Development of a Method To Measure DNA Methylation Levels by Using Methyl CpG-Binding Protein and Luciferase-Fused Zinc Finger Protein

Hiraoka, Daisuke; Yoshida, Wataru; Abe, Koichi; Wakeda, Hironobu; Hata, Kenichiro; Ikebukuro, Kazunori

doi:10.1021/ac3015774

Cited by 45 publications

(30 citation statements)

References 34 publications

(48 reference statements)

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“…As we observed changes in gene methylation and were aware that the AR has been reported to be methylated in DU145 cells (Hiraoka et al 2012), we therefore investigated whether there were any changes in AR abundance in the DU145 cells following exposure to different glucose levels. We could not detect AR in the DU145 cells regardless of the level of glucose in the media.…”

Section: Discussionmentioning

confidence: 99%

Hyperglycaemia-induced chemoresistance of prostate cancer cells due to IGFBP2

Biernacka¹,

Uzoh²,

Zeng³

et al. 2013

Endocrine-Related Cancer

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Clinically relevant prostate cancer (PCa) is more frequent in Westernised societies and increasingly men have co-morbidities associated with a Western lifestyle, primarily diabetes, characterised by hyperinsulinaemia and hyperglycaemia. IGFs and their binding proteins (IGFBPs) are important mediators of the effects of nutrition on growth and play a key role in the development of PCa. We used DU145, PC3 and LNCaP PCa cell lines to examine how hyperglycaemia altered their response to docetaxel. Trypan Blue dye-exclusion assay was used to determine the percentage of cell death. Protein abundance was determined using western immunoblotting. Levels of IGFBP2 were measured using an ELISA. IGFBP2 gene silencing was achieved using siRNA technology. DNA methylation was assessed using combined bisulphide restriction analysis. Acetylation status of histones H3 and H4 associated with IGFBP2 gene was assessed using chromatin immunoprecipitation assay. Hyperglycaemia reduced docetaxel-induced apoptosis by 40% for DU145 cells and by 88% for LNCaP cells. This reduced cell death was mediated by a glucose-induced up-regulation of IGFBP2, as silencing IGFBP2 negated the survival effect of high glucose. Glucose increased IGFBP2 via increasing the acetylation of histones associated with the IGFBP2 gene promoter. This finding could have important implications in relation to therapeutic strategies as epigenetic modulation could be reversible.

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Section: Discussionmentioning

confidence: 99%

Hyperglycaemia-induced chemoresistance of prostate cancer cells due to IGFBP2

Biernacka¹,

Uzoh²,

Zeng³

et al. 2013

Endocrine-Related Cancer

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“…Some biomolecules, such as MBD, anti-5-methylcytosine IgG1 antibody, zinc finger (ZF) protein, show inherent capability of methylation-loci specificity and can be directly used for DNAmethylation analysis [46][47][48][49]58]. In 2004, Shiraishi proposed an MBDbased method for screening and identifying 5-mC in the genome [46].…”

Section: Bioaffinity Reactionmentioning

confidence: 99%

“…ZF protein, as another popular DNA-binding protein in mammals [59], was applied to methylation analysis in 2012 [47]. DNA strands containing the target region were first isolated from the sample by sonication.…”

Section: Bioaffinity Reactionmentioning

confidence: 99%

Development of techniques for DNA-methylation analysis

Zhang

Xiao

et al. 2015

TrAC Trends in Analytical Chemistry

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“…Among all the MBD family members (MBD1, MBD2, MBD3, MBD4 and MeCP2), MBD2b protein, a small fraction of the total MBD2 proteins lacking 140 N-terminal aa [5,6], has the highest affinity for methylated DNA and displays the greatest capacity to differentiate between methylated and unmethylated DNA [9]. Based on the binding property of MBD, a series of analytical methods for detection of DNA methylation were established, such as methylated-CpG island recovery assay (MIRA) [10], MBD-isolated genome sequencing (MiGS) [11], and MBD column chromatography [12][13][14][15].…”

Section: Introductionmentioning

confidence: 99%

Engineered SNAP-MBD2b proteins for specific recognition of methylated DNA

et al. 2014

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Methyl-CpG-binding domain (MBD) proteins can specifically recognize and bind methylated CpG sites of DNA, thus repress gene transcription. In this study, we designed and expressed two recombinant proteins, MBD2b and SNAP-MBD2b, in E. coli. An optimized protocol was developed to purify the proteins using Ni-NTA affinity cartridge and cation exchange resin. The engineered proteins purified by this method exhibited more than 93% purity and high binding avidity. We found that both SNAP-MBD2b and MBD2b were prone to aggregate during dialysis. However, this could be prevented by the use of 0.3 mol/L NaCl. The fusion of SNAP-tag with MBD2b significantly enhanced the expression of MBD2b protein in E. coli and reduced the adsorption of MBD2b on solid interfaces involved in protein purification and immobilization. The engineered proteins can be used for the study of interaction with methylated DNA and the assays for DNA methylation.

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Development of a Method To Measure DNA Methylation Levels by Using Methyl CpG-Binding Protein and Luciferase-Fused Zinc Finger Protein

Cited by 45 publications

References 34 publications

Hyperglycaemia-induced chemoresistance of prostate cancer cells due to IGFBP2

Hyperglycaemia-induced chemoresistance of prostate cancer cells due to IGFBP2

Development of techniques for DNA-methylation analysis

Engineered SNAP-MBD2b proteins for specific recognition of methylated DNA

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