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A candidate mammalian DNA methyltransferase related to pmt1p of fission yeast

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Abstract

Trace levels of 5-methylcytosine persist in the DNA of mouse embryonic stem cells that are homozygous for null mutations in Dnmt1 , the gene for the one previously recognized metazoan DNA methyltransferase. This residual 5-methylcytosine may be the product of a candidate second DNA methyltransferase, Dnmt2, that has now been identified in human and mouse. Dnmt2 contains all the sequence motifs diagnostic of DNA (cytosine-5)-methyltransferases but appears to lack the large N-terminal regulatory domain common to other eukaryotic methyltransferases. Dnmt2 is more similar to a putative DNA methyltransferase of the fission yeast Schizosaccharomyces pombe than to Dnmt1. Dnmt2 produces multiple mRNA species that are present at low levels in all tissues of human and mouse and is not restricted to those cell types known to be active in de novo methylation. The human DNMT2 gene was mapped to chromosome 10p12-10p14 in a panel of radiation hybrids. Dnmt2 is a candidate for the activity that methylates newly integrated retroviral DNA and maintains trace levels of 5-methylcytosine in the DNA of embryonic stem cells homozygous for null mutations in Dnmt1.

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... Methylation of cytosine residues (m 5 C) in transfer RNAs (tRNAs) is a conserved modification found in this class of non-coding RNAs across eukaryotes (Helm, 2006;Motorin et al., 2009). It has been described in the nuclear tRNAs in lower and higher eukaryotes including the single-celled green algae and the multicellular flowering plants (Wilkinson et al., 1995;Okano et al., 1998;Yoder and Bestor, 1998;Goll et al., 2006;Burgess et al., 2015). Methylation of tRNAs is known to stabilize RNA secondary structures and prevent its cleavage by ribonucleases under a variety of stress conditions and also affect developmental processes in both plants and animals (Rai et al., 2007;Schaefer et al., 2010;Tuorto et al., 2012;Burgess et al., 2015;David et al., 2017). ...
... It differs from other proteins in this family in lacking the N-terminal regulatory domains, being shorter in length and in methylating cytosines in both DNA and tRNAs (Wilkinson et al., 1995;Goll et al., 2006). It was first identified in the fission yeast, Schizosaccharomyces pombe as pombe methyltransferase 1 (pmt1) and is known to be the only methyltransferase in the genomes of many model organisms including Drosophila melanogaster, Dictyostelium discoidium, and Entamoeba histolytica (Wilkinson et al., 1995;Okano et al., 1998;Yoder and Bestor, 1998). DNMT2 expression is developmentally regulated in these organisms (Hung et al., 1999;Kuhlmann et al., 2005;Rai et al., 2007;Müller et al., 2013). ...
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DNMT2 is a DNA/tRNA cytosine methyltransferase that is highly conserved in structure and function in eukaryotes. In plants however, limited information is available on the function of this methyltransferase. We have previously reported that in the moss Physcomitrella patens, DNMT2 plays a crucial role in stress recovery and tRNAAsp transcription/stability under salt stress. To further investigate the role of PpDNMT2 at genome level, in this study we have performed RNA sequencing of ppdnmt2. Transcriptome analysis reveals a number of genes and pathways to function differentially and suggests a close link between PpDNMT2 function and osmotic and ionic stress tolerance. We propose PpDNMT2 to play a pivotal role in regulating salt tolerance by affecting molecular networks involved in stress perception and signal transduction that underlie maintenance of ion homeostasis in cells. We also examined interactome of PpDNMT2 using affinity purification (AP) coupled to mass spectrometry (AP-MS). Quantitative proteomic analysis reveals several chloroplast proteins involved in light reactions and carbon assimilation and proteins involved in stress response and some not implicated in stress to co-immunoprecipitate with PpDNMT2. Comparison between transcriptome and interactome datasets has revealed novel association between PpDNMT2 activity and the antioxidant enzyme Superoxide dismutase (SOD), protein turnover mediated by the Ubiquitin-proteasome system and epigenetic gene regulation. PpDNMT2 possibly exists in complex with CuZn-SODs in vivo and the two proteins also directly interact in the yeast nucleus as observed by yeast two-hybrid assay. Taken together, the work presented in this study sheds light on diverse roles of PpDNMT2 in maintaining molecular and physiological homeostasis in P. patens. This is a first report describing transcriptome and interactome of DNMT2 in any land plant.
... Many CpGs are protected from methylation by being clustered into CpG islands (CGI), which are commonly found near the transcriptional start sites of genes and are normally unmethylated, except for CGI on the inactive X or on inactive imprinted alleles. DNMT1, a maintenance methyltransferase [2], is crucial to ensure the regular propagation of DNA methylation patterns to the daughter strand during replication [3]. This enzyme is predominantly found near replication foci [4] and preferentially targets hemi-methylated DNA [4][5][6] suggesting its main functions as a maintenance methyltransferase [7][8][9]. ...
... Recent work has delineated the gDMRs far more sharply since the original studies were carried out, and more quantitative techniques are now available. We aimed to investigate (1) whether deletion of Dnmt3ab gives comparable methylation loss at imprinted loci to Dnmt1 mutated cells; (2) whether imprints can be restored in 3abKO cells, unlike 1KO ESCs; (3) does loss of methylation result in dysregulated expression of imprinted genes; and (4) are there any exceptional imprinted gDMRs that do not regain methylation in rescued cells? ...
Article
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Background: Imprinted loci are paradigms of epigenetic regulation and are associated with a number of genetic disorders in human. A key characteristic of imprints is the presence of a gametic differentially methylated region (gDMR). Previous studies have indicated that DNA methylation lost from gDMRs could not be restored by DNMT1, or the de novo enzymes DNMT3A or 3B in stem cells, indicating that imprinted regions must instead undergo passage through the germline for reprogramming. However, previous studies were non-quantitative, were unclear on the requirement for DNMT3A/B and showed some inconsistencies. In addition, new putative gDMR has recently been described, along with an improved delineation of the existing gDMR locations. We therefore aimed to re-examine the dependence of methylation at gDMRs on the activities of the methyltransferases in mouse embryonic stem cells (ESCs). Results: We examined the most complete current set of imprinted gDMRs that could be assessed using quantitative pyrosequencing assays in two types of ESCs: those lacking DNMT1 (1KO) and cells lacking a combination of DNMT3A and DNMT3B (3abKO). We further verified results using clonal analysis and combined bisulfite and restriction analysis. Our results showed that loss of methylation was approximately equivalent in both cell types. 1KO cells rescued with a cDNA-expressing DNMT1 could not restore methylation at the imprinted gDMRs, confirming some previous observations. However, nearly all gDMRs were remethylated in 3abKO cells rescued with a DNMT3A2 expression construct (3abKO + 3a2). Transcriptional activity at theH19/Igf2locus also tracked with the methylation pattern, confirming functional reprogramming in the latter. Conclusions: These results suggested (1) a vital role for DNMT3A/B in methylation maintenance at imprints, (2) that loss of DNMT1 and DNMT3A/B had equivalent effects, (3) that rescue with DNMT3A2 can restore imprints in these cells. This may provide a useful system in which to explore factors influencing imprint reprogramming.
... This reaction is catalyzed by DNA methyltransferases (DNMT). The three DNA methyltransferases DNMT1, DNMT3A and DNMT3B form a highly conserved protein family and catalyze either "de novo" or "maintenance" methylation (Yoder and Bestor 1998;Okano et al. 1999;Bestor 2000;Cheng et al. 2008). DNMT1 is usually expressed in somatic cells, primarily during the S-phase and preferentially affects hemi-methylated DNA. ...
Thesis
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Among mental disorders, panic disorder (PD) is one of the most common anxiety disorders characterized by recurring and unexpected episodes of extreme fear i.e. panic attacks. PD displays lifetime prevalence rates in the general population between 2.1-4.7 % and in about 30 to 40 % occurs comorbid with major depressive disorder (MDD). Differential methylation levels of the monoamine oxidase A (MAOA) gene have previously been associated with the etiology of both PD and MDD. The TGFB-Inducible Early Growth Response Protein 2 (TIEG2; alias KLF11), an activating transcription factor of the MAOA gene, has been reported to be increased in MDD, but has not yet been investigated in PD on any level. Therefore, in an attempt to further define the role of an impaired TIEG2-MAOA pathway in anxiety and affective disorders, in the present thesis TIEG2 promoter DNA methylation was analyzed in two independent samples of I) PD patients with or without comorbid MDD in a case/control design and II) MDD patients with and without anxious depression. Additionally, in PD patients of sample I), TIEG2 methylation was correlated with Beck Depression Inventory (BDI-II) scores. Finally, in a third independent healthy control sample, correlation of TIEG2 promoter methylation levels with Anxiety Sensitivity Index (ASI) scores as a PD-related measure was analyzed. No overall association of TIEG2 promoter methylation with PD was detected. However, PD patients with comorbid MDD showed significant TIEG2 hypomethylation compared to PD patients without comorbid MDD (p=.008) as well as to healthy controls (p=.010). In addition, MDD patients without anxious features displayed a statistical trend in decreased TIEG2 methylation in comparison to MDD patients with anxious depression (p=.052). Furthermore, TIEG2 methylation was negatively correlated with BDI-II scores in PD patients (p=.013) and positively correlated with ASI scores in the healthy control sample (p=.043). In sum, the current study suggests TIEG2 promoter hypomethylation as a potential epigenetic marker of MDD comorbidity in PD or of non-anxious depression, respectively. If replicated and verified in future studies, altered TIEG2 methylation might therefore represent a differential pathomechanism of anxiety and mood disorders.
... This regulatory stamping of the genome is maintained by a family of enzymes termed as the DNA methylases, or DNMTs. There are three major DNMTs-DNMT1 and DNMT3 with two major isoforms, DNMT3a and DNMT3b [6,33,67,80,91]. The DNMTs transfer a methyl group from the methyl donor S-adenosylmethionine (SAM) to the cytosine residue in a dinucleotide CG or polynucleotide CGG CGG context, also referred to as CpG islands. ...
Article
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One of the most critical epigenetic signatures present in the genome of higher eukaryotes is the methylation of DNA at the C-5 position of the cytosine ring. Based on the sites of DNA methylation in a locus, it can serve as a repressive or activation mark for gene expression. In a crosstalk with histone modifiers, DNA methylation can consequently either inhibit binding of the transcription machinery or generate a landscape conducive for transcription. During developmental phases, the DNA methylation pattern in the genome undergoes alterations as a result of regulated balance between de novo DNA methylation and demethylation. Resultantly, differentiated cells inherit a unique DNA methylation pattern that fine tunes tissue-specific gene expression. Although apparently a stable epigenetic mark, DNA methylation is actually labile and is a complex reflection of interaction between epigenome, genome and environmental factors prior to birth and during progression of life. Recent findings indicate that levels of DNA methylation in an individual is a dynamic outcome, strongly influenced by the dietary environment during germ cell formation, embryogenesis and post birth exposures. Loss of balances in DNA methylation during developmental stages may result in imprinting disorders, while at any later stage may lead to increased predisposition to various diseases and abnormalities. This review aims to provide an outline of how our epigenome is uniquely guided by our lifetime of experiences beginning in the womb and how understanding it better holds future possibilities of improvised clinical applications.
... De novo m éthylation has previously been described at the RBI locus, in a subset of retinoblastomas (Ohtani Fujita et al, 1993;Sakai et al, 1991), The mechanism by which these de novo méthylation event occur remains unclear. Currently 5 DNA methyltransferases (DNA MTase) has been identified (Bestor et al, 1988;Dong et al, 2001;Leonhardt and Bestor, 1993;Yoder and Bestor, 1998), DNA Mtase has show n both maintenance and de novo methylating activity in vitro (Adams and Lindsay, 1993;Bestor, 1992), but only to have maintenance méthylation activity in vivo ), Yet, provocatively, DNA Mtase is over expressed in some tum our cells (el Deiry et al, 1991;Kautiainen and Jones, 1986), And more im portantly loss of DNA Mtase leads to chromosomal instability (Xu et al, 1999), A hum an genetic disorder (IGF syndrome) has recently been show n to be caused by m utations in the DNA methyltransferase 3B (DNMT3B) gene (Ehrlich et al, 2001), ...
Thesis
Human cancers are caused by mutations affecting the products of oncogenes, tumour suppressor genes and DNA repair genes. Identification of tumour suppressor genes that give rise to sporadic cancers has often been achieved by isolating rare familial cancer genes via a reverse genetic approach. Loss of heterozygosity (LOH) studies have suggested that somatic mutations of a tumour suppressor gene or genes on 3p are critical in the pathogenesis of non familial renal cell carcinoma (RCC). Different studies have implicated differing critical loci. To further investigate the role that 3p genes may have in the tumourigenesis of sporadic RCC, 55 paired normal-tumour DNA's were analysed for allele loss, and at regions of known or putative tumour suppressor genes on chromosomes 5, 11, 17 and 22. 64% (35/55) of informative tumours displayed LOH of at least one or more loci on 3p, compared with 13% at the p53 locus and 6% at 17q21. LOH at 5q21 and 22q was uncommon. The LOH study identified three critical regions of loss on chromosome 3, at 3p25-26, 3p21 and at 3p12-14. The von Hippel Lindau (VHL) tumour suppressor gene was isolated in 1993. It maps to chromosome 3p25-26, coincident with a region of LOH. VHL disease manifests as a variety of benign and malignant neoplasms, and is the most common cause of familial RCC. The role that mutations of the VHL gene may have in the pathogenesis of sporadic RCC was investigated. 99 primary RCC were analysed for SSCP and heteroduplex formations. Somatic mutations were identified in 46% (30/65) sporadic RCC's with 3p LOH and in 3% (1/34) with no 3p LOH. Histology was available for 59 tumours: 42% (18/43) of RCC with a clear cell phenotype had VHL mutations, whereas none of 16 with a non clear cell phenotype (8 chromophilic, 3 chromophobic, and 5 oncocytomas) had VHL mutations (χ2=7.77, p<0.025). These results confirm that mutations in the VHL gene are events that initiate the development of RCC. 3p allele loss is a frequent event in gonadal tumours. 60 gonadal (36 ovarian and 24 testicular) tumours were analysed for VHL gene mutations and 3p allele loss. 3p LOH was detected in 38% (10/26) of informative ovarian and 56% (7/13) testicular tumours, but no VHL mutations were found. This suggests that chromosome 3p tumour suppressor gene(s) other than VHL are involved in gonadal tumourigenesis.
... Not surprisingly, the same set of sequence motifs also occur in mammalian Dnmt2 (8)(9)(10), a tRNA 5mC MTase (11). The conservation in Dnmt sequence and structure reflects the conserved nature of SAM binding, which occurs so as to optimize the catalysis of methyl transfer onto cytosine-C5 in nucleic acids. ...
Article
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S-adenosyl-l-methionine dependent methyltransferases catalyze methyl transfers onto a wide variety of target molecules, including DNA and RNA. We discuss a family of methyltransferases, those that act on the amino groups of adenine or cytosine in DNA, have conserved motifs in a particular order in their amino acid sequence, and are referred to as class beta MTases. Members of this class include M.EcoGII and M.EcoP15I from Escherichia coli, Caulobacter crescentus cell cycle-regulated DNA methyltransferase (CcrM), the MTA1-MTA9 complex from the ciliate Oxytricha, and the mammalian MettL3-MettL14 complex. These methyltransferases all generate N6-methyladenine in DNA, with some members having activity on single-stranded DNA as well as RNA. The beta class of methyltransferases has a unique multimeric feature, forming either homo- or hetero-dimers, allowing the enzyme to use division of labor between two subunits in terms of substrate recognition and methylation. We suggest that M.EcoGII may represent an ancestral form of these enzymes, as its activity is independent of the nucleic acid type (RNA or DNA), its strandedness (single or double), and its sequence (aside from the target adenine).
... CpG sites in the parent strand and catalysis the addition of the methyl group in the corresponding CpG site in the daughter strand. Active localization of the enzyme to sites of DNA replication in dividing cells may facilitate a maintenance role of DNMT1 ( Leonhardt et al., 1992). Yoder and Bestor. (1998) identified one more methyltransferase -DNMT2 with unclear function. However initial studies already showed that this enzyme is not essential for de novo methylation in eukaryotic cells (Okano et al., 1998). ...
Thesis
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Introduction: The necessity for early detection and hence improving the outcome of treatment of hepatocellular carcinoma (HCC) is critical especially in Hepatitis C virus (HCV)-Genotype 4 induced cases. AIM: In our current work, we examined the miRNA152, DNMT1, GSTP1 and CDH1 expression and methylation patterns in chronic liver disease (CLD) due to HCV genotype 4 infection with/without cirrhosis and HCC patients in an attempt to evaluate the potential benefits of these new circulating, noninvasive, prognostic, epigenetic markers for liver cirrhosis and carcinogenesis of Egyptian patients. Patients: Hundred subjects were included in this study, divided into two groups; group I (50 patients) were classified into subgroup Ia (CLD without cirrhosis, n = 25) and subgroup Ib (CLD with cirrhosis, n = 25), group II (CLD patients with HCC, n = 25), and control (Healthy volunteer, n = 25). Methods: The expressions of the studied genes were analyzed using Real-Time PCR and MSP-PCR for methylation pattern detection. Results & Conclusion: there is a direct interaction between miRNA152 and DNMT1 regulating gene expression in cirrhotic and HCC patients. The upregulation of DNMT1 expression has a direct effect on increasing the incidence of methylation pattern in upstream and downstream regions of miRNA152 in HCC patients. Also a significant downregulation of TSGs (GSTP1 and CDH1) in HCC patients. In addition, the expression levels of miRNA152, GSTP1 and CDH1 could be considered as significant predictors associated with the changes of HCC development in those patients. Evidently, the hypermethylation in upstream and downstream regions for miRNA152 decrease its expression increasing the risk of HCC. The hypermethylation in promoter regions of GSTP1 and CDH1 encourage the downregulate/inhibition of its gene expressions leading to silencing of gene and therefore to HCC. This situation highlights the importance of methylation as a tumor marker. The expressions and methylation pattern of those genes could be used as noninvasive prognostic biomarkers and new therapeutic targets for HCV induced liver cirrhosis and HCC in HCV Genotype 4 infected patients.
... 15 However, its DNA methylation activity has been shown to be rather low. 15,16 In contrast, DNMT2 is located in the cytoplasm and displays tRNAAsp methylation activity. 17 In addition, recent findings support the view that the activity of DNMT2 is not limited to tRNAAsp but extends to other tRNAs that are methylated at C38. [18][19][20][21] In vivo and in vitro experiments have provided strong evidence for the role of DNMTs in prostate cancer progression, metastases, and therapy resistance. ...
Article
DNA methyltransferases (DNMTs) regulate gene expression by methylating cytosine residues within CpG dinucleotides. Aberrant methylation patterns have been shown in a variety of human tumours including prostate cancer. However, the expression of DNMTs in clinical samples across the spectrum of prostate cancer progression has not been studied before. Tissue microarrays were constructed from the prostatectomy specimens of 309 patients across the spectrum of prostate cancer progression: hormone-naïve low-grade prostate cancer (n=49), hormone-naïve high-grade prostate cancer (n=151), hormonally treated high-grade prostate cancer (n=65), and castrate-resistant prostate cancer (CRPC) including neuroendocrine carcinoma (n=44). Adjacent non-neoplastic parenchyma was also available in 100 patients. In 71 patients with high-grade carcinoma and lymph node metastasis, tissue from the metastasis was also available for analysis. Immunohistochemical staining was performed with antibodies against DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L. Our results showed that DNMT1 and DNMT3L were upregulated early in prostate cancer progression, whereas DNMT2 was upregulated as a response to androgen ablation. DNMT1, DNMT3A, and DNMT3B were higher in the late stages of prostate cancer progression, i.e., the emergence of castrate resistance and androgen-independent growth. Lastly, DNMT1, DNMT2, and DNMT3L were upregulated in lymph node metastases compared to primary carcinomas. Our results highlight a cascade of epigenetic events in prostate cancer progression.
... DNMT1 associates with the replication fork during DNA synthesis through binding to PCNA and UHRF1 and preferentially targets hemi-methylated DNA sites to restore 5mC on daughter strands; loss of either DNMT1 or UHRF1 triggers passive DNA demethylation (Bostick et al., 2007;Chuang et al., 1997;Sharif et al., 2007). The two remaining DNMT enzymes, DNMT3L and DNMT2, retain significant structural homology to the other DNMT enzymes but contain no detectable DNA methylation activity (Yoder and Bestor, 1998). DNMT2, now more commonly known as tRNA aspartic acid methyltransferase 1 (TRDMT1), instead functions as an RNA methyltransferase (Goll et al., 2006), whilst DNMT3L acts as a regulator of de novo methylation through its interaction with DNMT3A and DNMT3B (Chen et al., 2005;Jia et al., 2007;Suetake et al., 2004). ...
Conference Paper
5’-methylcytosine (5mC) plays a crucial role in the epigenetic regulation of gene expression and, until recently, was the only known epigenetic mark to result from the chemical modification of bases in mammalian deoxyribonucleic acid (DNA). The discovery of 5’-hydroxymethylcytosine (5hmC) at physiologically significant levels in a wide range of tissues, particularly those of the central nervous system, suggests that this novel epigenetic modification may have a similarly important function to 5mC in transcriptional regulation. The highest levels of 5hmC have been consistently found in fully differentiated cell types, whilst stem cells seem to be characterised by very low or insignificant levels of 5hmC. It therefore appears that loss of pluripotency is associated with a substantial increase in global 5hmC levels and this modification may play a crucial role in this switch in cell fate. The central aim of this project was to investigate the potential role of 5hmC by profiling both 5mC and 5hmC in parallel during the differentiation of embryonic stem cells (ESCs) down a neural lineage, allowing for a deeper understanding of the potential function of 5hmC in the genome. Analysis of genome-wide 5mC and 5hmC patterns in ESCs, neural stem cells (NSCs) and astrocytes supported the hypothesis that dynamic changes in the distribution of both modifications contribute to neural specification. Striking differences in 5hmC levels between in vitro- and in vivo-derived samples were observed, suggesting that cell culture models may not successfully recapitulate 5hmC profiles observed during normal development. Finally, a novel method was successfully developed and validated for genome-wide 5hmC profiling (oxBS-450K), allowing sensitive and reproducible detection of 5hmC at single-base resolution.
... 49 Chemotherapeutics that induce alkylation damage ( Figure 1B) make up one of the most common classes of agents used in cancer treatment 50,51 (Figure 1B), underscoring the clinical importance of an improved mechanistic foundation for RNA alkylation repair. Families of RNA-specific m 6 A and m 5 C Sadenosyl L-methionine (SAM)-dependent methyltransferases (MTases) such as METTL3, 5 2− 54 METTL14, 5 5, 5 6 METTL16, 57 and TRDMT1 58,59 have been identified as "writers" of RNA methylation. The m 6 A is reversible ( Figure 1C), and RNA demethylases or "erasers" belonging to the Fe(II)/2-oxoglutarate (2-OG)-dependent AlkB (alkylated DNA repair protein B) dioxygenase family, 45,60,61 such as FTO, 62−64 ALKBH1, 65,66 ALKBH3, 67−70 and ALKBH5, 71 have been shown to be essential for normal reversal of RNA methylation ( Figure 1C) as well as regulation of RNA alkylation. ...
Article
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An emerging molecular understanding of RNA alkylation and its removal are transforming our knowledge of RNA biology and its interplay with cancer chemotherapy responses. DNA modifications are known to perform critical functions depending on the genome template, including gene expression, DNA replication timing, and DNA damage protection. Yet, current results suggest that the chemical diversity of DNA modifications pales in comparison to those on RNA. Over 150 RNA modifications have been identified to date, and their complete functional implications are still being unveiled. These include intrinsic roles such as proper processing and RNA maturation; emerging evidence has furthermore uncovered RNA modification ‘readers’, seemingly analogous to those identified for histone modifications. These modification recognition factors may regulate mRNA stability, localization, and interaction with translation machinery, affecting gene expression. Not surprisingly, tumors differentially modulate factors involved in expressing these marks, contributing both to tumorigenesis and responses to alkylating chemotherapy. Here we describe current understanding of RNA modifications and their removal, with a focus primarily on methylation and alkylation as functionally relevant changes to the transcriptome. Intriguingly, some of the same RNA modifications elicited by physiological processes are also produced by alkylating agents, thus blurring the lines between what is a physiological mark versus a damage-induced modification. Furthermore, we find that high gene expression of enzymes with RNA dealkylation activity is a sensitive readout for poor survival in four different cancer types, underscoring the likely importance of examining RNA dealkylation mechanisms to cancer biology and for cancer treatment and prognosis.
... It remains possible, however, that DNA hypomethylation may enable the transcription of normally silent and methylated retrotransposable elements. DNA methylation is indeed the primary silencing force ensuring retrotransposon control in somatic cells (Walsh et al., 1998;Yoder and Bestor, 1998). If correct, this could lead to the reactivation of endogenous retroviral-like particles, which if detected by innate immune sensors, could in turn induce the anti-viral IFNα cytokine, a hallmark of AGS and SLE (Volkman and Stetson, 2014). ...
Article
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Aicardi–Goutières syndrome (AGS) is a severe childhood inflammatory disorder that shows clinical and genetic overlap with systemic lupus erythematosus (SLE). AGS is thought to arise from the accumulation of incompletely metabolized endogenous nucleic acid species owing to mutations in nucleic acid-degrading enzymes TREX1 (AGS1), RNase H2 (AGS2, 3 and 4), and SAMHD1 (AGS5). However, the identity and source of such immunogenic nucleic acid species remain undefined. Using genome-wide approaches, we show that fibroblasts from AGS patients with AGS1-5 mutations are burdened by excessive loads of RNA:DNA hybrids. Using MethylC-seq, we show that AGS fibroblasts display pronounced and global loss of DNA methylation and demonstrate that AGS-specific RNA:DNA hybrids often occur within DNA hypomethylated regions. Altogether, our data suggest that RNA:DNA hybrids may represent a common immunogenic form of nucleic acids in AGS and provide the first evidence of epigenetic perturbations in AGS, furthering the links between AGS and SLE.
... Heat map of the chosen 4 genes in 56 tissues using Genesis software, the colors depict intensity (log 2 expression ratio) from yellow (large negative) to black (zero). development (Chung et al. 2003;Huan et al. 2015); while in adult stage, the Dnmt2 present the significant expression (P<0.01) in all adult tissue and 30 and 45 day stage of fetus, which were consistent with the expression patterns in cattle (Golding and Westhusin 2003), and its expression patterns were very similar to those of Dnmt1 by gene cluster, which were consistent with the expression in human and mouse tissues (Yoder and Bestor 1998). ...
Article
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DNA methyltransferases (Dnmts) comprise a family of proteins which involved in the establishment and maintenance of DNA methylation patterns. In pig, the molecular characterization and tissue expression profile of Dnmt gene family are not clear. To solve this problem, reverse transcriptase PCR and rapid amplification of cDNA ends were used to clone the sequences of the porcine Dnmt2 and Dnmt3b genes. Furthermore, the mRNA expression profiles of Dnmt1, Dnmt2, Dnmt3a and Dnmt3b genes from 54 adult tissues and 2 entire fetuses of Rongchang pig were analyzed by quantitative real-time PCR (qRT-PCR). As a result, the lengths of porcine Dnmt2 and Dnmt3b gene cDNAs were 1 227 and 2 559 bp with cytosine-C5 specific DNA methylase domain, respectively. The four Dnmt genes were highly expressed in longissimus dorsi muscle (P<0.01). Dnmt1 is highly expressed in heart (P<0.01) and Dnmt 2 shows its preference in liver and seminal vesicle tissue (P<0.01). Dnmt3a and Dnmt3b are highly expressed in the two fetus stages (P<0.01). All these results suggested that each gene has its specific expression profile, and deeper study is required to dig more details between the methylation level and Dnmt family mRNA expressions in different tissues.
... In agreement with structural conservation, different methods in various systems have shown that DNMT2 has DNA methyltransferase activity [2][3][4][5]. DNMT2 was first identified in mice and humans and is likely conserved among eukaryotes [6,7]. This enzyme is the only DNMT found in dipterans, including Drosophila [8]. ...
Article
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The amino acid sequence of DNMT2 is very similar to the catalytic domains of bacterial and eukaryotic proteins. However, there is great variability in the region of recognition of the target sequence. While bacterial DNMT2 acts as a DNA methyltransferase, previous studies have indicated low DNA methylation activity in eukaryotic DNMT2, with preference by tRNA methylation. Drosophilids are known as DNMT2-only species and the DNA methylation phenomenon is a not elucidated case yet, as well as the ontogenetic and physiologic importance of DNMT2 for this species group. In addition, more recently study showed that methylation in the genome in Drosophila melanogaster is independent in relation to DNMT2. Despite these findings, Drosophilidae family has more than 4,200 species with great ecological diversity and historical evolution, thus we, therefore, aimed to examine the drosophilids DNMT2 in order to verify its conservation at the physicochemical and structural levels in a functional context. We examined the twenty-six DNMT2 models generated by molecular modelling and five crystallographic structures deposited in the Protein Data Bank (PDB) using different approaches. Our results showed that despite sequence and structural similarity between species close related, we found outstanding differences when they are analyzed in the context of surface distribution of electrostatic properties. The differences found in the electrostatic potentials may be linked with different affinities and processivity of DNMT2 for its different substrates (DNA, RNA or tRNA) and even for interactions with other proteins involved in the epigenetic mechanisms.
... The DNMT3 family also includes DNMT3-Like protein (DNMT3L) that has not been shown to possess methyltransferase activity (Bourc'his, Xu, Lin, Bollman, & Bestor, 2001) but instead increases the ability of DNMT3A and DNMT3B to bind to methyl groups (Bird, 2002;Jin et al., 2011). Organisms that contain members of the DNMT1 and DNMT3 families also express DNMT2, which displays weak DNMT activity (Okano, Xie, & Li, 1998;Yoder & Bestor, 1998). ...
Chapter
Drug addiction is a chronic relapsing disorder that is characterized by compulsive, uncontrollable drug use despite negative consequences. In recent years, an increasing number of reports have provided crucial evidence that epigenetic modifications, such as DNA methylation, in mesolimbic brain areas may alter psychostimulant-induced transcriptional and behavioral changes. Accumulating data now suggest that psychostimulants (e.g., cocaine, amphetamine) can directly alter enzymes that modify DNA methylation and demethylation and gene expression that are involved in brain maladaptation. In addition, different environmental factors via the DNA methylation and demethylation may alter subject vulnerability to drug abuse. In this review, we give an overview of DNA methylation and demethylation processes. We shortly describe the connections between DNA methylation and demethylation processes with other epigenetic modifications. We also summarize the latest findings from both molecular and behavioral experiments elucidating the potential role of DNA methylation and demethylation in the pathogenesis of psychostimulant-induced drug addiction.
... DNA methylation is a process mediated by enzymes known as DNA methyltransferases (DNMT). There are a total of four mammalian DNMTs reported, which are structurally and functionally distinct from each other: DNMT1 (Bestor 1988), DNMT2 (Yoder and Bestor, 1998), DNMT3A and DNMT3B (Xie et al., 1999). DNMTase can be separated into two different categories based on their function (i) de novo methyltransferases ...
Thesis
Severe forms of malaria in humans is caused by Plasmodium falciparum and Plasmodium vivax.Malaria infection is the results of complex membrane sorting and signaling. Erythrocyte membrane lipid rafts proteins regulate membrane sorting and signaling processes and hence lipid raft proteins (Gαs and β2AR) and the interacting proteins (ADORA2A and GRK5) can influence pathogen entry and consequently the erythrocyte phase of the infection that is crucial to the pathogenesis of malaria. It is also known that the entry of malaria parasite in patients induces the synthesis of inflammatory cytokines such as TNF-a, IL-1, IL-6 and IL-10 and altered gene expression known to be regulated by epigenetic mechanisms. We demonstrated that malaria susceptibility or its severity may be influenced by the SNPs in GNAS, ADRB2, ADORA2A, GRK5and ABCB1genes; and epigenetic changes at the critical CpG sites in the promoter region of ABCB1and ADRB2genes. We also showed that global DNA methylation variants could discriminate the malaria phenotypes. Our study provides evidence for the proposed role of host genes mediated mechanisms in the etiology of malaria susceptibility. For the effective control of malaria, the development of sensitive, accurate and rapid assay is essential. We developed the single-step amplification and non-amplification based assays with better sensitivity than existing assays. Thesis link: http://hdl.handle.net/10603/148259
... DNA methylation at CpG dinucleotides (mCG) has historically been associated with gene repression; however, recent advances have revealed a complex role for DNA methylation in regards to its dynamic turnover, cell type specific distribution patterns and effect on transcriptional regulation (Suzuki and Bird, 2008). DNA is methylated by the de novo methyltransferases DNMT3A and DNMT3B (Okano et al., 1999), in association with DNMT3L (Bourc'his et al., 2001), and the mark is maintained through cell division by the maintenance methyltransferase DNMT1 (Yoder and Bestor, 1998). Passive demethylation occurs in the absence of DNMT activity as cells divide without maintenance methylation (Chen et al., 2003). ...
Article
Methylation of cytosine is an epigenetic mark essential for many cellular and developmental processes. How methylation is interpreted into transcriptional regulation is not fully understood, but previous studies have found that this process involves the methyl-CpG binding domain (MBD) family of proteins. Three MBD proteins, MeCP2, MBD1 and MBD2, specifically bind methylated cytosines and recruit different co-repressor complexes to regulate transcription and chromatin states. Genetic studies also linked MeCP2 and MBD1 to neurodevelopmental disorders in humans and mice. However, a role for MBD2 in the brain has not been described. In this work, we characterized the phenotypes of mice lacking MBD2. We found that, unlike MeCP2 and MBD1, Mbd2 null mice behave similarly to wildtype littermates, with the exception of mildly altered nesting and locomotor activity and reduced body weight. To investigate the underlying cause of different functional requirements for the MBDs, we generated knockin mice in which endogenous MBD2 and MBD1 are biotin-tagged. We systematically compared the spatiotemporal expression patterns of the MBDs and found that MeCP2, MBD1 and MBD3 are primarily expressed in the brain. In contrast, MBD2 is widely expressed throughout the body at young and adult ages. In addition, the expression of MBD2 is upregulated in adult spleen and small intestine compared to younger ages, while MBD1 and MBD3 are only enriched at early ages in the brain. We also determined that MBD2 interacts with the NuRD complex ubiquitously across tissues. We conclude that MBD2 is likely dispensable for brain function and instead may mediate NuRDrelated functions primarily in peripheral tissues. Our study provides novel genetic tools and reveals new directions to investigate MBD2 functions in vivo.
... DNA methylation mostly occurs at CpG dinucleotides through adding methyl groups and converting cytosine to 5-methylcytosine by DNA methyltransferases (DNMTs) [2,3]. DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L are C(5)-cytosine-specific DNA methyltransferases [4][5][6]. A research has shown that promoters with more 5-methylcytosines have lower transcriptional activity [7]. ...
Article
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Background Oomycetes are a group of fungus-like eukaryotes with diverse microorganisms living in marine, freshwater and terrestrial environments. Many of them are important pathogens of plants and animals, causing severe economic losses. Based on previous study, gene expression in eukaryotic cells is regulated by epigenetic mechanisms such as DNA methylation and histone modification. However, little is known about epigenetic mechanisms of oomycetes. ResultsIn this study, we investigated the candidate genes in regulating histone acetylation in oomycetes genomes through bioinformatics approaches and identified a group of diverse histone acetyltransferases (HATs) and histone deacetylases (HDACs), along with three putative novel HATs. Phylogenetic analyses suggested that most of these oomycetes HATs and HDACs derived from distinct evolutionary ancestors. Phylogenetic based analysis revealed the complex and distinct patterns of duplications and losses of HATs and HDACs in oomycetes. Moreover, gene expression analysis unveiled the specific expression patterns of the 33 HATs and 11 HDACs of Phytophthora infestans during the stages of development, infection and stress response. Conclusions In this study, we reveal the structure, diversity and the phylogeny of HATs and HDACs of oomycetes. By analyzing the expression data, we provide an overview of the specific biological stages of these genes involved. Our datasets provide useful inputs to help explore the epigenetic mechanisms and the relationship between genomes and phenotypes of oomycetes.
... Results from genetic studies of Dnmt1 prompted the search for more DNA methyltransferase genes. In 1998, several groups reported the identifi cation of a second putative DNA methyltransferase gene, named Dnmt2 , which encodes a protein of 391 amino acids in human or 415 amino acids in mouse (Okano et al. 1998b ;Van den Wyngaert et al. 1998 ;Yoder and Bestor 1998 ). Despite the presence of all the conserved motifs shared by known prokaryotic and eukaryotic DNA cytosine methyltransferases, Dnmt2 has no detectable DNA methyltransferase activity in standard in vitro assays. ...
Chapter
Full-text available
Cytosine methylation at the C5-position, generating 5-methylcytosine (5mC), is a DNA modification found in many eukaryotic organisms, including fungi, plants, invertebrates, and vertebrates, albeit its levels vary greatly in different organisms. In mammals, cytosine methylation occurs predominantly in the context of CpG dinucleotides, with the majority (60–80 %) of CpG sites in their genomes being methylated. DNA methylation plays crucial roles in the regulation of chromatin structure and gene expression and is essential for mammalian development. Aberrant changes in DNA methylation levels and patterns are associated with various human diseases, including cancer and developmental disorders. DNA methylation is mediated by three active DNA methyltransferases (Dnmts), namely, Dnmt1, Dnmt3a, and Dnmt3b, in mammals. Over the last two decades, genetic manipulations of these enzymes, as well as their regulators, in mice have greatly contributed to our understanding of the biological functions of DNA methylation in mammals. In this chapter, we discuss genetic studies on mammalian Dnmts, focusing on their roles in embryogenesis, cellular differentiation, genomic imprinting, and X-chromosome inactivation.
... DNA methylation, one of several epigenetic modifications, is involved in many cellular processes and plays a crucial role in genomic imprinting for dynamic regulation of gene expression, in particular, gene silencing (possibly mediated by methyl-CpG binding proteins) in the processes of dosage compensation, mono-allelic expression, and transcriptional silencing of retroviruses and retrotransposons (Bourc'his and Bestor, 2004;Bestor et al., 2015). DNA methylation usually occurs in CpG dinucleotides in mammals, and methylation patterns are established and maintained by DNA methyltransferases and associated proteins through cell division (Yoder and Bestor, 1998;Okano et al., 1999;Bourc'his et al., 2001). In addition, methylation can be removed, which is dependent on the active demethylation by the ten-eleven translocation family in vertebrates (Tahiliani et al., 2009). ...
Article
DIO3 gene encoding type 3 iodothyronine deiodinase is an imprinted gene, located in the DLK1-DIO3 (delta-like 1 homolog-type 3 iodothyronine deiodinase) imprinted domain, and is potentially involved in degrading excessive amounts of thyroid hormone to protect embryogenesis. However, the underlying regulatory mechanism of the imprinted DIO3 gene expression during fetal and neonatal development in goats has not been elucidated. In this study, we explored the DNA methylation patterns of the caprine DIO3 intragenic CpG island and quantified gene expression level in six tissues from Chinese Nanjiang Yellow 3-day old kids. The expression of the DIO3 gene was determined using quantitative reverse transcription-polymerase chain reactions (qRT-PCRs), while the identification of methylation patterns was determined using bisulfite-sequencing PCRs. Modest, and non-significant (P > 0.05), methylation patterns were noted for the DIO3 CpG island methylation in the brain, heart, liver, kidney, lung, and longissimus dorsi tissues (ranging from 26.48 to 34.92%). The expression level of the DIO3 mRNA was significantly higher (P < 0.05) in the liver tissue than in the other five tissues. Pearson’s correlation analysis revealed that there was no significant relationship between methylation and gene expression (P > 0.05), which indicated that the expression of the caprine DIO3 gene was likely modified by other regulatory elements. This study identified DNA methylation and expression patterns of the DIO3 gene in goats and provided insights into further regulatory mechanisms of expression and imprinting in the DLK1-DIO3 domain.
... Once the patterns are established, they are maintained throughout cell generations by Dnmt1 (Bestor et al., 1988;Li et al., 1992). Unlike Dnmt1 and Dnmt3a/3b, Dnmt2 is a RNA methyltransferase rather than a DNA methyltransferase (Okano et al., 1998;Yoder and Bestor, 1998;Goll et al., 2006). A summary of the mouse Dnmt protein family and their domains is shown in Figure 1 and a summary of the respective knockout mice phenotypes is shown in Table 1. ...
Article
Full-text available
Cytosine base modifications in mammals underwent a recent expansion with the addition of several naturally occurring further modifications of methylcytosine in the last years. This expansion was accompanied by the identification of the respective enzymes and proteins reading and translating the different modifications into chromatin higher order organization as well as genome activity and stability, leading to the hypothesisof a cytosine code. Here, we summarize the current state-of-the-art on DNA modifications, the enzyme families setting the cytosine modifications and the protein families reading and translating the different modifications with emphasis on the mouse protein homologs. Throughout this review, we focus on functional and mechanistic studies performed on mammalian cells, corresponding mouse models and associated human diseases.
... The group of Dnmt2 orthologs presents an unusual evolutionary history. Initially, Dnmt2 enzymes were assigned as putative DNA methyltransferase due to their sequence similarity to other mammalian and bacterial 5mC DNA methyltransferases 7,8 . The Dnmt2 enzymes are highly conserved and are present in almost all eukaryotic organisms 9, 10 and a handful of bacterial species, which most likely acquired them via horizontal gene transfer from a eukaryote 11 . ...
Article
Full-text available
A group of homologous nucleic acid modification enzymes called Dnmt2, Trdmt1, Pmt1, DnmA, and Ehmet in different model organisms catalyze the transfer of a methyl group from the cofactor S-adenosyl-methionine (SAM) to the carbon-5 of cytosine residues. Originally considered as DNA MTases, these enzymes were shown to be tRNA methyltransferases about a decade ago. Between the presumed involvement in DNA modification-related epigenetics, and the recent foray into the RNA modification field, significant progress has characterized Dnmt2-related research. Here, we review this progress in its diverse facets including molecular evolution, structural biology, biochemistry, chemical biology, cell biology and epigenetics.
... The DNA methylation process has been hypothesized to be evolved to silence evolutionarily accumulated selfish genes (Yoder and Bestor 1998) and transposable elements (Zemach et al. 2010), and to regulate the transcription of genes (Razin and Riggs 1980). In addition, it has been posited to regulate gene splicing (Shukla et al. 2011), X-inactivation, and parental genomic imprinting (Li et al. 1993). ...
... The first mammalian DNMT2 identified using a cDNA database search (Yoder and Bestor, 1998) shares sequence similarity to pmt1 + of Schizosaccharomyces pombe, an organism that is believed not to methylate its DNA (Wilkinson et al., 1995). A DNMT2 knockout in mouse is a homologue of pmt1 + and had no obvious effect on genomic methylation patterns in embryonic stem cells or on newly integrated retroviral DNA (Okano et al., 1998b), indicating that DNMT2 is not required for de novo methylation or maintenance of methylation patterns. ...
Thesis
DNA methylation is a common epigenetic mark that affects gene regulation, genomic stability and chromatin structure. In mammals, methylation is mainly found in the CpG dinucleotides. The CpG methylation signals can be recognised by the Methyl-CpG-Binding Protein (MBP) family which includes MeCP2, MBD1, MBD2, MBD3, MBD4 and Kiaso. MeCP2 and MBD1-4 (except mammalian MBD3) recognise methyl-CpG via their MBD domain whereas Kaiso interprets methylation through its Zn finger DNA binding domain. The TRD domains of MeCP2, MBD1 and MBD2 have been reported to recruit transcriptional co-repressors to the methylated DNA. A thymine DNA glycosylase domain is located at the C-terminal region of MBD4. This study concerns the molecular details of the methyl-CpG recognition by the MBD domain of MeCP2. To achieve this, the MeCP2 MBD domain has been expressed, purified and co-crystallised with a 20 bp DNA fragment from the BDNF promoter. The DNA-protein cocrystal diffracted X-rays to a maximum resolution of 2.5Å using synchrotron sources. It belongs to space group C2 with unit cell dimensions: a = 79.71Å, b = 53.60Å, c = 65.73Å, and β = 132.1°. The X-ray structure of the MeCP2 MBD-DNA complex was solved using the SAD method. Structural analyses of the refined X-ray structure reveal that the methyl groups of the DNA make contact with a predominantly hydrophilic surface that includes tightly bound water molecules. From a structure of the MBD domain in MBD1, established by NMR, the binding specificity of the MBD domain had been thought to depend on hydrophobic interactions between the cytosine methyl groups and a hydrophobic patch within the MBD domain. The findings of this study suggest that MeCP2 recognises the hydration pattern of the major groove of methylated DNA rather than cytosine methylation per se. The X-ray structure also identifies a unique role of T158 and R106, the sites of the two most frequent Rett missense mutations. Both residues stabilise the tandem Asx-ST motif at the C-terminal region of MBD domain. Disruption of this tandem motif destabilises the DNA-protein interaction. The BDNF sequence in this study contains an AT run which displays unique properties of AT tract DNA. Previously, mutation of the AT run has been reported to decrease MeCP2 binding specificity. This study however demonstrated that a significant reduction can only be observed when both AT runs close to the methyl-CpG have been mutated. The X-ray structure of the MeCP2 MBD-DNA complex in this study rationalises the effects of the most common Rett mutations and provides a general model for methylated DNA binding that is dependent on structured water molecules.
... Members of the DNA methyltransferase enzyme (DNMT) family establish and maintain DNA methylation. The first member of the family identified was DNMT1, closely followed by DNMT2, DNMT3A, DNMT3B and DNMT3L (Bestor et al, 1988;Gruenbaum et al, 1982;Okano et al, 1998b;Van den Wyngaert et al, 1998;Yoder & Bestor, 1998) (Figure 1.1B). With the exception of DNMT3L, all these enzymes, contain C-terminal conserved catalytic domains which have been shown to be essential for their activity as methyltransferases (Cheng, 1995;Cheng & Blumenthal, 2008;Jeltsch, 2002;Jurkowska et al, 2011). ...
Thesis
Although the many cells within a mammal share the same DNA sequence, their gene expression programmes are highly heterogeneous, and their functions correspondingly diverse. This heterogeneity within an isogenic population of cells arises in part from the ability of each cell to respond to its immediate surroundings via a network of signalling pathways. However, this is not sufficient to explain many of the transcriptional and functional differences between cells, particularly those that are more stable, or, indeed, differences in expression between parental alleles within the same cell. This conundrum lead to the emergence of the field of epigenetics - the study of heritable changes in gene expression independent of DNA sequence. Such changes are dependent on “epigenetic modifications”, of which DNA methylation is one of the best characterised, and is associated with gene silencing. The establishment of correct DNA methylation patterns is particularly important during early development, leading to cell type specific and parental allele specific gene regulation. Besides DNA methyltransferases, various other proteins have recently been implicated in DNA methylation. The absence of these proteins leads to defects in DNA methylation and development that can be even more severe than those in DNA methyltransferase knockouts themselves. In this study I focus on three such accessory proteins: LSH (a putative chromatin remodelling ATPase), G9a (a histone lysine methyltransferase) and SmcHD1 (a structural maintenance of chromosomes protein). To compare DNA methylation between WT cells and cells knocked out for each of these proteins, I used whole genome methylated DNA affinity purification and subsequent hybridization to promoter microarrays. This enabled me to compare the requirement for each protein in DNA methylation at specific genomic regions. The absence of LSH in mouse embryonic fibroblasts (MEFs) resulted in the loss of DNA methylation at 20% of usually methylated promoters, and the misregulation of associated protein coding genes. This revealed a requirement for LSH in the establishment of DNA methylation at promoters normally methylated during pre-implantation as well as post-implantation development. Secondly, I identified hypomethylation at 26% of normally methylated promoters in G9a-/- compared to WT ES cells. Strikingly, this revealed that G9a is required for maintenance of DNA methylation at maternal as well as paternal imprinting control regions (ICRs). This is accompanied by expression defects of imprinted genes regulated by these ICRs. Finally, in collaboration with the Brockdorff lab at the University of Oxford I identified a role for SmcHD1 in establishing DNA methylation at promoters on the X chromosome normally methylated slowly during X chromosome inactivation. Interestingly, SmcHD1 was also required for DNA methylation at autosomal gene promoters, contrary to previous reports that it is mainly involved in X chromosome methylation. I conclude that different accessory proteins are required to facilitate correct DNA methylation and gene repression at distinct regions of the genome, as well as at different times during development. This function of accessory proteins may be in part dependent on the prior establishment of specific chromatin signatures and developmental signals, together comprising a precisely regulated system to establish and maintain appropriate DNA methylation throughout development.
... AgNPs also induced DNMT2 protein expression, which may be considered as a part of stress response. The role of DNMT2 in both DNA methylation and RNA methylation has been proposed [64,69,70]. DNA methyltransferase activity of human DNMT2 and Drosophila Dnmt2 has been reported [71,72]. ...
Article
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It is widely accepted that silver nanoparticles (AgNPs) are toxic to biological systems. However, little is known about their actions at molecular level and the cytophysiological effects after AgNP removal. As nanoparticles are suggested a promising tool to transport drugs to the brain for use in neurological conditions, we used HT22 mouse hippocampal neuronal cells as a model to study AgNP-mediated effects after their removal from the cell culture medium. We selected a relatively low concentration of AgNPs, 5 μg/ml, treated the cells for 48 h, and evaluated AgNP-induced cytophysiological effects after 96 h of AgNP removal. AgNP removal did not result in cytotoxicity. In contrast, AgNPs modulated HT22 cell cycle and proliferation and induced oxidative stress and 53BP1 recruitment, which were accompanied by elevated levels of p53 and p21. AgNP-associated diminution in lamin B1 pools did not significantly affect the structure of the nucleus. No disruption in F-actin dynamics was observed upon AgNP treatment. Moreover, we showed for the first time that AgNPs stimulated changes in DNA methylation: the augmentation in 5-methylcytosine (5-mC) and DNMT1, DNMT2, DNMT3a, and DNMT3b levels were observed. The upregulation of DNMT2 may be a part of cellular stress response to AgNP treatment. Taken together, AgNP removal resulted in p53/p21-mediated inhibition of cell proliferation, oxidant-based DNA damage response, and changes in DNA methylation patterns, which suggests that more attention should be paid to the possible outcomes in individuals exposed to nano-sized biomaterials.
Chapter
Cytosine methylation at the C5-position—generating 5-methylcytosine (5mC)—is a DNA modification found in many eukaryotic organisms, including fungi, plants, invertebrates, and vertebrates, albeit its levels vary greatly in different organisms. In mammals, cytosine methylation occurs predominantly in the context of CpG dinucleotides, with the majority (60–80%) of CpG sites in their genomes being methylated. DNA methylation plays crucial roles in the regulation of chromatin structure and gene expression and is essential for mammalian development. Aberrant changes in DNA methylation and genetic alterations in enzymes and regulators involved in DNA methylation are associated with various human diseases, including cancer and developmental disorders. In mammals, DNA methylation is mediated by two families of DNA methyltransferases (Dnmts), namely Dnmt1 and Dnmt3 proteins. Over the last three decades, genetic manipulations of these enzymes, as well as their regulators, in mice have greatly contributed to our understanding of the biological functions of DNA methylation in mammals. In this chapter, we discuss genetic studies on mammalian Dnmts, focusing on their roles in embryogenesis, cellular differentiation, genomic imprinting, and human diseases.KeywordsDNA methylationDnmt1Dnmt3aDnmt3bDnmt3cDnmt3LUhrf1Genomic imprinting
Chapter
Nucleic acid methylation is a fundamental epigenetic mechanism that impinges upon several cellular attributes, including metabolism and energy production. The dysregulation of deoxyribonucleic acid (DNA)/ribonucleic acid (RNA) methylation can lead to metabolic rewiring in the cell, which in turn facilitates tumor development. Here, we review the current knowledge on the interplay between DNA/RNA methylation and metabolic programs in cancer cells. We also discuss the mechanistic role of these pathways in tumor development and progression.KeywordsDNA methylationRNA methylationCpG5mCm6AWarburg effectTumor metabolismCancer
Article
Selective manipulation of the epitranscriptome could be beneficial for the treatment of cancer and also broaden the understanding of epigenetic inheritance. Inhibitors of the tRNA methyltransferase DNMT2, the enzyme catalyzing the S-adenosylmethionine-dependent methylation of cytidine 38 to 5-methylcytidine, were designed, synthesized, and analyzed for their enzyme-binding and -inhibiting properties. For rapid screening of potential DNMT2 binders, a microscale thermophoresis assay was established. Besides the natural inhibitors S-adenosyl-l-homocysteine (SAH) and sinefungin (SFG), we identified new synthetic inhibitors based on the structure of N-adenosyl-2,4-diaminobutyric acid (Dab). Structure-activity relationship studies revealed the amino acid side chain and a Y-shaped substitution pattern at the 4-position of Dab as crucial for DNMT2 inhibition. The most potent inhibitors are alkyne-substituted derivatives, exhibiting similar binding and inhibitory potencies as the natural compounds SAH and SFG. CaCo-2 assays revealed that poor membrane permeabilities of the acids and rapid hydrolysis of an ethylester prodrug might be the reasons for the insufficient activity in cellulo.
Article
In 1925, 5-methylcytosine was first reported in bacteria. However, its biological importance was not intuitive for several decades. After this initial lag, the ubiquitous presence of this methylated base emerged across all domains of life and revealed a range of essential biological functions. Today, we are armed with the knowledge of the key factors that establish, maintain, and remove DNA methylation and have access to a staggering and rapidly growing number of base-resolution methylation maps. Despite this, several fundamental details about the precise role and interpretation of DNA methylation patterns remain under investigation. Here, we review the field of DNA methylation from its beginning to present day, with an emphasis on findings in mammalian systems, and point the reader to select experiments that form the foundation of this field.
Thesis
Les cellules stromales lymphoïdes (CSL) tiennent un rôle central dans la physiologie des organes lymphoïde secondaire. Elles sont nécessaires à la migration et la régulation des cellules immunitaires via la sécrétion de chimiokines, telles que CCL19, CCL21 ou CXCL13, et l’expression de protéines d’adhésions, tel que PDPN, ICAM-1 et VCAM-1. La différenciation et l’activation de ces cellules dépendent de deux facteurs indispensables : le TNF alpha et la lymphotoxine alpha1beta2. Cependant, les précurseurs mésenchymateux dont elles dérivent sont à ce jour peu décrits. De manière intéressante, ces précurseurs mésenchymateux peuvent se différencier en d’autres types cellulaires sous le contrôle de mécanismes épigénétiques, soulevant l’intérêt d’étudier l’intervention de tels mécanismes lors la différenciation stromale lymphoïde. Dans ce travail de recherche, nous mettons en évidence la surexpression d’un facteur épigénétique, KDM6B, lors de la polarisation in vitro d’immunofibroblastes et lors de la formation de structure lymphoïde tertiaire chez la souris, en lien avec l’acquisition d’un phénotype de type stroma lymphoïde. De plus, l’expression de KDM6B est associée à une modification précoce de la marque histone H3K27ac, au cours de cette polarisation, au niveau des régions régulatrices de gènes immunorégulateurs tels que ICAM-1, PDPN, CCL2 ou encore CCL5. L’inhibition de ce facteur bloque l’acquisition du phénotype lymphoïde et limite ainsi les propriétés fonctionnelles de ces cellules, telles que le recrutement de monocytes. Ces résultats mettent ainsi en avant KDM6B comme une cible thérapeutique potentielle afin de bloquer l’apparition d’un stroma lymphoïde de soutien lors de pathologie auto-immune ou lors de cancers.
Thesis
At the root of the hematopoietic hierarchy reside the Janus-faced hematopoietic stem cells (HSC), capable of both self-renewal and differentiation. Aging impairs HSC function, leading to increased self-renewal, reduced homing ability and a myeloid differentiation bias. In addition, hematopoietic cells acquire somatic mutations as they age, frequently affecting epigenetic modifier genes. In this dissertation work, I provide a comprehensive characterization of epigenomic changes during normal human HSC aging and demonstrate that aged HSCs undergo widespread reduction in H3K27ac, H3K4me1 and H3K4me3, with little change in H3K27me3. Age-associated loss of enrichment of the activating histone marks H3K27ac and H3K4me3 was particularly prominent at active enhancers and bivalent promoters, respectively. Functional annotation of enhancers lost with age suggests that enhancer deregulation may contribute to HSC myeloid bias and the immune impairments observed in older individuals. Focal changes in DNA methylation were also observed with age, affecting WNT and cadherin associated pathways, and at regions that may predispose to leukemogenesis. DNA 5-hydroxymethylation displayed age-related gains, targeting GATA and KLF binding sites. Concurrent with these epigenetic changes were transcriptional downregulation and mis-splicing of epigenetic modifiers, spliceosome components, transcription factors, including many in the KLF family, and LMNA, which is mutated in Hutchinson-Gilford progeria syndrome. Together, these results establish that multiple levels of epigenetic deregulation with age converge on key hematopoietic regulatory genes and pathways contributing to aged HSC dysfunction.
Article
Human TRDMT1 is a transfer RNA (tRNA) methyltransferase for cytosine-5 methylation and has been suggested to be involved in the regulation of numerous developmental processes. However, little is known about the molecular mechanisms or their biological significance. In this study, we investigated the effects of CRISPR-based TRDMT1 knockdown on phenotypes, mRNA m5C modifications and gene expression changes in HEK293 cells. We found that knockdown of TRDMT1 significantly inhibited cell proliferation and migration but had no effect on clonogenic potential. The inhibitory effects could be attenuated by re-expression of TRDMT1 in HEK293 cells. RNA sequencing (RNA-Seq) and RNA bisulfite sequencing (RNA-BisSeq) were performed in TRDMT1 knockdown and wild-type HEK293 cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that the differentially expressed genes were associated with the cell cycle, RNA transport, and RNA degradation and were enriched in cancer and Notch signaling pathways. We also found that TRDMT1 knockdown could change mRNA methylation levels. For the first time, these findings clarify the role of TRDMT1 in regulating mRNA methylation and inhibiting the proliferation and migration of HEK293 cells. These results provide new insights into a new function of TRDMT1 and elucidate the molecular mechanisms of aberrant RNA m5C during tumorigenesis.
Article
Trichloroacetic acid (TCA) is one of the major metabolites of trichloroethylene (TCE) as the significant factor of environmental and occupational pollution. TCA has been shown to induce a series of epigenetic mutation in mouse liver. However, the epigenetic cytotoxicity of TCA is still in infancy. In this study, we explored the cellular biological characteristics, the genome DNA methylation status and the expression profile of DNA methyltransferases in human hepatic L-02 cells treated with TCA with certain time and dose effects. The cell cycle measured by flow cytometry revealed an increasing S + G2 (M) phase of TCA (0.9 mM 24 h, 48 h and 72 h) treated cells after a recovery day, and sub-G1 phase was not appeared. The levels of 5 -mC were decreased in TCA (0.9 mM 24 h and 72 h) treated cells by 5-mC immunolocalization process and HPCE (decreased from 27.2% to 50.1% respectively). Meanwhile, the mCpG% in normal L-02 cells and TCA (0.9 mM 48 h) treated cells was 79.6% ± 6.5% and 50.8% ± 3.8%, respectively (P < 0.05). It also revealed that treatment of L-02 cells with TCA induced decreased in DNMT1 and DNMT3a mRNA and protein levels with a time-dependent manner and a dose-response relationship, while DNMT3b had no obvious change. These results establish a link between DNA methyltransferases and Genome DNA hypomethylation, which is associated with TCA exposure.
Chapter
The two major biochemical pathways of epigenetic gene regulation are DNA methylation and posttranslational modifications of amino acid side chains in histone proteins, around which the DNA is wrapped. Multiple enzymes are involved that deposit these modifications and remove them as well as protein domains, which can recognize them and recruit further effector proteins. DNA methylation refers to the enzymatic addition of a methyl group directly to nucleotide bases. In the case of DNA methylation, the biochemical modification is occurring directly on the DNA, but the base pairing of cytosine to the guanine base on the complementary DNA strand is not altered upon methylation, and, thus, the genetic code is preserved. DNA nucleotide methyltransferases (DNMT1) was the first cytosine methyltrans‐ferase identified and is closely associated with the DNA replication process. It transfers the DNA methylation patterns from the parent strand to the newly synthesized strand.
Article
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The DNA methyltransferase 2 (DNMT2) protein is the most conserved member of the DNA methyltransferase family. Nevertheless, its substrate specificity is still controversial and elusive. The genomic role and determinants of DNA methylation are poorly understood in invertebrates, and several mechanisms and associations are suggested. In Drosophila, the only known DNMT gene is Dnmt2. Here we present our findings from a wide search for Dnmt2 homologs in 68 species of Drosophilidae. We investigated its molecular evolution, and in our phylogenetic analyses the main clades of Drosophilidae species were recovered. We tested whether the Dnmt2 has evolved neutrally or under positive selection along the subgenera Drosophila and Sophophora and investigated positive selection in relation to several physicochemical properties. Despite of a major selective constraint on Dnmt2, we detected six sites under positive selection. Regarding the DNMT2 protein, 12 sites under positive-destabilizing selection were found, which suggests a selection that favors structural and functional shifts in the protein. The search for new potential protein partners with DNMT2 revealed 15 proteins with high evolutionary rate covariation (ERC), indicating a plurality of DNMT2 functions in different pathways. These events might represent signs of molecular adaptation, with molecular peculiarities arising from the diversity of evolutionary histories experienced by drosophilids.
Article
An emerging paradigm shift for disease diagnosis is to rely on molecular characterization beyond traditional clinical and symptom-based examinations. Although genetic alterations and transcription signature were first introduced as potential biomarkers, clinical implementations of these markers are limited due to low reproducibility and accuracy. Instead, epigenetic changes are considered as an alternative approach to disease diagnosis. Complex epigenetic regulation is required for normal biological functions and it has been shown that distinctive epigenetic disruptions could contribute to disease pathogenesis. Disease-specific epigenetic changes, especially DNA methylation, have been observed, suggesting its potential as disease biomarkers for diagnosis. In addition to specificity, the feasibility of detecting disease-associated methylation marks in the biological specimens collected non-invasively, such as blood samples, has driven the clinical studies to validate disease-specific DNA methylation changes as a diagnostic biomarker. Here, we highlight the advantages of DNA methylation signature for diagnosis in different diseases and discuss the statistical and technical challenges to be overcome before clinical implementation.
Article
Epigenetics oftenly described as the heritable changes in gene expression independent of changes in DNA sequence. Various environmental factors such as nutrition-dietary components, lifestyle, exercise, physical activity, toxins, and other contributing factors remodel the genome either in a constructive or detrimental way. Since epigenetic changes are reversible and nutrition is one of the many epigenetic regulators that modify gene expression without changing the DNA sequence, dietary nutrients and bioactive food components contribute to epigenetic phenomena either by directly suppressing DNA methylation or histone catalyzing enzymes or by changing the availability of substrates required for enzymatic reactions. Diets that contain catechol-dominant polyphenols are reported to suppress enzyme activity and activate epigenetically silenced genes. Furthermore, several dietary nutrients play a crucial role in one-carbon metabolism including folate, cobalamin, riboflavin, pyridoxine, and methionine by directly affecting S-adenosyl-l-methionine. Soy polyphenols block DNA methyltransferases and histone deacetylases to reverse aberrant CpG island methylation. Organosulfur rich compounds such as the sulforaphane found in broccoli appear to normalize DNA methylation and activate miR-140 expression, which represses SOX9 and ALDH1 and decreases tumor growth. The purpose of this short communication is to overview the epigenetic regulatory mechanisms of diet and other environmental factors. We discuss the epigenetic contributions of dietary components with a particular focus on nutritional polyphenols and flavonoids as epigenetic mediators that modify epigenetic tags and control gene expression. These mechanisms provide new insights to better understand the influence of dietary nutrients on epigenetic modifications and gene expression.
Article
The DNA methyltransferase (DNMT) family comprises a conserved set of DNA-modifying enzymes that have a central role in epigenetic gene regulation. Recent studies have shown that the functions of the canonical DNMT enzymes — DNMT1, DNMT3A and DNMT3B — go beyond their traditional roles of establishing and maintaining DNA methylation patterns. This Review analyses how molecular interactions and changes in gene copy numbers modulate the activity of DNMTs in diverse gene regulatory functions, including transcriptional silencing, transcriptional activation and post-transcriptional regulation by DNMT2-dependent tRNA methylation. This mechanistic diversity enables the DNMT family to function as a versatile toolkit for epigenetic regulation.
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In mammals, cytosine in CpG sequences in genomic DNA is often methylated at the 5th position. DNA methylation acts as a regulator of gene expression, and is crucial for development, especially in higher eukaryotes. Three DNA (cytosine-5)-methyltransferases, Dnmt1, Dnmt3a, and Dnmt3b, have been identified. Dnmt3a and Dnmt3b are mainly responsible for establishing DNA methylation patterns in the genome. Factors interacting with Dnmt3a or Dnmt3b, histone modifications, and their timing of expression act as determinants for sites to be methylated. Once DNA methylation patterns are established, the patterns are maintained by Dnmt1, which favors methylation of hemi-methylated DNA (where only one DNA strand is methylated) after DNA replication and repair. For maintenance DNA methylation, interacting factors and histone modifications are also necessary in vivo. In this chapter, the function of DNA methylation and the molecular mechanisms to establish and maintain DNA methylation are described.
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Enzymes of the Dnmt2 family of methyltransferases have yielded a number of unexpected discoveries. The first surprise came more than ten years ago when it was realized that, rather than being DNA methyltransferases, Dnmt2 enzymes actually are transfer RNA (tRNA) methyltransferases for cytosine-5 methylation, foremost C38 (m5C38) of tRNAAsp. The second unanticipated finding was our recent discovery of a nutritional regulation of Dnmt2 in the fission yeast Schizosaccharomyces pombe. Significantly, the presence of the nucleotide queuosine in tRNAAsp strongly stimulates Dnmt2 activity both in vivo and in vitro in S. pombe. Queuine, the respective base, is a hypermodified guanine analog that is synthesized from guanosine-5’-triphosphate (GTP) by bacteria. Interestingly, most eukaryotes have queuosine in their tRNA. However, they cannot synthesize it themselves, but rather salvage it from food or from gut microbes. The queuine obtained from these sources comes from the breakdown of tRNAs, where the queuine ultimately was synthesized by bacteria. Queuine thus has been termed a micronutrient. This review summarizes the current knowledge of Dnmt2 methylation and queuosine modification with respect to translation as well as the organismal consequences of the absence of these modifications. Models for the functional cooperation between these modifications and its wider implications are discussed.
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In mammals, more than 70 % of the CpG sequences in the genome are methylated at the 5th position of cytosine bases. DNA methylation acts as a regulator of gene expression and is crucial for development, especially in higher eukaryotes. In mammals, three DNA (cytosine-5-)-methyltransferases, Dnmt1, Dnmt3a, and Dnmt3b, have been identified. Dnmt3a and Dnmt3b are mainly responsible for establishing DNA methylation patterns in the genome. For the establishment of DNA methylation patterns, interacting or associating factors that take Dnmt3a or Dnmt3b to the site of methylation, the timing of expression, and the substrate DNA with higher ordered structures (chromatin states) are the determinants. Dnmt1 favors methylation of hemi-methylated DNA, which appears just after replication or repair, and thus is responsible for maintaining the methylation patterns during replication and after repair. Recently, it was found that Uhrf1 and histone ubiquitylation are necessary factors for maintenance DNA methylation in vivo. In this chapter, the establishment and maintenance of DNA methylation by Dnmt3a, Dnmt3b, and Dnmt1 are described.
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Dnmt2 is the most strongly conserved cytosine DNA methyltransferase in eukaryotes. It has been found in all organisms possessing methyltransferases of the Dnmt1 and Dnmt3 families, whereas in many others Dnmt2 is the sole cytosine DNA methyltransferase. The Dnmt2 molecule contains all conserved motifs of cytosine DNA methyltransferases. It forms 3D complexes with DNA very similar to those of bacterial DNA methyltransferases and performs cytosine methylation by a catalytic mechanism common to all cytosine DNA methyltransferases. Catalytic activity of the purified Dnmt2 with DNA substrates is very low and could hardly be detected in direct biochemical assays. Dnmt2 is the sole cytosine DNA methyltransferase in Drosophila and other dipteran insects. Its overexpression as a transgene leads to DNA hypermethylation in all sequence contexts and to an extended life span. On the contrary, a null-mutation of the Dnmt2 gene leads to a diminished life span, though no evident anomalies in development are observed. Dnmt2 is also the sole cytosine DNA methyltransferase in several protists. Similar to Drosophila these protists have a very low level of DNA methylation. Some limited genome compartments, such as transposable sequences, are probably the methylation targets in these organisms. Dnmt2 does not participate in genome methylation in mammals, but seems to be an RNA methyltransferase modifying the 38th cytosine residue in anticodon loop of certain tRNAs. This modification enhances stability of tRNAs, especially in stressful conditions. Dnmt2 is the only enzyme known to perform RNA methylation by a catalytic mechanism characteristic of DNA methyltransferases. The Dnmt2 activity has been shown in mice to be necessary for paramutation establishment, though the precise mechanisms of its participation in this form of epigenetic heredity are unknown. It seems likely, that either of the two Dnmt2 activities could become a predominant one during the evolution of different species. The high level of the Dnmt2 evolutionary conservation proves its activity to have a significant adaptive value in natural environment.
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It has been known for a long time that tumor development coincides with changes in DNA methylation. Epigenetic mechanisms such as DNA methylation and specialized chromatin structures are capable of stably modulating gene expression over many cell generations. Thus, an epigenetic mutation can have the same effect like a classical, genetic mutation, i.e., loss- or gain-of-function of any given gene. Sensitive methods for the detection of epigenetic mutations have been developed in the past years and have been used to demonstrate a role of epigenetic mechanisms in tumorigenesis. Recent progress in our understanding of these mechanisms also allowed for the development of epigenetic cancer therapies. These concepts have a high potential for increasing the efficiency of conventional chemotherapy.
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There is increasing evidence that epigenetic modifications play a crucial role in determining the dynamic structure and function of chromatin and that the epigenetic changes at the chromatin level control the ordered flow of decisions in cells. Postsynthetic modifications of both DNA and chromatin proteins are involved in achieving a regulated access to the underlying DNA to perform DNA replication, initiate appropriate transcriptional programs, and carry out the repair of DNA damage. DNA methylation is a specific postsynthetic, enzymatic modification of DNA that, in eukaryotic cells, appears to play an important role in the epigenetic modulation of gene expression. The effect of DNA methylation is exerted through direct or indirect changes in a chromatin structure. The connection between epigenetic factors and human disease has been unraveled. The new understanding has given rise to numerous new questions that need to be addressed experimentally. The complexity of the phenomena requires the concerted action of many laboratories worldwide and the application of new methodologies.
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Wendell W Weber is professor Emeritus (active) in Pharmacology at the University of Michigan. His teaching and research interests have centered on pharmocogenetics for more than 40 years. He has concentrated mainly on hereditary traits affecting human drug response and cancer susceptibility, particularly the metabolic polymorphisms in humans and experimental animal models. He is the author or coauthor of more than 175 research papers and book chapters, and has written two books on pharmacogenetics: The Acetylator Genes and Drug Response (Oxford University Press, 1987) and Pharmacogenetics (Oxford University Press, 1997).
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DNA methylation is required for normal development and is frequently deregulated in cancer. The highly coordinated pattern of the methylation marks on the DNA is established and maintained by DNA methyltransferases (DNMTs). Much of what is known today about the functional role of DNA methylation came from the analyses of mice in which DNMT activity had been pharmacologically or genetically decreased. These approaches could clearly demonstrate a causal role for DNA methylation in cancer development and very recently uncovered its unique role in the coordination of normal and malignant stem cell potential. This chapter introduces the mouse models currently available to study DNA methylation functions in mammals and discusses the recent advances in our understanding of how this epigenetic process drives stem cell functions in development, tissue homeostasis, and cancer. © 2014 Springer-Verlag Berlin Heidelberg. All rights are reserved.
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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.
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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.
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A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
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A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
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A 0.5 kb fragment of chicken DNA methyltransferase cDNA was PCR-amplified using a set of degenerate primers. A clone harboring a 5 kb insert was isolated from a cDNA library by screening with the PCR-amplified cDNA fragment as a probe. The elucidated nucleotide sequence gave a 4,614 nucleotide open reading frame, and the predicted protein was highly homologous to the mouse and human DNA methyltransferases, especially in the amino acid sequence of the catalytic domain in the carboxyl-terminal region. The cysteine-rich region and Lys-Gly repeat first found in the mouse sequence were also conserved in chicken. However, about 250 amino acid residues in the amino-terminal portion of chicken DNA methyltransferase diverged from the amino-terminus of the mouse or human sequence. Northern blot analysis showed that the message of chicken DNA methyltransferase was expressed at high levels in the testis, in the lung and in Marek's virus-transformed chicken T-lymphoma cells. Expression of the chicken DNA methyltransferase in COS1 cells demonstrated that the enzyme is a so-called maintenance-type methylase. When poly(dG-dC)-poly(dG-dC) was used as the methyl acceptor, to provide a measure of de novo methylase activity, the Km value for S-adenosyl L-methionine was about 5 microM, which was 10 times higher than that when poly(dI-dC)-poly(dI-dC) was used. The affinity of DNA methyltransferase for S-adenosyl L-methionine in catalyzing de novo-type methylation activity was lower than that in catalyzing maintenance-type activity, though it was still high enough for the enzyme to work as a de novo-type methylase under physiological conditions.
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DNA (cytosine-5)-methyltransferases (EC 2.1.1.37) maintain patterns of methylated cytosine residues in the mammalian genome; faithful maintenance of methylation patterns is required for normal development of mice, and aberrant methylation patterns are associated with certain human tumors and developmental abnormalities. The organization of coding sequences at the 5′-end of the murine and human DNA methyltransferase genes was investigated, and the DNA methyltransferase open reading frame was found to be longer than previously suspected. Expression of the complete open reading frame by in vitro transcription-translation and by transfection of expression constructs into COS7 cells resulted in the production of an active DNA methyltransferase of the same apparent mass as the endogenous protein, while translation from the second in-frame ATG codon produced a slightly smaller but fully active protein. Characterization of mRNA 5′ sequences and the intron-exon structure of the 5′ region of the murine and human genes indicated that a previously described promoter element (Rouleau, J., Tanigawa, G., and Szyf, M. (1992) J. Biol. Chem. 267, 7368-7377) actually lies in an intron that is more than 5 kilobases downstream of the transcription start sites.
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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.
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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.
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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
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
A cDNA encoding DNA (cytosine-5)-methyltransferase (DNA MeTase) of mouse cells has been cloned and sequenced. The nucleotide sequence contains an open reading frame sufficient to encode a polypeptide of 1573 amino acid residues, which is close to the apparent size of the largest species of DNA MeTase found in mouse cells. The carboxylterminal 570 amino acid residues of the inferred protein sequence shows striking similarities to bacterial type II DNA cytosine methyltransferases and appears to represent a catalytic methyltransferase domain. The amino-terminal portion of the molecule may be involved in regulating the activity of the carboxyl-terminal methyltransferase domain, since antibodies directed against a peptide sequence located within this region inhibits transmethylase activity in vitro. A 5200 base DNA MeTase-specific mRNA was found to be expressed in all mouse cell types tested, and cell lines known to have different genomic methylation patterns were found to contain DNA MeTase proteins of similar or identical sizes and de novo sequence specificities. The implications of these findings for an understanding of the mechanisms involved in the establishment and maintenance of methylation patterns are discussed.
Article
The int-1 proto-oncogene is transcriptionally activated in mammary tumors by mouse mammary tumor virus insertion mutations and is normally expressed only in adult mouse testes and mid-gestational embryos. We have used anatomical dissection of embryos, germ-cell fractionation, peripuberal expression studies, and spermatogenesis mutants to identify more precisely the tissues and cells that contain int-1 RNA. In the testis, int-1 RNA is detected only in postmeiotic germ cells undergoing differentiation from round spermatids into mature spermatozoa. In embryos 11-15 days after conception, expression of the gene is restricted to the developing central nervous system in regions of the neural tube other than the telencephalon. Our findings suggest that int-1 mediates developmental events at these two sites.
Article
Dye-ligand chromatography on Cibacron blue F3GA-agarose has been used to resolve two species of DNA (cytosine-5-)-methyltransferase from nuclear extracts of uninduced Friend murine erythroleukemia cells. Each species has been highly purified; the activities in the first and second peaks were associated with polypeptides of Mr 150,000 and 175,000, respectively. Analysis of substrate specificity with synthetic DNAs and restriction fragments of phi X174 replicative form DNA and pBR322 DNA showed that neither enzyme had dependence on the sequence context of CpG dinucleotides; poly(dG-dC) had the greatest methyl-accepting activity of any unmethylated DNA substrate tested. De novo methylation by both enzymes was inefficient relative to methylation of hemimethylated sites. Methyl-accepting activity was strongly dependent on DNA chain length. This observation suggests that binding to DNA, followed by one-dimensional diffusion of enzyme along the DNA molecule, is important in the mechanism by which DNA methyltransferase locates its recognition sites.
Article
The mouse Xist gene, which is expressed only from the inactive X chromosome, is thought to play a role in the initiation of X inactivation. The 5' end of this gene is fully methylated on the active X chromosome and completely demethylated on the inactive X chromosome, suggesting that DNA methylation may be involved in controlling allele-specific transcription of this gene. To directly investigate the importance of DNA methylation in the control of Xist expression, we have examined its methylation patterns and expression in ES cells and embryos that are deficient in DNA methyltransferase activity. We report here that demethylation of the Xist locus in male mutant embryos induces Xist expression, thus establishing a direct link between demethylation and expression of the Xist gene in the postgastrulation embryo. The transcriptional activity of Xist in undifferentiated ES cells, however, appears to be independent of its methylation status. These results suggest that methylation may only become essential for Xist repression after ES cells have differentiated or after the embryo has undergone gastrulation.
Article
DNA methylation of cytosine residues is a widespread phenomenon and has been implicated in a number of biological processes in both prokaryotes and eukaryotes. This methylation occurs at the 5-position of cytosine and is catalyzed by a distinct family of conserved enzymes, the cytosine-5 methyltransferases (m5C-MTases). We have cloned a fission yeast gene pmt1+ (pombe methyl transferase) which encodes a protein that shares significant homology with both prokaryotic and eukaryotic m5C-MTases. All 10 conserved domains found in these enzymes are present in the pmt1 protein. This is the first m5C-MTase homologue cloned from a fungal species. Its presence is surprising, given the inability to detect DNA methylation in yeasts. Haploid cells lacking the pmt1+ gene are viable, indicating that pmt1+ is not an essential gene. Purified, bacterially produced pmt1 protein does not possess obvious methyltransferase activity in vitro. Thus the biological significance of this m5C-MTase homologue in fission yeast is currently unclear.
Article
Mammals have long been known to tag their DNA by the addition of methyl groups to cytosine residues. Only quite recently, however, has the functional significance of DNA methylation established a firm footing. Evidence now indicates that DNA methylation is essential for development, and is involved in both programmed and ectopic gene inactivation. Recent structural and mechanistic work on bacterial cytosine-5-methyltransferases has provided much insight into the function of the carboxy-terminal catalytic domain of eukaryotic cytosine-5-methyltransferases; evidence is emerging that the amino-terminal domain targets the enzyme to the replication machinery and may be involved in sensing the pre-existing methylation state of the DNA.
Article
The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order to downweight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new program, CLUSTAL W which is freely available.
Article
The paternal and maternal genomes are not equivalent and both are required for mammalian development. The difference between the parental genomes is believed to be due to gamete-specific differential modification, a process known as genomic imprinting. The study of transgene methylation has shown that methylation patterns can be inherited in a parent-of-origin-specific manner, suggesting that DNA methylation may play a role in genomic imprinting. The functional significance of DNA methylation in genomic imprinting was strengthened by the recent finding that CpG islands (or sites) in three imprinted genes, H19, insulin-like growth factor 2 (Igf-2), and Igf-2 receptor (Igf-2r), are differentially methylated depending on their parental origin. We have examined the expression of these three imprinted genes in mutant mice that are deficient in DNA methyltransferase activity. We report here that expression of all three genes was affected in mutant embryos: the normally silent paternal allele of the H19 gene was activated, whereas the normally active paternal allele of the Igf-2 gene and the active maternal allele of the Igf-2r gene were repressed. Our results demonstrate that a normal level of DNA methylation is required for controlling differential expression of the paternal and maternal alleles of imprinted genes.
Article
The crystal structure has been determined at 2.8 A resolution for a chemically-trapped covalent reaction intermediate between the HhaI DNA cytosine-5-methyltransferase, S-adenosyl-L-homocysteine, and a duplex 13-mer DNA oligonucleotide containing methylated 5-fluorocytosine at its target. The DNA is located in a cleft between the two domains of the protein and has the characteristic conformation of B-form DNA, except for a disrupted G-C base pair that contains the target cytosine. The cytosine residue has swung completely out of the DNA helix and is positioned in the active site, which itself has undergone a large conformational change. The DNA is contacted from both the major and the minor grooves, but almost all base-specific interactions between the enzyme and the recognition bases occur in the major groove, through two glycine-rich loops from the small domain. The structure suggests how the active nucleophile reaches its target, directly supports the proposed mechanism for cytosine-5 DNA methylation, and illustrates a novel mode of sequence-specific DNA recognition.
Article
A plant cytosine methyltransferase cDNA was isolated using degenerate oligonucleotides, based on homology between prokaryote and mouse methyltransferases, and PCR to amplify a short fragment of a methyltransferase gene. A fragment of the predicted size was amplified from genomlc DNA from Arabidopsis thaliana. Overlapping cDNA clones, some with homology to the PCR amplified fragment, were Identified and sequenced. The assembled nucleic acid sequence Is 4720 bp and encodes a protein of 1534 amlno acids which has significant homology to prokaryote and mammalian cytosine methyltransferases. Like mammalian methylases, this enzyme has a C terminal methyltransferase domain linked to a second larger domain. The Arabidopsis methylase has eight of the ten conserved sequence motifs found in prokaryote cytoslne-5 methyltransferases and shows 50% homology to the murlne enzyme in the methyltransferase domain. The amlno terminal domain is only 24% homologous to the murlne enzyme and lacks the zinc binding region that has been found in methyltransferases from both mouse and man. In contrast to mouse where a single methyltransferase gene has been identified, a small multigene family with homology to the region amplified in PCR has been identified in Arabidopsis thaliana
Article
We describe an effort to share resources such that the maximum amount of gene-related data is obtained with the last redundancy. Specifically, we describe the I.M.A.G.E. Consortium effort to share arrayed cDNA libraries, to have the data derived from the use of these common reagents placed in public databases, and to use these data to create master arrays containing a representative cDNA clone from each gene. 8 refs.
Article
There are two biological properties of genomic methylation patterns that can be regarded as established. First, methylation of 5'-CpG-3' dinucleotides within promoters represses transcription, often to undetectable levels. Second, in most cases methylation patterns are subject to clonal inheritance. These properties suit methylation patterns for a number of biological roles, although none of the current hypotheses can be regarded as proved or disproved. One hypothesis suggests that the activity of parasitic sequence elements is repressed by selective methylation. Features of invasive sequences that might allow their identification and inactivation are discussed in terms of the genome defense hypothesis. Identification of the cues that direct de novo methylation may reveal the biological role (or roles) of genomic methylation patterns.
Article
The biological methylation cytosine bases in DNA is central to such diverse phenomena as restriction and modification in bacteria, repeat induced point-mutation (RIPing) in fungi and for programming gene expression patterns in vertebrates. Structural studies on HhaI DNA methyltransferase, together with the sequence comparisons of around 40 cytosine-specific DNA methyltransferases, have recently provided a molecular framework for understanding the mechanism of action of the related group of enzymes that catalyse this base modification. There are, however, a number of organisms, including Saccharomyces cerevisiae, Schizosaccharomyces pombe and Drosophila melanogaster, which have no detectable DNA methylation. Here we report that the product of the pmt1 gene recently identified in S. pombe, which contains most of the primary structure elements of a typical cytosine-specific DNA methyltransferase, is catalytically inert owing to the insertion of a Ser residue between the Pro-Cys motif found at the active site of all such DNA methyltransferases. Following deletion of this Ser residue, catalytic activity is restored and, using a range of DNA binding experiments, it is shown that the enzyme recognises and methylates the sequence CC(A/T)GG, the same sequence that is modified by the product of the Escherichia coli dcm gene. The pmt gene of S. pombe therefore encodes a pseudo DNA methyltranferase, which we have called psiM.SpoI.
Article
Conservation of genomic organization in different mammalian species has long been recognized, but only recently has it been possible to examine these relationships systematically on a genome-wide scale in some detail. Mapping of several mammalian species in progressing rapidly, but by far the most detailed information is still to be found in the human and mouse databases. Perhaps the most important aspect of recent progress in genome mapping data. With mapping databases continuing to expand at a greater than linear rate, any attempt at a comprehensive comparative map is doomed to be out of date by the time it is published. However, we feel that it is valuable to provide a summary that is as nearly up to date as possible. We have made a particular effort to include recent human physical mapping data and to identify those mouse genes that have been well-mapped with respect to each other by virtue of having been examined in the same cross. As the human-mouse comparative map becomes more dense, it is not surprising that the observed number of conserved linkage groups continues to increase. Nadeau et al. placed 425 loci on both maps, which delineated over 100 conserved linkage groups. Copeland et al. put a total of 917 markers on both the human and the mouse maps, marking 101 segments of conserved linkage groups. In the present summary, we have placed 1416 loci, and these define at least 181 different conserved linkage groups. 47 refs., 1 fig.
Article
Xist and other X-linked gene expression was examined by fluorescence in situ hybridization in cells of wild type and DNA methyltranferase (Dnmt) mutant embryos and embryonic stem (ES) cells to determine whether demethylation-induced Xist expression leads to inappropriate X chromosome inactivation. In undifferentiated ES cells low-level Xist expression was detected from the single active X chromosome (Xa) in male cells and on both Xa's in female cells. Upon differentiation Xist expression was detected only in female cells, in which Xist RNA colocalized with the entire inactive X chromosome (Xi). Differentiated Dnmt mutant ES cells or cells of mutant postgastrulation embryos showed aberrant patterns of Xist expression: Xist transcripts colocalized with the single X chromosome in male cells and with both X chromosomes in female cells. X-linked gene expression was not detected from chromosomes coated with Xist RNA. These results suggest that ectopic Xist expression, induced by DNA hypomethylation, may lead to the inactivation of X-linked genes. We conclude that Xist-mediated X chromosome inactivation can occur in the absence of DNA methylation, arguing that DNA methylation may be required to repress Xist expression for the maintenance of a transcriptionally active Xa. In differentiated Dnmt mutant ES cells the activation of Xist expression correlated with a dramatic increase in apoptotic bodies, suggesting that Xist-mediated X chromosome inactivation may result in cell death and contribute to the embryonic lethality of the Dnmt mutation.
Article
It has been a controversial issue as to how many DNA cytosine methyltransferase mammalian cells have and whether de novo methylation and maintenance methylation activities are encoded by a single gene or two different genes. To address these questions, we have generated a null mutation of the only known mammalian DNA methyltransferase gene through homologous recombination in mouse embryonic stem cells and found that the development of the homozygous embryos is arrested prior to the 8-somite stage. Surprisingly, the null mutant embryonic stem cells are viable and contain low but stable levels of methyl cytosine and methyltransferase activity, suggesting the existence of a second DNA methyltransferase in mammalian cells. Further studies indicate that de novo methylation activity is not impaired by the mutation as integrated provirus DNA in MoMuLV-infected homozygous embryonic stem cells become methylated at a similar rate as in wild-type cells. Differentiation of mutant cells results in further reduction of methyl cytosine levels, consistent with the de novo methylation activity being down regulated in differentiated cells. These results provide the first evidence that an independently encoded DNA methyltransferase is present in mammalian cells which is capable of de novo methylating cellular and viral DNA in vivo.
Article
A Xenopus DNA methyltransferase cDNA was isolated from a Xenopus oocyte cDNA library by screening with the mouse DNA methyltransferase cDNA as a probe. The elucidated nucleotide sequence gave a 4,470 nucleotide open reading frame, and the predicted protein was composed of 1,490 amino acid residues, showing high homology to animal DNA methyltransferases, especially in the catalytic domain in the carboxyl terminal region. The cysteine-rich region and the Lys-Gly repeat which were first found in the mouse sequence were conserved in Xenopus. However, 200 amino acid residues at the amino-terminus of Xenopus DNA methyl transferase were quite different from those of mouse and human, but showed 70% homology with those of chicken. The cloned Xenopus DNA methyltransferase cDNA expressed in COS1 cells showed a significant DNA methyltransferase activity. The size of the translation product of Xenopus DNA methyltransferase cDNA expressed in COS1 cells was identical with that of the endogenous DNA methyltransferase in Xenopus A6 cells and also with the size of newly synthesized DNA methyltransferase in Xenopus oocytes. However, a slightly larger immunoreactlve band of about 205 kDa, and a small iminunoreactive band of about 100 kDa, which were poorly labeled by short incubation with radiolabeled amino acids, were the main bands in stage I–III and stage IV–VI oocytes, respectively.
Article
Apparent alterations in DNA methylation have been observed in many cancers, but whether such alterations represent a persistent alteration in the normal methylation process is not known. In this study, we report a striking difference in the expression of exogenously introduced retroviral genes in various colorectal cancer cell lines. Extinguished expression was associated with DNA methylation and could be reversed by treatment with the demethylating agent 5-azacytidine. A striking correlation between genetic instability and methylation capacity suggested that methylation abnormalities may play a role in chromosome segregation processes in cancer cells.
Article
Most of the 5-methylcytosine in mammalian DNA resides in transposons, which are specialized intragenomic parasites that represent at least 35% of the genome. Transposon promoters are inactive when methylated and, over time, C-->T transition mutations at methylated sites destroy many transposons. Apart from that subset of genes subject to X inactivation and genomic imprinting, no cellular gene in a non-expressing tissue has been proven to be methylated in a pattern that prevents transcription. It has become increasingly difficult to hold that reversible promoter methylation is commonly involved in developmental gene control; instead, suppression of parasitic sequence elements appears to be the primary function of cytosine methylation, with crucial secondary roles in allele-specific gene expression as seen in X inactivation and genomic imprinting.
Article
Epigenetic mechanisms can serve as genome defense systems. In haploid nuclei of special sexual cells of fungi, such as Neurospora and Ascobolus, duplicated genes are silenced by hypermutation, DNA methylation, or both. In some cases, DNA introduced into the genome of Neurospora cells by transformation can also inhibit homologous genes by a silencing mechanism that does not involve DNA pairing or methylation and appears to be post-transcriptional. Transforming DNA can also trigger de novo methylation in vegetative cells, which then causes transcriptional silencing. The rules governing silencing in vegetative cells of fungi are undefined, but repeated sequences seem particularly susceptible to these processes. Thus, fungi exhibit both repeat-induced and repeat-associated silencing mechanisms. Additionally, some native genes depend on homologous pairing in the diplophase for proper regulation. Together, these processes should limit the proliferation of transposable elements and serve to preserve the overall structure of the genome.
A gene map of the human genome NOTE ADDED IN PROOF Malagnac and colleagues have cloned a candidate eukaryotic DNA methyltransferase, masc1, from the ascomycete fungus, Ascobolus immersus, which appears to be only distantly related to Dnmt1 and Dnmt2
  • G D Schuler
Schuler, G.D. et al. (1996) A gene map of the human genome. Science, 274, 540–546. NOTE ADDED IN PROOF Malagnac and colleagues have cloned a candidate eukaryotic DNA methyltransferase, masc1, from the ascomycete fungus, Ascobolus immersus, which appears to be only distantly related to Dnmt1 and Dnmt2 (Cell, 91, 281–290).
Malagnac and colleagues have cloned a candidate eukaryotic DNA methyltransferase, masc1, from the ascomycete fungus, Ascobolus immersus, which appears to be only distantly related to Dnmt1 and Dnmt2
  • Note Added
  • In Proof
NOTE ADDED IN PROOF Malagnac and colleagues have cloned a candidate eukaryotic DNA methyltransferase, masc1, from the ascomycete fungus, Ascobolus immersus, which appears to be only distantly related to Dnmt1 and Dnmt2 (Cell, 91, 281–290).