Using a medium-throughput comet assay to evaluate the global DNA methylation status of single cells

Abstract Background Epigenetics refers to an alteration in gene expression without alteration in the sequence of DNA and this process may be affected by environmental factors and lifestyle like cigarette smoking. This study was designed to evaluate the potential effect of cigarette smoking on the global DNA methylation status and the transcription level of protamine 1 and protamine 2 in human spermatozoa. A total of 188 semen samples were collected from men with a mean age of 34.9 ± 5.8 years old (98 heavy smokers and 90 non-smokers). The DNA and RNA were isolated from purified spermatozoa, then the status of global DNA methylation and the transcription level of protamine 1 and protamine 2 were evaluated using ELISA and qPCR, respectively. The chromatin non-condensation and DNA fragmentation in human spermatozoa were evaluated using chromomycin A3 staining and TUNEL assay, respectively. Results A significant increase has been found in the status of global DNA methylation in spermatozoa of heavy smokers compared to non-smokers (7.69 ± 0.69 ng/μl vs. 4.90 ± 0.40 ng/μl, P < 0.001). Additionally, a significant reduction has been found in transcription level of protamine 1 (25.49 ± 0.31 vs. 23.94 ± 0.40, P < 0.001) and protamine 2 (28.27 ± 0.39 vs. 23.45 ± 0.30, P < 0.001) in heavy smokers. A downregulation has been found in the transcription level of protamine 1 and protamine 2 with a fold change of 0.497 and 0.047, respectively. A significant increase has been shown in the level of DNA fragmentation and chromatin non-condensation in heavy smokers compared to non-smokers (P < 0.001). On the other hand, a significant positive correlation has been found between sperm chromatin non-condensation, sperm DNA fragmentation, transcription level of protamine 1, transcription level of protamine 2, and global DNA methylation status (r = 0.304, P < 0.001; r = 0.399, P < 0.001; r = 0.216, P = 0.003; r = 0.494, P < 0.001, respectively). Conclusion Tobacco cigarette smoking has a potential influence on the global DNA methylation and the transcription level of protamine genes in human spermatozoa, and consequently, affect negatively on the semen parameters.

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The development and validation of EpiComet-Chip, a modified high-throughput comet assay for the assessment of DNA methylation status

Environmental and Molecular Mutagenesis ◽

10.1002/em.22101 ◽

2017 ◽

Vol 58 (7) ◽

pp. 508-521 ◽

Cited By ~ 17

Author(s):

Todd A. Townsend ◽

Marcus C. Parrish ◽

Bevin P. Engelward ◽

Mugimane G. Manjanatha

Keyword(s):

Dna Methylation ◽

Comet Assay ◽

High Throughput ◽

Methylation Status ◽

Development And Validation

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Overexpression of CDKN2B (p15INK4B) and altered global DNA methylation status in mesenchymal stem cells of high-risk myelodysplastic syndromes

Leukemia ◽

10.1038/leu.2014.197 ◽

2014 ◽

Vol 28 (11) ◽

pp. 2241-2244 ◽

Cited By ~ 8

Author(s):

A Poloni ◽

G Maurizi ◽

D Mattiucci ◽

S Amatori ◽

B Fogliardi ◽

...

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Mesenchymal Stem Cells ◽

High Risk ◽

Myelodysplastic Syndromes ◽

Methylation Status ◽

Global Dna Methylation

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Repeats as global DNA methylation marker in bovine preimplantation embryos

Czech Journal of Animal Science ◽

10.17221/29/2016-cjas ◽

2017 ◽

Vol 62 (No. 2) ◽

pp. 43-50 ◽

Cited By ~ 1

Author(s):

W. Li ◽

A. Van Soom ◽

L. Peelman

Keyword(s):

Dna Methylation ◽

Mutation Rate ◽

Methylation Level ◽

Methylation Status ◽

Repetitive Sequences ◽

Cell Types ◽

Blastocyst Stage ◽

Repeat Sequence ◽

Preimplantation Embryos ◽

Global Dna Methylation

DNA methylation undergoes dynamic changes and is a crucial part of the epigenetic regulation during mammalian early development. To determine the DNA methylation levels in bovine embryos, we applied a bisulfite sequencing based method aimed at repetitive sequences including three retrotransposons (L1_BT, BovB, and ERV1-1-I_BT) and Satellite I. A more accurate estimate of the global DNA methylation level compared to previous methods using only one repeat sequence, like Alu, could be made by calculation of the weighted arithmetic mean of multiple repetitive sequences, considering the copy number of each repetitive sequence. Satellite I and L1_BT showed significant methylation reduction at the blastocyst stage, while BovB and ERV1-1-I_BT showed no difference. The mean methylation level of the repetitive sequences during preimplantation development was the lowest at the blastocyst stage. No methylation difference was found between embryos cultured in 5% and 20% O<sub>2</sub>. Because mutations of CpGs negatively influence the calculation accuracy, we checked the mutation rate of the sequenced CpG sites. Satellite I and L1_BT showed a relatively low mutation rate (1.92 and 3.72% respectively) while that of ERV1-1-I_BT and BovB was higher (11.95 and 24% respectively). Therefore we suggest using a combination of repeats with low mutation rate, taking into account the proportion of each sequence, as a relatively quick marker for the global DNA methylation status of preimplantation stages and possibly also for other cell types.

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Altered Adipose Tissue DNA Methylation Status in Metabolic Syndrome: Relationships Between Global DNA Methylation and Specific Methylation at Adipogenic, Lipid Metabolism and Inflammatory Candidate Genes and Metabolic Variables

Journal of Clinical Medicine ◽

10.3390/jcm8010087 ◽

2019 ◽

Vol 8 (1) ◽

pp. 87 ◽

Cited By ~ 23

Author(s):

Daniel Castellano-Castillo ◽

Isabel Moreno-Indias ◽

Lidia Sanchez-Alcoholado ◽

Bruno Ramos-Molina ◽

Juan Alcaide-Torres ◽

...

Keyword(s):

Metabolic Syndrome ◽

Dna Methylation ◽

Adipose Tissue ◽

Lipid Metabolism ◽

Environmental Factors ◽

Metabolic Diseases ◽

Methylation Status ◽

Serum Triglyceride ◽

Positive Association ◽

Global Dna Methylation

Metabolic syndrome (MetS) has been postulated to increase the risk for type 2 diabetes, cardiovascular disease and cancer. Adipose tissue (AT) plays an important role in metabolic homeostasis, and AT dysfunction has an active role in metabolic diseases. MetS is closely related to lifestyle and environmental factors. Epigenetics has emerged as an interesting landscape to evaluate the possible interconnection between AT and metabolic disease, since it can be modulated by environmental factors and metabolic status. The aim of this study was to determine whether MetS has an impact on the global DNA methylation pattern and the DNA methylation of several genes related to adipogenesis (PPARG, PPARA), lipid metabolism (RXRA, SREBF2, SREBF1, SCD, LPL, LXRb), and inflammation (LRP1 C3, LEP and TNF) in visceral adipose tissue. LPL and TNF DNA methylation values were significantly different in the control-case comparisons, with higher and lower methylation respectively in the MetS group. Negative correlations were found between global DNA methylation (measured by LINE-1 methylation levels) and the metabolic deterioration and glucose levels. There were associations among variables of MetS, BMI, and HOMA-IR with DNA methylation at several CpG positions for the studied genes. In particular, there was a strong positive association between serum triglyceride levels (TG) with PPARA and LPL methylation levels. TNF methylation was negatively associated with the metabolic worsening and could be an important factor in preventing MetS occurrence according to logistic regression analysis. Therefore, global DNA methylation and methylation at specific genes related to adipogenesis, lipid metabolism and inflammation are related to the etiology of MetS and might explain in part some of the features associated to metabolic disorders.

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Alteration in global DNA methylation status following preconditioning injury influences axon growth competence of the sensory neurons

Experimental Neurology ◽

10.1016/j.expneurol.2020.113177 ◽

2020 ◽

Vol 326 ◽

pp. 113177

Author(s):

Hae Young Shin ◽

Kyung Kim ◽

Min Jung Kwon ◽

Young Joo Oh ◽

Eun Hye Kim ◽

...

Keyword(s):

Dna Methylation ◽

Sensory Neurons ◽

Methylation Status ◽

Axon Growth ◽

Global Dna Methylation

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Intertumor and Intratumor NY-ESO-1 Expression Heterogeneity Is Associated with Promoter-Specific and Global DNA Methylation Status in Ovarian Cancer

Clinical Cancer Research ◽

10.1158/1078-0432.ccr-07-5279 ◽

2008 ◽

Vol 14 (11) ◽

pp. 3283-3290 ◽

Cited By ~ 67

Author(s):

Anna Woloszynska-Read ◽

Paulette Mhawech-Fauceglia ◽

Jihnhee Yu ◽

Kunle Odunsi ◽

Adam R. Karpf

Keyword(s):

Ovarian Cancer ◽

Dna Methylation ◽

Methylation Status ◽

Global Dna Methylation

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Chrysin Modulates Aberrant Epigenetic Variations and Hampers Migratory Behavior of Human Cervical (HeLa) Cells

Frontiers in Genetics ◽

10.3389/fgene.2021.768130 ◽

2022 ◽

Vol 12 ◽

Author(s):

Ritu Raina ◽

Abdulmajeed G. Almutary ◽

Sali Abubaker Bagabir ◽

Nazia Afroze ◽

Sharmila Fagoonee ◽

...

Keyword(s):

Dna Methylation ◽

Tumor Suppressor ◽

Histone Modification ◽

Hela Cells ◽

Tumor Suppressor Genes ◽

Methylation Status ◽

Global Dna Methylation ◽

Dependent Manner ◽

Suppressor Genes ◽

Protein Levels

Purpose: Plant-derived phytochemicals have shown epigenetic modulatory effect in different types of cancer by reversing the pattern of DNA methylation and chromatin modulation, thereby restoring the function of silenced tumor-suppressor genes. In the present study, attempts have been made to explore chrysin-mediated epigenetic alterations in HeLa cells.Methods: Colony formation and migration assays followed by methylation-specific PCR for examining the methylation status of CpG promoters of various tumor-suppressor genes (TSGs) and the expression of these TSGs at the transcript and protein levels were performed. Furthermore, global DNA methylation; biochemical activities of DNA methyltransferases (DNMTs), histone methyl transferases (HMTs), histone deacetylases (HDACs), and histone acetyl transferases (HATs) along with the expression analysis of chromatin-modifying enzymes; and H3 and H4 histone modification marks analyses were performed after chrysin treatment.Results: The experimental analyses revealed that chrysin treatment encourages cytostatic behavior as well as inhibits the migration capacity of HeLa cells in a time- and dose-dependent manner. Chrysin reduces the methylation of various tumor-suppressor genes, leading to their reactivation at mRNA and protein levels. The expression levels of various chromatin-modifying enzymes viz DNMTs, HMTs, HDACs, and HATS were found to be decreased, and H3 and H4 histone modification marks were modulated too. Also, reduced global DNA methylation was observed following the treatment of chrysin.Conclusion: This study concludes that chrysin can be used as a potential epigenetic modifier for cancer treatment and warrants for further experimental validation.

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