A nuclear protein that binds preferentially to methylated DNA in vitro may play a role in the inaccessibility of methylated CpGs in mammalian nuclei

1990 ◽  
Vol 326 (1235) ◽  
pp. 199-205 ◽  
Keyword(s):  
Dna Methylation ◽  
Chromatin Structure ◽  
Nuclear Protein ◽  
Cpg Islands ◽  
Methylated Dna ◽  
Cpg Sites ◽  
Restriction Sites ◽  
Specific Proteins

The effects of DNA methylation on gene expression and chromatin structure suggest the existence of a mechanism in the nucleus capable of distinguishing methylated and non-methylated sequences. We report the finding of a nuclear protein in several vertebrate tissues and cell lines that binds preferentially to methylated DNA in vitro . Its lack of sequence-specific requirements makes it potentially capable of binding to any methylated sequence in mammalian nuclei. An in vivo counterpart of these results is that methylated CpGs are inaccessible to nucleases within nuclei. In contrast, non-methylated CpG sites, located mainly at CpG islands, and restriction sites not containing this dinucleotide, are relatively accessible. The possibility that DNA methylation acts through binding to specific proteins that could alter chromatin structure is discussed.

scholarly journals PGC7 suppresses TET3 for protecting DNA methylation

2013 ◽  
Vol 42 (5) ◽  
pp. 2893-2905 ◽  
Author(s):  
Chunjing Bian ◽  
Xiaochun Yu
Keyword(s):  
Dna Methylation ◽  
Molecular Mechanism ◽  
Biological Process ◽  
Cpg Islands ◽  
Binding Motifs ◽  
Genome Wide Analysis ◽  
Dna Motif ◽  
Genome Wide

Abstract Ten-eleven translocation (TET) family enzymes convert 5-methylcytosine to 5-hydroxylmethylcytosine. However, the molecular mechanism that regulates this biological process is not clear. Here, we show the evidence that PGC7 (also known as Dppa3 or Stella) interacts with TET2 and TET3 both in vitro and in vivo to suppress the enzymatic activity of TET2 and TET3. Moreover, lacking PGC7 induces the loss of DNA methylation at imprinting loci. Genome-wide analysis of PGC7 reveals a consensus DNA motif that is recognized by PGC7. The CpG islands surrounding the PGC7-binding motifs are hypermethylated. Taken together, our study demonstrates a molecular mechanism by which PGC7 protects DNA methylation from TET family enzyme-dependent oxidation.

DNA methylation in lowbush blueberry (Vaccinium angustifolium Ait.) propagated by softwood cutting and tissue culture

2018 ◽  
Vol 98 (5) ◽  
pp. 1035-1044 ◽  
Author(s):  
Juran C. Goyali ◽  
Abir U. Igamberdiev ◽  
Samir C. Debnath
Keyword(s):  
Dna Methylation ◽  
Tissue Culture ◽  
Growth Conditions ◽  
Vaccinium Angustifolium ◽  
Lowbush Blueberry ◽  
Restriction Sites ◽  
Methylation Sensitive Amplification Polymorphism ◽  
Softwood Cutting

Plant DNA methylation is one of the frequent epigenetic variations induced by tissue culture. Global DNA methylation was evaluated in lowbush blueberry (Vaccinium angustifolium Ait.) wild clone QB9C and cultivar Fundy propagated by conventional softwood cutting (SC) and tissue culture (TC) using the methylation-sensitive amplification polymorphism (MSAP) technique. In all, 106 and 107 DNA fragments were amplified using 16 selective primer combinations in SC plants of QB9C and Fundy, respectively. In micropropagated QB9C and Fundy plants, there were 105 and 109 amplified fragments, respectively. Overall, 25% of restriction sites were methylated at the cytosine nucleotide in QB9C plants propagated by SC compared with 19% in Fundy. In contrast, a total of 29% and 20% of restriction sites were methylated at cytosine in micropropagated QB9C and Fundy plants, respectively. Tissue culture plants demonstrated higher methylation events than SC plants in both genotypes. Previously, methylation polymorphism has been detected in TC plants but not in SC counterparts. Different patterns of DNA methylation and polymorphism in the plants propagated in in vitro and in vivo conditions suggest the possibility of involvement of these fragments in the processes of regulating plant growth and development under prevailing growth conditions.

scholarly journals A DNA methylation reader complex that enhances gene transcription

Science ◽  
2018 ◽  
Vol 362 (6419) ◽  
pp. 1182-1186 ◽  
Author(s):  
C. Jake Harris ◽  
Marion Scheibe ◽  
Somsakul Pop Wongpalee ◽  
Wanlu Liu ◽  
Evan M. Cornett ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Arabidopsis Thaliana ◽  
Gene Transcription ◽  
Methylated Dna ◽  
Transposon Insertion ◽  
Activate Gene

DNA methylation generally functions as a repressive transcriptional signal, but it is also known to activate gene expression. In either case, the downstream factors remain largely unknown. By using comparative interactomics, we isolated proteins in Arabidopsis thaliana that associate with methylated DNA. Two SU(VAR)3-9 homologs, the transcriptional antisilencing factor SUVH1, and SUVH3, were among the methyl reader candidates. SUVH1 and SUVH3 bound methylated DNA in vitro, were associated with euchromatic methylation in vivo, and formed a complex with two DNAJ domain-containing homologs, DNAJ1 and DNAJ2. Ectopic recruitment of DNAJ1 enhanced gene transcription in plants, yeast, and mammals. Thus, the SUVH proteins bind to methylated DNA and recruit the DNAJ proteins to enhance proximal gene expression, thereby counteracting the repressive effects of transposon insertion near genes.

scholarly journals DNA Methylation Pyrosequencing Assay Is Applicable for the Assessment of Epigenetic Active Environmental or Clinical Relevant Chemicals

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Ana-Maria Florea
Keyword(s):  
Dna Methylation ◽  
Tumor Cells ◽  
Trichostatin A ◽  
Methylation Status ◽  
Positive Control ◽  
Cpg Sites ◽  
Line1 Methylation ◽  
Negative Controls

Exposure of cells and organisms to stressors might result in epigenetic changes. Here it is shown that investigation of DNA methylation using pyrosequencing is an alternative forin vitroandin vivotoxicological testing of epigenetic effects induced by chemicals and drugs. Anin vitroevaluation of global and CpG site specific DNA methylation upon treatment of cells with chemicals/drugs is shown. Bisulfite genomic sequencing of methylation controls showed high methylation of LINE1 in methylation positive control and low methylation in the negative controls. The CpG sites within the LINE1 element are methylated at different levels.In vitrocell cultures show a methylation level ranging from 56% to 49%. Cultures of drug resistant tumor cells show significant hypomethylation as compared with the originating nonresistant tumor cells. Thein vitrotesting of epigenetically active chemicals (5-methyl-2’-deoxycytidine and trichostatin A) revealed a significant change of LINE1 methylation status upon treatment, while specific CpG sites were more prone to demethylation than others (focal methylation). In conclusion, DNA methylation using pyrosequencing might be used not only for testing epigenetic toxins/drugs but also in risk assessment of drugs, food, and environmental relevant pollutants.

TMOD-02. GENERATION OF A NOVEL MOUSE MODEL FOR BRAIN TUMORS OF THE DNA METHYLATION CLASS “GBM MYCN”

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi215-vi216
Author(s):  
Melanie Schoof ◽  
Carolin Göbel ◽  
Dörthe Holdhof ◽  
Sina Al-Kershi ◽  
Ulrich Schüller
Keyword(s):  
Dna Methylation ◽  
Brain Tumors ◽  
Molecular Mechanisms ◽  
Treatment Options ◽  
Tumor Development ◽  
Model Systems ◽  
Preclinical Research ◽  
Human Gbm

Abstract DNA methylation based classification of brain tumors has revealed a high heterogeneity between tumors and led to the description of multiple distinct subclasses. The increasing subdivision of tumors can help to understand molecular mechanisms of tumor development and to improve therapy if appropriate model systems for preclinical research are available. Multiple recent publications have described a subgroup of pediatric glioblastoma which is clearly separable from other pediatric and adult glioblastoma in its DNA methylation profile (GBM MYCN). Many cases in this group are driven by MYCN amplifications and harbor TP53 mutations. These tumors almost exclusively occur in children and were further described as highly aggressive with a median overall survival of only 14 months. In order to further investigate the biology and treatment options of these tumors, we generated hGFAP-cre::TP53 Fl/Fl ::lsl-MYCN mice. These mice carry a loss of TP53 and show aberrant MYCN expression in neural precursors of the central nervous system. The animals develop large forebrain tumors within the first 80 days of life with 100 % penetrance. These tumors resemble human GBM MYCN tumors histologically and are sensitive to AURKA and ATR inhibitors in vitro. We believe that further characterization of the model and in vivo treatment studies will pave the way to improve treatment of patients with these highly aggressive tumors.

scholarly journals DNA methylation specifies chromosomal localization of MeCP2.

1996 ◽  
Vol 16 (1) ◽  
pp. 414-421 ◽  
Author(s):  
X Nan ◽  
P Tate ◽  
E Li ◽  
A Bird
Keyword(s):  
Dna Methylation ◽  
Cultured Cells ◽  
Centromeric Heterochromatin ◽  
Intact Protein ◽  
Chromosomal Protein ◽  
Mouse Cells ◽  
Low Levels ◽  
Mutant Cells

MeCP2 is a chromosomal protein that is concentrated in the centromeric heterochromatin of mouse cells. In vitro, the protein binds preferentially to DNA containing a single symmetrically methylated CpG. To find out whether the heterochromatic localization of MeCP2 depended on DNA methylation, we transiently expressed MeCP2-LacZ fusion proteins in cultured cells. Intact protein was targeted to heterochromatin in wild-type cells but was inefficiently localized in mutant cells with low levels of genomic DNA methylation. Deletions within MeCP2 showed that localization to heterochromatin required the 85-amino-acid methyl-CpG binding domain but not the remainder of the protein. Thus MeCP2 is a methyl-CpG-binding protein in vivo and is likely to be a major mediator of downstream consequences of DNA methylation.

scholarly journals Characterization of human gastric carcinoma-related methylation of 9 miR CpG islands and repression of their expressions in vitro and in vivo

BMC Cancer ◽  
2012 ◽  
Vol 12 (1) ◽  
Author(s):  
Yantao Du ◽  
Zhaojun Liu ◽  
Liankun Gu ◽  
Jing Zhou ◽  
Bu-dong Zhu ◽  
...  
Keyword(s):  
Gastric Carcinoma ◽  
Cpg Islands ◽  
Human Gastric Carcinoma

scholarly journals Two Isoforms of Npap60 (Nup50) Differentially Regulate Nuclear Protein Import

2010 ◽  
Vol 21 (4) ◽  
pp. 630-638 ◽  
Author(s):  
Yutaka Ogawa ◽  
Yoichi Miyamoto ◽  
Munehiro Asally ◽  
Masahiro Oka ◽  
Yoshinari Yasuda ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Protein ◽  
Time Lapse ◽  
Binding Assays ◽  
Vitro Binding ◽  
Importin Α ◽  
Nuclear Protein Import

Npap60 (Nup50) is a nucleoporin that binds directly to importin α. In humans, there are two Npap60 isoforms: the long (Npap60L) and short (Npap60S) forms. In this study, we provide both in vitro and in vivo evidence that Npap60L and Npap60S function differently in nuclear protein import. In vitro binding assays revealed that Npap60S stabilizes the binding of importin α to classical NLS-cargo, whereas Npap60L promotes the release of NLS-cargo from importin α. In vivo time-lapse experiments showed that when the Npap60 protein level is controlled, allowing CAS to efficiently promote the dissociation of the Npap60/importin α complex, Npap60S and Npap60L suppress and accelerate the nuclear import of NLS-cargo, respectively. These results demonstrate that Npap60L and Npap60S have opposing functions and suggest that Npap60L and Npap60S levels must be carefully controlled for efficient nuclear import of classical NLS-cargo in humans. This study provides novel evidence that nucleoporin expression levels regulate nuclear import efficiency.

Epigenetic approaches to cancer therapy

2004 ◽  
Vol 32 (6) ◽  
pp. 1095-1097 ◽  
Author(s):  
J.A. Plumb ◽  
N. Steele ◽  
P.W. Finn ◽  
R. Brown
Keyword(s):  
Dna Methylation ◽  
Cell Line ◽  
Gene Promoter ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Tumour Suppressor Genes ◽  
Control Of Gene Expression ◽  
Hmlh1 Gene

Histone deacetylation and DNA methylation have a central role in the control of gene expression, including transcriptional repression of tumour suppressor genes. Loss of DNA mismatch repair due to methylation of the hMLH1 gene promoter results in resistance to cisplatin in vitro and in vivo. The cisplatin-resistant cell line A2780/cp70 is 8-fold more resistant to cisplatin than the non-resistant cell line, and has the hMLH1 gene methylated. Treatment with an inhibitor of DNA methyltransferase, DAC (2-deoxy-5′-azacytidine), results in a partial reversal of DNA methylation, re-expression of MLH1 (mutL homologue 1) and sensitization to cisplatin both in vitro and in vivo. PXD101 is a novel hydroxamate type histone deacetylase inhibitor that shows antitumour activity in vivo and is currently in phase I clinical evaluation. Treatment of A2780/cp70 tumour-bearing mice with DAC followed by PXD101 results in a marked increase in the number of cells that re-express MLH1. Since the clinical use of DAC may be limited by toxicity and eventual re-methylation of genes, we suggest that the combination of DAC and PXD101 could have a role in increasing the efficacy of chemotherapy in patients with tumours that lack MLH1 expression due to hMLH1 gene promoter methylation.

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