Gene reactivation: a tool for the isolation of mammalian DNA methylation mutants.
TL;DR: It is proposed that the phenotype of tsm cells is due to a mutation involved in the regulation of DNA methylation, and the further characterization of this and other mammalian mutants should help to clarify the physiological role of DNAmethylation, as well as its regulation.
Abstract: We report the isolation and characterization of a mammalian strain (tsm) that has a temperature-sensitive mutation in DNA methylation. The isolation procedure was based on the observation that treatment of a CHO TK- MT- cell line with demethylating agents introduces up to 46% demethylation, resulting in phenotypic reversion and transcriptional activation of the thymidine kinase (TK) and metallothionein (MT) genes at frequencies ranging from 1% to 59%. Seven thousand individual colonies from an EMS-mutagenized CHO TK- MT- population were screened for spontaneous reversion to TK+ phenotype after treatment at 39 degrees C. Successful isolates were subsequently examined for MT+ reversion. A single clone (tsm) was obtained that showed temperature-dependent reactivation of both TK and MT genes at frequencies of 7.2 X 10(-4) and 6 X 10(-4), respectively. The tsm cells were viable at 39 degrees C and showed no increased mutation frequency. Reactivation correlated with transcriptional activation of the respective genes, whereas backreversion to the TK- phenotype was associated with transcriptional inactivation. TK- backrevertants were reactivable again with demethylating agents. Although demethylation in tsm cells was not detectable by HPLC, Southern blot analysis revealed that reactivants, irrespective of their mode of generation, showed specific demethylation of both TK and MT genes. Also, after about 150 cell generations after treatment, reactivants from both temperature-induced tsm and cells exposed to demethylating agents gained 60% and 23%, respectively, in 5-methylcytosine (5mC). It is proposed that the phenotype of tsm cells is due to a mutation involved in the regulation of DNA methylation. The further characterization of this and other mammalian mutants should help to clarify the physiological role of DNA methylation, as well as its regulation.
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TL;DR: It is proposed that epigenetic defects in germline cells due to loss of methylation can be repaired by recombination at meiosis but that some are transmitted to offspring.
Abstract: Evidence from many sources shows that the control of gene expression in higher organisms is related to the methylation of cytosine in DNA, and that the pattern of methylation is inherited. Loss of methylation, which can result from DNA damage, will lead to heritable abnormalities in gene expression, and these may be important in oncogenesis and aging. Transformed permanent lines often lose gene activity through de novo methylation. It is proposed that epigenetic defects in germline cells due to loss of methylation can be repaired by recombination at meiosis but that some are transmitted to offspring.
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TL;DR: It is suggested that mutation-like gene inactivation due to CpG island methylation is widespread in many cell lines and could explain the loss of cell type-specific functions in culture.
691 citations
TL;DR: The possibility that the ‘histone code’ and the DNA cytosine methylation pattern are closely linked is suggested, suggesting ways in which DNA methylation patterns may be established during normal development.
Abstract: There is tremendous ferment in the field of epigenetics as the relationships between chromatin structure and DNA methylation patterns become clearer. Central to this activity is the realization that the 'histone code', which involves the post-translational modification of histones and which has important ramifications for chromatin structure, may be linked to the DNA cytosine methylation pattern. New discoveries have suggested that histone lysine 9 methylation is implicated in the spread of heterochromatin in Drosophila and other organisms. Very recently it has been found that histone lysine 9 methylation is also necessary for some DNA methylation in Neurospora and plants. There is therefore the possibility that these two processes are closely linked, suggesting ways in which DNA methylation patterns may be established during normal development. Understanding these processes is fundamental to understanding what goes awry during the process of aging and carcinogenesis where DNA methylation patterns become substantially altered and contribute to the malignant phenotype.
297 citations
TL;DR: The evidence available for the role epigenetics on host- Pathogen interactions, and the utility and versatility of the epigenetic technologies available that can be cross-applied to host-pathogen studies are reviewed are reviewed.
Abstract: A growing body of evidence points towards epigenetic mechanisms being responsible for a wide range of biological phenomena, from the plasticity of plant growth and development to the nutritional control of caste determination in honeybees and the etiology of human disease (e.g., cancer). With the (partial) elucidation of the molecular basis of epigenetic variation and the heritability of certain of these changes, the field of evolutionary epigenetics is flourishing. Despite this, the role of epigenetics in shaping host–pathogen interactions has received comparatively little attention. Yet there is plenty of evidence supporting the implication of epigenetic mechanisms in the modulation of the biological interaction between hosts and pathogens. The phenotypic plasticity of many key parasite life-history traits appears to be under epigenetic control. Moreover, pathogen-induced effects in host phenotype may have transgenerational consequences, and the bases of these changes and their heritability probably have an epigenetic component. The significance of epigenetic modifications may, however, go beyond providing a mechanistic basis for host and pathogen plasticity. Epigenetic epidemiology has recently emerged as a promising area for future research on infectious diseases. In addition, the incorporation of epigenetic inheritance and epigenetic plasticity mechanisms to evolutionary models and empirical studies of host–pathogen interactions will provide new insights into the evolution and coevolution of these associations. Here, we review the evidence available for the role epigenetics on host–pathogen interactions, and the utility and versatility of the epigenetic technologies available that can be cross-applied to host–pathogen studies. We conclude with recommendations and directions for future research on the burgeoning field of epigenetics as applied to host–pathogen interactions.
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TL;DR: Analysis of chromatin structure and DNA methylation of the ev-1 provirus in 1836 cells revealed the presence (in a fraction of the proviruses) of both DNase I hypersensitive sites in the long terminal repeats and in gag and a pattern of cleavage sites for methyl-sensitive restriction endonuclease not found in a nonexpressing sibling, lending strong support to the role ofDNA methylation in the control of gene expression.
Abstract: The endogenous avian provirus ev-1 is widespread in white leghorn chickens. Although it has no major structural defects, ev-1 has not been associated with any phenotype and is ordinarily expressed at a very low level. In this report, we describe a chicken embryo (Number 1836) cell culture containing both ev-1 and ev-6 which spontaneously expressed the ev-1 provirus. This culture released a high level of noninfectious virions containing a full complement of virion structural (gag) proteins but devoid of reverse transcriptase activity or antigen. These virions contained 70S RNA closely related to the genome of Rous-associated virus type 0, but identifiable as the ev-1 genome by oligonucleotide mapping. A fraction of the RNA molecules in the 70S complex were unusual in that they were polyadenylated 100 to 200 nucleotides downstream of the usual polyadenylation site. Eight sibling embryo cultures did not share this unusual phenotype with 1836, indicating that it was not inherited. However, an identical phenotype was inducible in the sibling cultures by treatment with 5-azacytidine, an inhibitor of DNA methylation, and the induced expression was stable for more than 10 generations. Analysis of chromatin structure and DNA methylation of the ev-1 provirus in 1836 cells revealed the presence (in a fraction of the proviruses) of both DNase I hypersensitive sites in the long terminal repeats and in gag and a pattern of cleavage sites for methyl-sensitive restriction endonuclease not found in a nonexpressing sibling. These results lend strong support to the role of DNA methylation in the control of gene expression. Additionally, they explain the lack of phenotype associated with ev-1 as due to a combination of its low expression and defectiveness in pol and env.
70 citations
TL;DR: Interestingly, an 8 nucleotide sequence (TGTAAATA) has been conserved in sequence and position in the 3' untranslated regions of each metallothionein mRNA sequenced thus far.
Abstract: Polyadenylated RNA was extracted from a cadmium resistant Chinese hamster (CHO) cell line, enriched for metal-induced, abundant RNA sequences and cloned as double-stranded cDNA in the plasmid pBR322. Two cDNA clones, pCHMT1 and pCHMT2, encoding two Chinese hamster isometallothioneins were identified, and the nucleotide sequence of each insert was determined. The two Chinese hamster metallothioneins show nucleotide sequence homologies of 80% in the protein coding region and approximately 35% in both the 5' and 3' untranslated regions. Interestingly, an 8 nucleotide sequence (TGTAAATA) has been conserved in sequence and position in the 3' untranslated regions of each metallothionein mRNA sequenced thus far. Estimated nucleotide substitution rates derived from interspecies comparisons were used to calculate a metallothionein gene duplication time of 45 to 120 million years ago.
63 citations
TL;DR: Data support a causative relationship between DNA methylation and decreased gene expression in herpes simplex patients and all TK+ reexpressors examined had DNA rearrangements involving TK DNA.
Abstract: Plasmid pTKx-1, containing the herpes simplex virus gene for thymidine kinase (TK) inserted into the BamHI site of plasmid pBR322, was introduced into Ltk- cells by calcium phosphate precipitation in the absence of carrier DNA. Line 101 is a TK+ derivative of Ltk- that contains multiple copies of pTKx-1 in a multimeric structure. A derivative of 101 that retained but no longer expressed the herpes simplex TK genes (termed 101BU1) and derivatives of line 101BU1 that reexpressed the genes (termed 101H1, 101HC, and 101HG) were selected. The TK genes in 101BU1 were hypermethylated relative to those in the TK+ parent and derivatives. Growth of 101BU1 in the presence of the methylation inhibitor 5-azacytidine resulted in an average 13-fold increase in the number of TK+ reexpressors, DNA from 101BU1 was inactive in secondary gene transfer, whereas DNA from 101 and from TK+ reexpressors was active. These data support a causative relationship between DNA methylation and decreased gene expression. All TK+ reexpressors examined had DNA rearrangements involving TK DNA.
59 citations
TL;DR: Biochemical and genetic data indicated that the TK(-) clones derived from one revertant are phenotypically different, and the phenotypes displayed by these cell lines are stable and do not depend upon the continued presence of the selective agent.
Abstract: In order to obtain a large collection of Chinese hamster cell clones defective in thymidine kinase (TK - ), BrdU r selection experiments have been performed on wild-type and revertant TK + cell lines. No clones ( -9 ) were obtained from the wild-type TK + cell line by single-step selection. In contrast, revertant TK + clones readily gave rise to stable TK - derivatives (1 - 2 × 10 -4 ). Both wild-type and revertant TK + clones spontaneously yielded 8-AG r colonies with the same frequency (1 - 5 × 10 -6 ), suggesting that the differences between wild-type and revertant cell lines specifically affected selection of the TK - phenotype. The increased frequency of TK - clones reflects perhaps the number (ploidy) or character of the autosomal TK loci in TK + revertants, or perhaps the mechanisms which regulate expression of the TK genes. Several mutagens, EMS, MNNG and UV, stimulated the TK + revertants9 frequency of TK - subclones only slightly ( - clones derived from one revertant are phenotypically different. The phenotypes displayed by these cell lines are stable and do not depend upon the continued presence of the selective agent.
53 citations
TL;DR: It is concluded that CHO cells are at least diploid at the ODC locus, but that only one allele is active, and the possibility that ethyl methane sulfonate is not just a classical mutagen but may also induce gene inactivations that are revertible by 5-azacytidine is suggested.
Abstract: A group of Chinese hamster ovary (CHO) cell mutants deficient in ornithine decarboxylase (ODC) activity are described and compared to the prototype mutant reported previously (21). Although all mutants belong to the same complementation group, they can be divided into two classes: those with some residual enzyme activity and those with no activity. All mutants are putrescine auxotrophs, but they differ in their ability to utilize the enzyme's substrate, ornithine, a property which correlates with the amount of residual enzyme activity. The mutants also differ in their frequency of reversion to prototrophy. The leaky mutants revert at a high rate by overproducing a partially defective enzyme by a gene amplification mechanism similar to that leading to the ornithine analog-resistant mutants which have elevated enzyme levels. Spontaneous reversion in the null mutants is rare. However, one null mutant, which was induced with ethyl methane sulfonate and which makes ODC mRNA but no active enzyme, is nevertheless revertible with 5-azacytidine. We conclude that CHO cells are at least diploid at the ODC locus, but that only one allele is active. Further studies suggest the possibility that ethyl methane sulfonate is not just a classical mutagen but may also induce gene inactivations that are revertible by 5-azacytidine.
52 citations