Changes of the methylation pattern of the c-myc gene during in vitro aging of IMR90 human embryonic fibroblasts

Halle, Joern-Peter; Schmidt, Claudia C.; Adam, Gerold

doi:10.1016/0921-8734(95)90002-0

Cited by 11 publications

(7 citation statements)

References 47 publications

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“…Nuclear labeling with 5-methylcytosine and 5-methylcytidine has been used to assess methylation status in many reports (e.g., Halle et al , 1995, Havlis and Trbusek, 2002). In addition, both of these markers have also been reported to be associated with ribosomal RNA (rRNA) (Dunn, 1960, Tantravahi et al , 1981, Obara et al , 1982, Negre et al , 1989).…”

Section: Resultsmentioning

confidence: 99%

Epigenetic changes in Alzheimer's disease: Decrements in DNA methylation

Mastroeni

Grover

Delvaux

et al. 2010

Neurobiology of Aging

340

253

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DNA methylation is a vital component of the epigenetic machinery that orchestrates changes in multiple genes and helps regulate gene expression in all known vertebrates. We evaluated immunoreactivity for two markers of DNA methylation and eight methylation maintenance factors in entorhinal cortex layer II, a region exhibiting substantial Alzheimer's disease (AD) pathology in which expression changes have been reported for a wide variety of genes. We show, for the first time, neuronal immunoreactivity for all 10 of the epigenetic markers and factors, with highly significant decrements in AD cases. These decrements were particularly marked in PHF1/PS396 immunoreactive, neurofibrillary tangle-bearing neurons. In addition, two of the DNA methylation maintenance factors, DNMT1 and MBD2, have been reported also to interact with ribosomal RNAs and ribosome synthesis. Consistent with these findings, DNMT1 and MBD2, as well as p66α, exhibited punctate cytoplasmic immunoreactivity that co-localized with the ribosome markers RPL26 and 5.8s rRNA in ND neurons. By contrast, AD neurons generally lacked such staining, and there was a qualitative decrease in RPL26 and 5.8s rRNA immunoreactivity. Collectively, these findings suggest epigenetic dysfunction in AD-vulnerable neurons.

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Section: Resultsmentioning

confidence: 99%

Epigenetic changes in Alzheimer's disease: Decrements in DNA methylation

Mastroeni

Grover

Delvaux

et al. 2010

Neurobiology of Aging

340

253

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“…IMR 90 (human lung fibroblasts; ATCC No. CCL 186) were cultivated in DMEM with 10% FCS (both Gibco, Eggenstein, FRG) and subcultivated as described before (33). Cells were seeded at 3 ϫ 10 3 cells/cm 2 and for every protein preparation the cell density number (cells/cm 2 ) was determined.…”

Section: Methodsmentioning

confidence: 99%

“…Moore et al measured 6300 total c-Myc protein mol-ecules per cell in serum-stimulated human MRC5 fibroblasts (33). This smaller amount could be cell type specific.…”

Section: Determination Of the Number Of C-myc Protein Molecules Per Cmentioning

confidence: 99%

Determination of Copy Number of c-Myc Protein per Cell by Quantitative Western Blotting

Rudolph

Adam²,

Simm

1999

Analytical Biochemistry

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“…Previous studies using methylation-sensitive restriction endonucleases did not detect m CpGs until ϳ5 kb downstream of this region (84 -86), suggesting that this CpG island is exceptional, because it is completely demethylated over a much larger range than previously reported for CpG islands (usually about 1 kb (87)). A small subset of CpG dinucleotides downstream of the promoter (84,86,88,89) as well as within the promoter region in mouse and human cells (85,90) have been examined by methylation-sensitive restriction endonucleases and found to be unmethylated. Six CpG dinucleotides within the origin region (positions 1036 -1156) were found to be unmethylated using the hydrazine method (85), but this method would not detect methylation in less than 25% of the molecules (53,85).…”

Section: Dna Methylation At Mammalian Replication Originsmentioning

confidence: 99%

“…A small subset of CpG dinucleotides downstream of the promoter (84,86,88,89) as well as within the promoter region in mouse and human cells (85,90) have been examined by methylation-sensitive restriction endonucleases and found to be unmethylated. Six CpG dinucleotides within the origin region (positions 1036 -1156) were found to be unmethylated using the hydrazine method (85), but this method would not detect methylation in less than 25% of the molecules (53,85). Another study using the bisulfite method also concluded that the human c-myc origin was unmethylated (28), but this study examined only 10% of the CpGs (positions 764 -1095), and these were located in the region of lowest CpG density (Fig.…”

Section: Dna Methylation At Mammalian Replication Originsmentioning

confidence: 99%

DNA Methylation at Mammalian Replication Origins

Rein

Kobayashi²,

Malott³

et al. 1999

Journal of Biological Chemistry

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In Escherichia coli, DNA methylation regulates both origin usage and the time required to reassemble prereplication complexes at replication origins. In mammals, at least three replication origins are associated with a high density cluster of methylated CpG dinucleotides, and others whose methylation status has not yet been characterized have the potential to exhibit a similar DNA methylation pattern. One of these origins is found within the ϳ2-kilobase pair region upstream of the human c-myc gene that contains 86 CpGs. Application of the bisulfite method for detecting 5-methylcytosines at specific DNA sequences revealed that this region was not methylated in either total genomic DNA or newly synthesized DNA. Therefore, DNA methylation is not a universal component of mammalian replication origins. To determine whether or not DNA methylation plays a role in regulating the activity of origins that are methylated, the rate of remethylation and the effect of hypomethylation were determined at origin ␤ (ori-␤), downstream of the hamster DHFR gene. Remethylation at ori-␤ did not begin until ϳ500 base pairs of DNA was synthesized, but it was then completed by the time that 4 kilobase pairs of DNA was synthesized (<3 min after release into S phase). Thus, DNA methylation cannot play a significant role in regulating reassembly of prereplication complexes in mammalian cells, as it does in E. coli. To determine whether or not DNA methylation plays any role in origin activity, hypomethylated hamster cells were examined for ori-␤ activity. Cells that were >50% reduced in methylation at ori-␤ no longer selectively activated ori-␤. Therefore, at some loci, DNA methylation either directly or indirectly determines where replication begins.In early embryos undergoing rapid cell cleavage (e.g. frogs, flies, sea urchin, fish), initiation of DNA replication appears neither to require specific DNA sequences nor to occur at specific DNA sites. However, as development progresses past the blastula stage, initiation of DNA replication begins to occur at specific sites (1, 2). Thus, it is not surprising that initiation sites for DNA replication in cultured mammalian cells occur at specific genomic loci (3). For example, a 200-kb 1 region at the human ␤-globin gene (4, 5) and a 500-kb region at the mouse IgH gene (6) are both replicated from a single initiation locus. Moreover, what previously had been viewed as random initiation events distributed throughout "initiation zones" in Schizosaccharomyces pombe and hamster cells more likely reflects the presence of several strongly preferred initiation sites (7,8).Nevertheless, the precise size and composition of these initiation loci, as well as the parameters that define them remain the subject of intense investigation.The fact that specific sites for initiation of DNA replication can be developmentally acquired makes it clear that initiation sites in the metazoa are determined at least in part by epigenetic parameters. These parameters include nuclear structure, chromatin structure, and even...

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Changes of the methylation pattern of the c-myc gene during in vitro aging of IMR90 human embryonic fibroblasts

Cited by 11 publications

References 47 publications

Epigenetic changes in Alzheimer's disease: Decrements in DNA methylation

Epigenetic changes in Alzheimer's disease: Decrements in DNA methylation

Determination of Copy Number of c-Myc Protein per Cell by Quantitative Western Blotting

DNA Methylation at Mammalian Replication Origins

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