DNA methylation in chicken brain and liver

Summary Adult chicken brain DNA is subject to a developmental alteration of methylation atHpaII sites flanking 4, 1, 0.8 and 0.7 kilobase sequences. This change is not evident in adult chicken liver DNA and 1-day-old chick brain and liver DNAs, suggesting tissue specificity and age-dependency in DNA methylation.     

Differential expression of glucose transporters during chick embryogenesis

The patterns of Glut1 and Glut3 glucose transporter protein and mRNA expression were assessed during embryogenesis of chicken brain and skeletal muscle, Glut4 protein levels were also evaluated in skeletal muscle and heart, and Glut1 was examined in the developing heart and liver. Glut1 protein expression was detectable throughout brain ontogeny but was highest during early development. Glut1 mRNA levels in the brain remained very high throughout development. Glut3 protein was highest very early and very late and mRNA was highest during the last half of development. In embryonic skeletal muscle, the levels of Glut1and Glut3 proteins and mRNA were highest very early, and declined severely by mid-development. Glut1 protein and mRNA in the heart also peaked early and then decreased steadily. Although Glut1 mRNA levels were consistently high in the embryonic liver, Glut1 protein expression was not detected. These results suggest that (1) Glut1 is developmentally regulated in chick brain, skeletal muscle, and heart, (2) Glut1 mRNA is present in liver but does not appear to be translated, (3) Glut3 in brain increases developmentally but is virtually absent in muscle, and (4) Glut4 protein and mRNA appear to be absent from chick heart and skeletal muscle. Received 11 January 2001; accepted 14 February 2001     

Biochemistry and biology of mammalian DNA methyltransferases

DNA methylation is a stable but not irreversible epigenetic signal that silences gene expression. It has a variety of important functions in mammals, including control of gene expression, cellular differentiation and development, preservation of chromosomal integrity, parental imprinting and X-chromosome inactivation. In addition, it has been implicated in brain function and the development of the immune system. Somatic alterations in genomic methylation patterns contribute to the etiology of human cancers and ageing. It is tightly interwoven with the modification of histone tails and other epigenetic signals. Here we review our current understanding of the molecular enzymology of the mammalian DNA methyltransferases Dnmt1, Dnmt3a, Dnmt3b and Dnmt2 and the roles of the enzymes in the above-mentioned biological processes.     

Effects of d1-methadone and morphine on developing chick embryo

Summary Injections of methadone into the air space of fertile chicken eggs affected development of the embryo. Both methadone and morphine caused decreases in liver weight and brain protein, and morphine increased liver protein levels.Acknowledgment. We thank E. Sutherland for technical assistance. Supported by a grant from the Department of Health and Welfare, Canada, and the British Columbia Ministry of Health, Alcohol and Drug Commission.     

The role of genetics in the establishment and maintenance of the epigenome

Epigenetic mechanisms play an important role in gene regulation during development. DNA methylation, which is probably the most important and best-studied epigenetic mechanism, can be abnormally regulated in common pathologies, but the origin of altered DNA methylation remains unknown. Recent research suggests that these epigenetic alterations could depend, at least in part, on genetic mutations or polymorphisms in DNA methyltransferases and certain genes encoding enzymes of the one-carbon metabolism pathway. Indeed, the de novo methyltransferase 3B (DNMT3B) has been recently found to be mutated in several types of cancer and in the immunodeficiency, centromeric region instability and facial anomalies syndrome (ICF), in which these mutations could be related to the loss of global DNA methylation. In addition, mutations in glycine-N-methyltransferase (GNMT) could be associated with a higher risk of hepatocellular carcinoma and liver disease due to an unbalanced S-adenosylmethionine (SAM)/S-adenosylhomocysteine (SAH) ratio, which leads to aberrant methylation reactions. Also, genetic variants of chromatin remodeling proteins and histone tail modifiers are involved in genetic disorders like α thalassemia X-linked mental retardation syndrome, CHARGE syndrome, Cockayne syndrome, Rett syndrome, systemic lupus erythematous, Rubinstein–Taybi syndrome, Coffin–Lowry syndrome, Sotos syndrome, and facioescapulohumeral syndrome, among others. Here, we review the potential genetic alterations with a possible role on epigenetic factors and discuss their contribution to human disease.     

Ultrastructural localization of 5-methylcytosine on DNA and RNA

DNA methylation is the major epigenetic modification and it is involved in the negative regulation of gene expression. Its alteration can lead to neoplastic transformation. Several biomolecular approaches are nowadays used to study this modification on DNA, but also on RNA molecules, which are known to play a role in different biological processes. RNA methylation is one of the most common RNA modifications and 5-methylcytosine presence has recently been suggested in mRNA. However, an analysis of nucleic acid methylation at electron microscope is still lacking. Therefore, we visualized DNA methylation status and RNA methylation sites in the interphase nucleus of HeLa cells and rat hepatocytes by ultrastructural immunocytochemistry and cytochemical staining. This approach represents an efficient alternative to study nucleic acid methylation. In particular, this ultrastructural method makes the visualization of this epigenetic modification on a single RNA molecule possible, thus overcoming the technical limitations for a (pre-)mRNA methylation analysis.     

Emerging connections between DNA methylation and histone acetylation

Modifications of both DNA and chromatin can affect gene expression and lead to gene silencing. Evidence of links between DNA methylation and histone hypoacetylation is accumulating. Several proteins that specifically bind to methylated DNA are associated with complexes that include histone deacetylases (HDACs). In addition, DNA methyltransferases of mammals appear to interact with HDACs. Experiments with animal cells have shown that HDACs are responsible for part of the repressive effect of DNA methylation. Evidence was found in Neurospora that protein acetylation can in some cases affect DNA methylation. The available data suggest that the roles of DNA methylation and histone hypoacetylation, and their relationship with each other, can vary, even within an organism. Some open questions in this emerging field that should be answered in the near future are discussed.     

Epigenetic tinkering and evolution: is there any continuity in the role of cytosine methylation from invertebrates to vertebrates?

The function of DNA methylation has been investigated in depth in vertebrate and plant genomes, establishing that it is involved in gene silencing and transposon control. Data regarding insect methylation, even if still scanty, apparently argue against evolutionary conservation of DNA methylation functions. Cytosine methylation, therefore, proves to be an epigenetic tool repeatedly used to accomplish different functions in different taxa according to a sort of epigenetic tinkering occurring during evolution.     

DNA hypomethylation in the origin and pathogenesis of human diseases

The pathogenesis of any given human disease is a complex multifactorial process characterized by many biologically significant and interdependent alterations. One of these changes, specific to a wide range of human pathologies, is DNA hypomethylation. DNA hypomethylation signifies one of the major DNA methylation states that refers to a relative decrease from the “normal” methylation level. It is clear that disease by itself can induce hypomethylation of DNA; however, a decrease in DNA methylation can also have an impact on the predisposition to pathological states and disease development. This review presents evidence suggesting the involvement of DNA hypomethylation in the pathogenesis of several major human pathologies, including cancer, atherosclerosis, Alzheimer’s disease, and psychiatric disorders. The views expressed in this paper do not necessarily represent those of the US Food and Drug Administration.     

Blastomere biopsy influences epigenetic reprogramming during early embryo development,which impacts neural development and function in resulting mice

Blastomere biopsy is used in preimplantation genetic diagnosis; however, the long-term implications on the offspring are poorly characterized. We previously reported a high risk of memory defects in adult biopsied mice. Here, we assessed nervous function of aged biopsied mice and further investigated the mechanism of neural impairment after biopsy. We found that aged biopsied mice had poorer spatial learning ability, increased neuron degeneration, and altered expression of proteins involved in neural degeneration or dysfunction in the brain compared to aged control mice. Furthermore, the MeDIP assay indicated a genome-wide low methylation in the brains of adult biopsied mice when compared to the controls, and most of the genes containing differentially methylated loci in promoter regions were associated with neural disorders. When we further compared the genomic DNA methylation profiles of 7.5-days postconception (dpc) embryos between the biopsy and control group, we found the whole genome low methylation in the biopsied group, suggesting that blastomere biopsy was an obstacle to de novo methylation during early embryo development. Further analysis on mRNA profiles of 4.5-dpc embryos indicated that reduced expression of de novo methylation genes in biopsied embryos may impact de novo methylation. In conclusion, we demonstrate an abnormal neural development and function in mice generated after blastomere biopsy. The impaired epigenetic reprogramming during early embryo development may be the latent mechanism contributing to the impairment of the nervous system in the biopsied mice, which results in a hypomethylation status in their brains.     

Epigenetic mechanisms in mammals

DNA and histone methylation are linked and subjected to mitotic inheritance in mammals. Yet how methylation is propagated and maintained between successive cell divisions is not fully understood. A series of enzyme families that can add methylation marks to cytosine nucleobases, and lysine and arginine amino acid residues has been discovered. Apart from methyltransferases, there are also histone modification enzymes and accessory proteins, which can facilitate and/or target epigenetic marks. Several lysine and arginine demethylases have been discovered recently, and the presence of an active DNA demethylase is speculated in mammalian cells. A mammalian methyl DNA binding protein MBD2 and de novo DNA methyltransferase DNMT3A and DNMT3B are shown experimentally to possess DNA demethylase activity. Thus, complex mammalian epigenetic mechanisms appear to be dynamic yet reversible along with a well-choreographed set of events that take place during mammalian development.     

DNA damage repair and transcription

Silencing of DNA repair genes plays a critical role in the development of the cancer because these genes, functioning normally, would prevent the accumulation of mutations leading to carcinogenesis. Epigenetic gene silencing is an alternative mechanism to genetic gene aberration, inactivating those genes in cancer. DNA methylation and histone modification are the major factors for epigenetic regulation of gene expression. Here, we describe recent advances in understanding of epigenetic silencing of DNA repair genes and their epigenetic mechanisms involving DNA methylation and histone modification.     

Biological aspects of cytosine methylation in eukaryotic cells.

The existence in eukaryotes of a fifth base, 5-methylcytosine, and of tissue-specific methylation patterns have been known for many years, but except for a general association with inactive genes and chromatin the exact function of this DNA modification has remained elusive. The different hypotheses regarding the role of DNA methylation in regulation of gene expression, chromatin structure, development, and diseases, including cancer are summarized, and the experimental evidence for them is discussed. Structural and functional properties of the eukaryotic DNA cytosine methyltransferase are also reviewed.     

Biological aspects of cytosine methylation in eukaryotic cells

The existence in eukaryotes of a fifth base, 5-methylcytosine, and of tissue-specific methylation patterns have been known for many years, but except for a general association with inactive genes and chromatin the exact function of this DNA modification has remained elusive. The different hypotheses regarding the role of DNA methylation in regulation of gene expression, chromatin structure, development, and diseases, including cancer are summarized, and the experimental evidence for them is discussed. Structural and functional properties of the eukaryotic DNA cytosine methyltransferase are also reviewed.