Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.1.1.37 (DNA methyltransferase)
4,983 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In Xenopus laevis zygotic transcription begins at the midblastula transition (MBT). Prior to this the genome is organized into chromatin that facilitates rapid cycles of DNA replication but not transcription. Here we demonstrate that DNA methylation contributes to the overall transcriptional silencing before MBT. Transient depletion of the maternal DNA methyltransferase (xDnmt1) by anti sense RNA during cleavage stages is associated with a decrease in the genomic 5-methyl-cytosine content and leads to the activation of zygotic transcription approximately two cell cycles earlier than normal. Hypomethylation allows the early expression of mesodermal marker genes such as Xbra, Cerberus, and Otx2, which are subsequently down-regulated during gastrulation of the xDnmt1-depleted embryos. The temporal switch in gene expression may account for the appearance of body plan defects that we observe. Loss of xDnmt1 can be rescued by the coinjection of mouse or human Dnmt1 protein. These results demonstrate that DNA methylation has a role in the regulation of immediately early genes in Xenopus at MBT.
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PMID:Transient depletion of xDnmt1 leads to premature gene activation in Xenopus embryos. 1067 3

Dnmt1 is the predominant DNA methyltransferase (MTase) in mammals. The C-terminal domain of Dnmt1 clearly shares sequence similarity with many prokaryotic 5mC methyltransferases, and had been proposed to be sufficient for catalytic activity. We show here by deletion analysis that the C-terminal domain alone is not sufficient for methylating activity, but that a large part of the N-terminal domain is required in addition. Since this complex structure of Dnmt1 raises issues about its evolutionary origin, we have compared several eukaryotic MTases and have determined the genomic organization of the mouse Dnmt1 gene. The 5' most part of the N-terminal domain is dispensible for enzyme activity, includes the major nuclear import signal and comprises tissue-specific exons. Interestingly, the functional subdivision of Dnmt1 correlates well with the structure of the Dnmt1 gene in terms of intron/exon size distribution as well as sequence conservation. Our results, based on functional, structural and sequence comparison data, suggest that the gene has evolved from the fusion of at least three genes.
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PMID:Structure and function of the mouse DNA methyltransferase gene: Dnmt1 shows a tripartite structure. 1071 1

Tissue- and gene-specific patterns of cytosine-DNA methylation are characteristic features of vertebrate genomes. The generation and proper maintenance of DNA methylation patterns are essential for embryonic development, as demonstrated by the lethal phenotypes of mice with either a targeted disruption of Dnmt1, the gene responsible for the maintenance of DNA methylation, or targeted disruption of Dnmt3a or Dnmt3b, the genes involved in generation of newly formed methylation patterns. Recently, a novel mRNA, Dnmt1b, resulting from alternative splicing of Dnmt1 was identified (Hsu, D. W., Lin, M. J., Lee, T. L., Wen, S. C., Chen, X., and Shen, C. K., (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9751-9756). The abundance of Dnmt1b mRNA was estimated by semiquantitative reverse transcription polymerase chain reaction and was suggested to encode a major C-5 DNA methyltransferase isoform. Here we report characterization of this novel DNA methyltransferase transcript, Dnmt1b, and its protein product in human cell lines and in freshly isolated human peripheral blood mononuclear cells. The abundance of Dnmt1b transcript, as determined by quantitative RNase protection analysis, was determined to range from 6% to 25% of Dnmt1 in human cells. Second generation antisense inhibitors targeted to the 5'- and 3'-ends of Dnmt1 inhibited the accumulation of both Dnmt1 and Dnmt1b in cells. Dnmt1b protein purified from a baculovirus expression system was demonstrated to be a functional DNA methyltransferase, and to have Michaelis constants for both DNA and S-adenosyl-L-methionine similar to baculovirus-expressed Dnmt1. However, antibodies raised against Dnmt1b epitopes demonstrated that Dnmt1b protein was present at approximately 2-5% of the level of Dnmt1 and therefore represents only a minor DNA methyltransferase isoform in human cells.
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PMID:Characterization of the human DNA methyltransferase splice variant Dnmt1b. 1075 66

The mouse (cytosine-5) DNA methyltransferase (Dnmt1) consists of a regulatory N-terminal and a catalytic C-terminal domain, which are fused by a stretch of Gly-Lys dipeptide repeats. The C-terminal region contains all of the conserved motifs found in other cytosine-5 DNA methyltransferases including the relative position of the catalytic Pro-Cys dipeptide. In prokaryotes, the methyltransferases are simpler and lack the regulatory N-terminal domain. We constructed three hybrid methyltransferases, containing the intact N-terminus of the murine Dnmt1 and most of the coding sequences from M.HhaI (GCGC), M.HpaII (CCGG) or M.SssI (CG). These hybrids are biologically active when expressed in a baculovirus system and show the specificity of the parental C-terminal domain. Expression of these recombinant constructs leads to de novo methylation of both host and viral genomes in a sequence-specific manner. Steady-state kinetic analyses were performed on the murine Dnmt1-HhaI hybrid using poly(dG-dC).poly (dG-dC), unmethylated and hemimethylated oligonucleotides as substrates. The enzyme has a slow catalytic turnover number of 4.38 h(-1) for poly(dG-dC). poly(dG-dC), and exhibits 3-fold higher catalytic efficiency for hemimethylated substrates.
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PMID:Hybrid mouse-prokaryotic DNA (cytosine-5) methyltransferases retain the specificity of the parental C-terminal domain. 1079 Mar 76

The H19 gene is imprinted with preferential expression from the maternal allele. The putative imprinting control region for this locus is hypermethylated on the repressed paternal allele. Although maternal-specific expression of H19 is observed in mouse blastocysts that develop in vivo, biallelic expression has been documented in embryos and embryonic stem cells experimentally manipulated by in vitro culture conditions. In this study the effect of culture on imprinted H19 expression and methylation was determined. After culture of 2-cell embryos to the blastocyst stage in Whitten's medium, the normally silent paternal H19 allele was aberrantly expressed, whereas little paternal expression was observed following culture in KSOM containing amino acids (KSOM+AA). Analysis of the methylation status of a CpG dinucleotide located in the upstream imprinting control region revealed a loss in methylation in embryos cultured in Whitten's medium but not in embryos cultured in KSOM+AA. Thus, H19 expression and methylation were adversely affected by culture in Whitten's medium, while the response of H19 to culture in KSOM+AA approximated more closely the in vivo situation. It is unlikely that biallelic expression of H19 following culture in Whitten's medium is a generalized effect of lower methylation levels, since the amount of DNA methyltransferase activity and the spatial distribution of Dnmt1 protein were similar in in vivo-derived and cultured embryos. Moreover, imprinted expression of Snrpn was maintained following culture in either medium, indicating that not all imprinted genes are under the same stringent imprinting controls. The finding that culture conditions can dramatically, but selectively, affect the expression of imprinted genes provides a model system for further study of the linkage between DNA methylation and gene expression.
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PMID:Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. 1081 52

Modulation of gene expression by catalytic RNA requires accessible ribozyme cleavage sites in the target mRNA, and accessibility is determined by the secondary and tertiary structure of the target RNA, as affected by its interactions with cellular proteins. As we previously reported, an oligonucleotide-scanning approach using antisense oligonucleotides can be used to determine RNA accessibility in cell extracts. To test whether this method can be used to improve selection of ribozyme target sites, we designed ribozymes corresponding to the sites identified by oligonucleotide scanning and have evaluated their catalytic activities, first in cell extracts and then in transduced cell lines. As a target we used the mRNA of murine DNA (cytosine-5)-methyltransferase 1 (MTase). For intracellular studies, the ribozyme genes were inserted downstream of a Pol III tRNAVAL promoter, which in turn was cloned in the U3 region of a retroviral vector. We find that the efficiency of the ribozymes both in cell extracts and in vivo corresponds with the relative effectiveness predicted by the oligonucleotide-scanning assay. The best ribozyme causes a 70-80% reduction in the MTase mRNA levels in NIH 3T3 cells that are stably transduced with the retroviral constructs. This reduction in mRNA levels is accompanied by a small decrease in the methylation of repetitive intercisternal A particle DNA elements. Ribozyme expression also increased several-fold the reactivation frequency of a methylation-silenced green fluorescent protein (GFP) transgene. Both the reduction in methylation and reactivation of GFP were roughly equivalent to the effects obtained by treating NIH 3T3 cells with 2.5 microM 5-azacytidine, which gives an effect of about 10% of maximum. These results confirm the validity of the cell extract approach for ribozyme site selection and provide a potentially useful ribozyme for future study of DNA methyltransferase function.
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PMID:Oligonucleotide scanning of native mRNAs in extracts predicts intracellular ribozyme efficiency: ribozyme-mediated reduction of the murine DNA methyltransferase. 1089 25

Altered patterns of the 5-cytosine methylation of genomic DNA are associated with the development of a wide range of human cancers. We have studied the mechanisms and genetic pathways by which a targeted heterozygous deficiency in the murine 5-cytosine DNA methyltransferase gene (Dnmt1(N/+)) diminishes intestinal tumorigenesis in C57BL/6-multiple intestinal neoplasia (Min)/+ mice. We found that Dnmt1(N/+) retards the net growth rate of intestinal adenomas and reduces tumor multiplicity by approximately 50%. This tumor resistance affects the entire intestinal tract and is independent of the status of modifier of Min 1 and p53, two loci that have been found to confer strong resistance to Min-induced neoplasia Interestingly, Dnmt/(N/+) and modifier of Min 1 resistance interact synergistically, together virtually eliminating tumor incidence. This finding may provide an insight into potential combinatorial therapeutic approaches for treating human colon cancer.
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PMID:Dnmt1N/+ reduces the net growth rate and multiplicity of intestinal adenomas in C57BL/6-multiple intestinal neoplasia (Min)/+ mice independently of p53 but demonstrates strong synergy with the modifier of Min 1(AKR) resistance allele. 1091 75

Maintenance-type DNA methyltransferase (Dnmt1) is usually down-regulated in non-proliferating cells. In the present study, we detected significant expression of Dnmt1 protein in adult mouse brain where the majority of the cells are in a post-mitotic state. A significant amount of Dnmt1 protein was fractionated into the post-nuclear fraction for both cerebrum and cerebellum. The Dnmt1 in this fraction was enzymatically active. An immunofluorescence study revealed that Dnmt1 protein was mainly expressed in neurons and seemed to be localized in the cytoplasmic compartment. Primary culturing of neurons confirmed the expression and localization of Dnmt1 in the cytoplasmic compartment. The findings that the Dnmt1 transcript in the brain utilized the somatic-type exon and that the apparent size of the Dnmt1 protein in the cytoplasm was identical to that in proliferating culture cells indicate that the cytoplasmic Dnmt1 in neurons was of the somatic-type.
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PMID:Maintenance-type DNA methyltransferase is highly expressed in post-mitotic neurons and localized in the cytoplasmic compartment. 1092 Feb 68

DNA methylation is a major determinant in the epigenetic silencing of genes. The mechanisms underlying the targeting of DNA methylation and the subsequent repression of transcription are relevant to human development and disease, as well as for attempts at somatic gene therapy. The success of transgenic technologies in plants and animals is also compromised by DNA methylation-dependent silencing pathways. Recent biochemical experiments provide a mechanistic foundation for understanding the influence of DNA methylation on transcription. The DNA methyltransferase Dnmt1, and several methyl-CpG binding proteins, MeCP2, MBD2, and MBD3, all associate with histone deacetylase. These observations firmly connect DNA methylation with chromatin modifications. They also provide new pathways for the potential targeting of DNA methylation to repressive chromatin as well as the assembly of repressive chromatin on methylated DNA. Here we discuss the implications of the methylation-acetylation connection for human cancers and the developmental syndromes Fragile X and Rett, which involve a mistargeting of DNA methylation-dependent repression.
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PMID:DNA methylation and histone deacetylation in the control of gene expression: basic biochemistry to human development and disease. 1109 25

Cytosine methylation of mammalian DNA is essential for the proper epigenetic regulation of gene expression and maintenance of genomic integrity. To define the mechanism through which demethylated cells die, and to establish a paradigm for identifying genes regulated by DNA methylation, we have generated mice with a conditional allele for the maintenance DNA methyltransferase gene Dnmt1. Cre-mediated deletion of Dnmt1 causes demethylation of cultured fibroblasts and a uniform p53-dependent cell death. Mutational inactivation of Trp53 partially rescues the demethylated fibroblasts for up to five population doublings in culture. Oligonucleotide microarray analysis showed that up to 10% of genes are aberrantly expressed in demethylated fibroblasts. Our results demonstrate that loss of Dnmt1 causes cell-type-specific changes in gene expression that impinge on several pathways, including expression of imprinted genes, cell-cycle control, growth factor/receptor signal transduction and mobilization of retroelements.
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PMID:Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation. 1113 87


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