid sequence of mammalian DNA methyltransferase has been deduced from the nucleotide sequence of a cloned cDNA. It appears that the mammalian enzyme arose during evolution via fusion of a prokaryotic restriction methyltransferase gene and a second gene of unknown function. Mammalian DNA methyltransferase currently comprises an N-terminal domain of about 1000 amino acids that may have a regulatory role and a C-terminal 570 amino acid domain that retains similarities to bacterial restriction methyltransferases. The sequence similarities among mammalian and bacterial DNA cytosine methyltransferases suggest a common evolutionary origin. DNA methylation is uncommon among those eukaryotes having genomes of less than 10(8) base pairs, but nearly universal among large-genome eukaryotes. This and other considerations make it likely that sequence inactivation by DNA methylation has evolved to compensate for the expansion of the genome that has accompanied the development of higher plants and animals. As methylated sequences are usually propagated in the repressed, nuclease-insensitive state, it is likely that DNA methylation compartmentalizes the genome to facilitate gene regulation by reducing the total amount of DNA sequence that must be scanned by DNA-binding regulatory proteins. DNA methylation is involved in immune recognition in bacteria but appears to regulate the structure and expression of the genome in complex higher eukaryotes. I suggest that the DNA-methylating system of mammals was derived from that of bacteria by way of a hypothetical intermediate that carried out selective de novo methylation of exogenous DNA and propagated the methylated DNA in the repressed state within its own genome.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
Many transposable elements in maize alternate between active and inactive phases associated with the modification of their DNA. Elements in an inactive phase lose their ability to transpose, their ability to excise from reporter alleles and, in some cases, their ability to enhance or suppress mutant phenotypes caused by their insertion. The maize mutant hcf106 is a recessive pale green seedling lethal caused by the insertion of the transposable element Mu1. We show that the hcf106 mutant phenotype is suppressed in lines that have lost Mu activity. That is, homozygous hcf106 seedlings are dark green and viable when transposable elements belonging to the Robertson's Mutator family are modified in their terminal inverted repeats, a diagnostic feature of inactive lines. This property of the mutant phenotype has been used to follow clonal leaf sectors containing modified Mu elements that arise from single somatic cells during plant development. The distribution of these sectors indicates that epigenetic switches involving Mu DNA modification occur progressively as the meristem ages.
Article
A graphical method is presented for displaying the patterns in a set of aligned sequences. The characters representing the sequence are stacked on top of each other for each position in the aligned sequences. The height of each letter is made proportional to Its frequency, and the letters are sorted so the most common one is on top. The height of the entire stack is then adjusted to signify the information content of the sequences at that position. From these ‘sequence logos’, one can determine not only the consensus sequence but also the relative frequency of bases and the information content (measured In bits) at every position in a site or sequence. The logo displays both significant residues and subtle sequence patterns.
Article
The eukaryotic DNA cytosine-5-methyltransferase (E.C.2.1.1.37) is known to methylate cytosine in DNA mainly, but not exclusively in C-G. In the present study the minor, non-C-G recognition sequences of a rat DNA methyltransferase were analyzed by Maxam-Gilbert sequencing of in vitro methylated SV40 DNA. The enzyme methylates C-A and C-T at a 50-fold lower initial rate than C-G. Methylation of C-C at the 5'C was not observed in the piece of DNA sequenced. The methylation of C-A is very low in the trinucleotides ACA and CAC, the other C-A containing trinucleotides in DNA are much better methylacceptors. C-T was found methylated predominantly in the sequences CCTAA, ACTAA, and ACTGT. A comparison of the activity with different substrates is in favour of the enzyme making its recognition in the major groove of the DNA.
Article
Comparisons of the amino acid sequences of m5C DNA methyltransferases (Mtases) from 11 prokaryotes and one eukaryote reveal a very similar organization. Among all the enzymes one can distinguish highly conserved "core" sequences and "variable" regions. The core sequences apparently mediate steps of the methylation reaction that are common to all the enzymes. The major variable region has been shown in our previous studies on multispecific phage Mtases to contain the target-recognizing domains (TRDs) of these enzymes. Here we have compared the amino acid sequences of various TRDs from phage Mtases. This has revealed the presence of both highly conserved and variable amino acids. We postulate that the conserved residues represent a "consensus" sequence defining a TRD, whereas the specificity of the TRD is determined by the variable residues. We have observed similarity between this consensus sequence and sequences in the variable region of the monospecific Mtases. We predict that the regions thus identified represent part of the TRDs of monospecific Mtases.
Article
Thirteen bacterial DNA methyltransferases that catalyze the formation of 5-methylcytosine within specific DNA sequences possess related structures. Similar building blocks (motifs), containing invariant positions, can be found in the same order in all thirteen sequences. Five of these blocks are highly conserved while a further five contain weaker similarities. One block, which has the most invariant residues, contains the proline-cysteine dipeptide of the proposed catalytic site. A region in the second half of each sequence is unusually variable both in length and sequence composition. Those methyltransferases that exhibit significant homology in this region share common specificity in DNA recognition. The five highly conserved motifs can be used to discriminate the known 5-methylcytosine forming methyltransferases from all other methyltransferases of known sequence, and from all other identified proteins in the PIR, GenBank and EMBL databases. These five motifs occur in a mammalian methyltransferase responsible for the formation of 5-methylcytosine within CG dinucleotides. By searching the unidentified open reading frames present in the GenBank and EMBL databases, two potential 5-methylcytosine forming methyltransferases have been found.
Article
Analysis of the enzymatic methylation of oligodeoxynucleotides containing multiple C-G groups showed that hemimethylated sites in duplex oligomers are not significantly methylated by human or murine DNA methyltransferase unless those sites are capable of being methylated de novo in the single- or double-stranded oligomers. Thus, the primary sequence of the target strand, rather than the methylation pattern of the complementary strand, determines maintenance methylation. This suggests that de novo and maintenance methylation are the same process catalyzed by the same enzyme. In addition, the study revealed that complementary strands of oligodeoxynucleotides are methylated at different rates and in different patterns. Both primary DNA sequence and the spacing between C-G groups seem important since in one case studied, maximal methylation required a specific spacing of 13 to 17 nucleotides between C-G pairs.