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)

DNA methyltransferase I (Dnmt1), the maintenance enzyme for DNA cytosine methylation, is expressed at high levels in the CNS during embryogenesis and after birth. Because embryos deficient for Dnmt1 die at gastrulation, the role of Dnmt1 in the development and function of the nervous system could not be studied by using this mutation. We therefore used the cre/loxP system to produce conditional mutants that lack Dnmt1 in neuroblasts of embryonic day 12 embryos or in postmitotic neurons of the postnatal animal. Conditional deletion of the Dnmt1 gene resulted in rapid depletion of Dnmt1 proteins, indicating that the enzyme in postmitotic neurons turns over quickly. Dnmt1 deficiency in postmitotic neurons neither affected levels of global DNA methylation nor influenced cell survival during postnatal life. In contrast, Dnmt1 deficiency in mitotic CNS precursor cells resulted in DNA hypomethylation in daughter cells. Whereas mutant embryos carrying 95% hypomethylated cells in the brain died immediately after birth because of respiratory distress, mosaic animals with 30% hypomethylated CNS cells were viable into adulthood. However, these mutant cells were eliminated quickly from the brain within 3 weeks of postnatal life. Thus, hypomethylated CNS neurons were impaired functionally and were selected against at postnatal stages.
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PMID:DNA hypomethylation perturbs the function and survival of CNS neurons in postnatal animals. 1115 65

DNA methylation, chromatin structure, transcription, and cancer have traditionally been studied as separate phenomena. Recent data provide now direct physical and functional links between these processes revealing a complex network of interactions and mutual dependences. Methylated DNA is bound by methyl-CpG binding protein (MeCP) complexes that include histone deacetylases (HDACs). This recruitment of HDACs is suggested to promote local chromatin condensation and thereby repress gene expression. Most recently, also complexes of DNA methyltransferase (Dnmt1) with transcriptional repressors, DMAP1 and pRB, have been described providing a direct link to transcriptional regulation and tumor suppression. Inactivation of the DNA methyltransferase genes (Dnmt1, 3a, and 3b) was found to be lethal in mice and several human diseases (ICF and Rett syndrome) turned out to be linked to DNA methylation. In particular, global hypomethylation has been found in tumor samples together with cancer-type-specific, local hypermethylation. Taken together, these lines of evidence clearly underscore the central role of DNA methylation in the regulation of gene expression and chromatin structure during normal development and diseases like cancer. J. Cell. Biochem. Suppl. 35:78-83, 2000.
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PMID:DNA methylation, nuclear structure, gene expression and cancer. 1138 35

The mammalian DNA methyltransferase Dnmt1 is responsible for the maintenance of the pattern of DNA methylation in vivo. It is a large multidomain enzyme comprising 1620 amino acid residues. We have purified and characterized individual domains of Dnmt1 (NLS-containing domain, NlsD, amino acid residues: 1-343; replication foci-directing domain, 350-609; Zn-binding domain (ZnD), 613-748; polybromo domain, 746-1110; and the catalytic domain (CatD), 1124-1620). CatD, ZnD and NlsD bind to DNA, demonstrating the existence of three independent DNA-binding sites in Dnmt1. CatD shows a preference for binding to hemimethylated CpG-sites; ZnD prefers methylated CpGs; and NlsD specifically binds to CpG-sites, but does not discriminate between unmethylated and methylated DNA. These results are not compatible with the suggestion that the target recognition domain of Dnmt1 resides in the N terminus of the enzyme. We show by protein-protein interaction assays that ZnD and CatD interact with each other. The isolated catalytic domain does not methylate DNA, neither alone nor in combination with other domains. Full-length Dnmt1 was purified from baculovirus-infected insect cells. Under the experimental conditions, Dnmt1 has a strong (50-fold) preference for hemimethylated DNA. Dnmt1 is stimulated to methylate unmodified CpG sites by the addition of fully methylated DNA. This effect is dependent on Zn, suggesting that binding of methylated DNA to ZnD triggers the allosteric activation of the catalytic center of Dnmt1. The allosteric activation model can explain kinetic data obtained by others. It suggests that Dnmt1 might be responsible for spreading of methylation, a process that is observed during aging and carcenogenesis but may be important for de novo methylation of DNA.
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PMID:The activity of the murine DNA methyltransferase Dnmt1 is controlled by interaction of the catalytic domain with the N-terminal part of the enzyme leading to an allosteric activation of the enzyme after binding to methylated DNA. 1139 88

We present the first in vitro study investigating the catalytic properties of a mammalian de novo DNA methyltransferase. Dnmt3a from mouse was cloned and expressed in Escherichia coli. It was shown to be catalytically active in E. coli cells in vivo. The methylation activity of the purified protein was highest at pH 7.0 and 30 mM KCl. Our data show that recombinant Dnmt3a protein is indeed a de novo methyltransferase, as it catalyzes the transfer of methyl groups to unmethylated substrates with similar efficiency as to hemimethylated substrates. With oligonucleotide substrates, the catalytic activity of Dnmt3a is similar to that of Dnmt1: the K(m) values for the unmethylated and hemimethylated oligonucleotide substrates are 2.5 microM, and the k(cat) values are 0.05 h(-1) and 0.07 h(-1), respectively. The enzyme catalyzes the methylation of DNA in a distributive manner, suggesting that Dnmt3a and Dnmt1 may cooperate during de novo methylation of DNA. Further, we investigated the methylation activity of Dnmt3a at non-canonical sites. Even though the enzyme shows maximum activity at CpG sites, with oligonucleotide substrates, a high methylation activity was also found at CpA sites, which are modified only twofold slower than CpG sites. Therefore, the specificity of Dnmt3a is completely different from that of the maintenance methyltransferase Dnmt1, which shows a 40 to 50-fold preference for hemimethylated over unmethylated CpG sites and has almost no methylation activity at non-CpG sites.
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PMID:Enzymatic properties of recombinant Dnmt3a DNA methyltransferase from mouse: the enzyme modifies DNA in a non-processive manner and also methylates non-CpG [correction of non-CpA] sites. 1139 89

In cultured cells, the maintenance-type DNA methyltransferase (Dnmt1) is highly expressed during the proliferation stage. In the present study, we detected significant expression of Dnmt1 protein in the nuclear fraction of mouse small intestine. From its mobility in SDS polyacrylamide gel electrophoresis and the specific antibodies against the somatic cell-type Dnmt1, Dnmt1 was determined as a somatic cell type. Immunofluorescence study revealed that the Dnmt1 was highly expressed in the proliferating stem cells in crypts, and was localized in the nuclei. The present results indicate that the expression of Dnmt1 in vivo is also under the control of cell proliferation as in cultured cells.
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PMID:Proliferation stage-dependent expression of DNA methyltransferase (Dnmt1) in mouse small intestine. 1148 56

DNA methylation is a major epigenetic modification of the genome that regulates crucial aspects of its function. Genomic methylation patterns in somatic differentiated cells are generally stable and heritable. However, in mammals there are at least two developmental periods-in germ cells and in preimplantation embryos-in which methylation patterns are reprogrammed genome wide, generating cells with a broad developmental potential. Epigenetic reprogramming in germ cells is critical for imprinting; reprogramming in early embryos also affects imprinting. Reprogramming is likely to have a crucial role in establishing nuclear totipotency in normal development and in cloned animals, and in the erasure of acquired epigenetic information. A role of reprogramming in stem cell differentiation is also envisaged. DNA methylation is one of the best-studied epigenetic modifications of DNA in all unicellular and multicellular organisms. In mammals and other vertebrates, methylation occurs predominantly at the symmetrical dinucleotide CpG (1-4). Symmetrical methylation and the discovery of a DNA methyltransferase that prefers a hemimethylated substrate, Dnmt1 (4), suggested a mechanism by which specific patterns of methylation in the genome could be maintained. Patterns imposed on the genome at defined developmental time points in precursor cells could be maintained by Dnmt1, and would lead to predetermined programs of gene expression during development in descendants of the precursor cells (5, 6). This provided a means to explain how patterns of differentiation could be maintained by populations of cells. In addition, specific demethylation events in differentiated tissues could then lead to further changes in gene expression as needed. Neat and convincing as this model is, it is still largely unsubstantiated. While effects of methylation on expression of specific genes, particularly imprinted ones (7) and some retrotransposons (8), have been demonstrated in vivo, it is still unclear whether or not methylation is involved in the control of gene expression during normal development (9-13). Although enzymes have been identified that can methylate DNA de novo (Dnmt3a and Dnmt3b) (14), it is unknown how specific patterns of methylation are established in the genome. Mechanisms for active demethylation have been suggested, but no enzymes have been identified that carry out this function in vivo (15-17). Genomewide alterations in methylation-brought about, for example, by knockouts of the methylase genes-result in embryo lethality or developmental defects, but the basis for abnormal development still remains to be discovered (7, 14). What is clear, however, is that in mammals there are developmental periods of genomewide reprogramming of methylation patterns in vivo. Typically, a substantial part of the genome is demethylated, and after some time remethylated, in a cell- or tissue-specific pattern. The developmental dynamics of these reprogramming events, as well as some of the enzymatic mechanisms involved and the biological purposes, are beginning to be understood. Here we look at what is known about reprogramming in mammals and discuss how it might relate to developmental potency and imprinting.
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PMID:Epigenetic reprogramming in mammalian development. 1149 79

In mammalian cells, DNA methylation patterns are precisely maintained after DNA replication with defined changes occurring during development. The major DNA methyltransferase (Dnmt1) is associated with nuclear replication sites during S-phase, which is consistent with a role in maintenance methylation. The subcellular distribution of the recently discovered de novo DNA methyltransferases, Dnmt3a and Dnmt3b, was investigated by immunofluorescence and by epitope tagging. We now show that both Dnmt3a and Dnmt3b are distributed throughout the nucleoplasm but are not associated with nuclear DNA replication sites during S-phase. These results suggest that de novo methylation by Dnmt3a and Dnmt3b occurs independently of the replication process and might involve an alternative mechanism for accessing the target DNA. The different subcellular distribution of mammalian DNA methyltransferases might thus contribute to the regulation of DNA methylation.
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PMID:Mammalian DNA methyltransferases show different subnuclear distributions. 1159 6

Tumor suppressor gene inactivation is a crucial event in oncogenesis. Gene inactivation mechanisms include events resulting in loss of heterozygosity (LOH), gene mutation, and transcriptional silencing. The contribution of each of these different pathways varies among tumor suppressor genes and by cancer type. The factors that influence the relative utilization of gene inactivation pathways are poorly understood. In this study, we describe a detailed quantitative analysis of the three major gene inactivation mechanisms for a model gene at two different genomic integration sites in mouse embryonic stem (ES) cells. In addition, we targeted the major DNA methyltransferase gene, Dnmt1, to investigate the relative contribution of DNA methylation to these various competing gene inactivation pathways. Our data show that gene loss is the predominant mode of inactivation of a herpes simplex virus thymidine kinase neomycin phosphotransferase reporter gene (HSV-TKNeo) at the two integration sites tested and that this event is significantly reduced in Dnmt1-deficient cells. Gene silencing by promoter methylation requires Dnmt1, suggesting that the expression of Dnmt3a and Dnmt3b alone in ES cells is insufficient to achieve effective gene silencing. We used a novel assay to show that missense mutation rates are also substantially reduced in Dnmt1-deficient cells. This is the first direct demonstration that DNA methylation affects point mutation rates in mammalian cells. Surprisingly, the fraction of CpG transition mutations was not reduced in Dnmt1-deficient cells. Finally, we show that methyl group-deficient growth conditions do not cause an increase in missense mutation rates in Dnmt1-proficient cells, as predicted by methyltransferase-mediated mutagenesis models. We conclude that Dnmt1 deficiency and the accompanying genomic DNA hypomethylation result in a reduction of three major pathways of gene inactivation in our model system.
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PMID:Reduced rates of gene loss, gene silencing, and gene mutation in Dnmt1-deficient embryonic stem cells. 1160 95

DNA methylation is important for controlling gene expression and is catalyzed by DNA methyltransferase (Dnmt1) an enzyme abundant in brain. We recently demonstrated that mice expressing reduced levels of Dnmt1 are protected from cerebral ischemia. Here, we used the cre/loxP system to produce conditional mutants that lack Dnmt 1 in postmitotic neurons of the postnatal brain. We demonstrate that animals heterozygous for the conditional allele (Dnmt11lox/+) have significantly smaller infarcts following 1 h middle cerebral artery occlusion/reperfusion compared to their wildtype litters. Surprisingly, mice with a deletion of Dnmt1 in post-mitotic neurons (Dnmt11lox/c) were not protected. In conclusion, we demonstrate that reduced levels of Dnmt1, but not its absence, in post-mitotic neurons protect from ischemic brain injury.
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PMID:Effects of cerebral ischemia in mice lacking DNA methyltransferase 1 in post-mitotic neurons. 1172 90

The role of DNA methylation and of the maintenance DNA methyltransferase Dnmt1 in the epigenetic regulation of developmental stage- and cell lineage-specific gene expression in vivo is uncertain. This is addressed here through the generation of mice in which Dnmt1 was inactivated by Cre/loxP-mediated deletion at sequential stages of T cell development. Deletion of Dnmt1 in early double-negative thymocytes led to impaired survival of TCRalphabeta(+) cells and the generation of atypical CD8(+)TCRgammadelta(+) cells. Deletion of Dnmt1 in double-positive thymocytes impaired activation-induced proliferation but differentially enhanced cytokine mRNA expression by naive peripheral T cells. We conclude that Dnmt1 and DNA methylation are required for the proper expression of certain genes that define fate and determine function in T cells.
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PMID:A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival. 1172 38


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