Effects of parental environmental copper stress on offspring development: DNA methylation modification and responses of differentially methylated region-related genes in transcriptional expression

ABSTRACT Igf2 and H19 are coordinately regulated imprinted genes physically linked on the distal end of mouse chromosome 7. Genetic analyses demonstrate that the differentially methylated region (DMR) upstream of the H19 gene is necessary for three distinct functions: transcriptional insulation of the maternal Igf2 allele, transcriptional silencing of paternal H19 allele, and marking of the parental origin of the two chromosomes. To test the sufficiency of the DMR for the third function, we inserted DMR at two heterologous positions in the genome, downstream of H19 and at the alpha-fetoprotein locus on chromosome 5. Our results demonstrate that the DMR alone is sufficient to act as a mark of parental origin. Moreover, this activity is not dependent on germ line differences in DMR methylation. Thus, the DMR can mark its parental origin by a mechanism independent of its own DNA methylation.

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Epigenetic silencing of MEIS2 in prostate cancer recurrence

Clinical Epigenetics ◽

10.1186/s13148-019-0742-x ◽

2019 ◽

Vol 11 (1) ◽

Cited By ~ 3

Author(s):

Maibritt Nørgaard ◽

Christa Haldrup ◽

Marianne Trier Bjerre ◽

Søren Høyer ◽

Benedicte Ulhøi ◽

...

Keyword(s):

Prostate Cancer ◽

Dna Methylation ◽

Prostate Tissue ◽

Rank Test ◽

Transcriptional Expression ◽

Tissue Samples ◽

Whitney Test ◽

Log Rank Test ◽

Mann Whitney Test ◽

Malignant Prostate

Abstract Background Current diagnostic and prognostic tools for prostate cancer (PC) are suboptimal, resulting in overdiagnosis and overtreatment of clinically insignificant tumors. Thus, to improve the management of PC, novel biomarkers are urgently needed. Results In this study, we integrated genome-wide methylome (Illumina 450K DNA methylation array (450K)) and RNA sequencing (RNAseq) data performed in a discovery set of 27 PC and 15 adjacent normal (AN) prostate tissue samples to identify candidate driver genes involved in PC development and/or progression. We found significant enrichment for homeobox genes among the most aberrantly methylated and transcriptionally dysregulated genes in PC. Specifically, homeobox gene MEIS2 (Myeloid Ecotropic viral Insertion Site 2) was significantly hypermethylated (p < 0.0001, Mann-Whitney test) and transcriptionally downregulated (p < 0.0001, Mann-Whitney test) in PC compared to non-malignant prostate tissue in our discovery sample set, which was also confirmed in an independent validation set including > 500 PC and AN tissue samples in total (TCGA cohort analyzed by 450K and RNAseq). Furthermore, in three independent radical prostatectomy (RP) cohorts (n > 700 patients in total), low MEIS2 transcriptional expression was significantly associated with poor biochemical recurrence (BCR) free survival (p = 0.0084, 0.0001, and 0.0191, respectively; log-rank test). Next, we analyzed another RP cohort consisting of > 200 PC, AN, and benign prostatic hyperplasia (BPH) samples by quantitative methylation-specific PCR (qMSP) and found that MEIS2 was significantly hypermethylated (p < 0.0001, Mann-Whitney test) in PC compared to non-malignant prostate tissue samples (AN and BPH) with an AUC > 0.84. Moreover, in this cohort, aberrant MEIS2 hypermethylation was significantly associated with post-operative BCR (p = 0.0068, log-rank test), which was subsequently confirmed (p = 0.0067; log-rank test) in the independent TCGA validation cohort (497 RP patients; 450K data). Conclusions To the best of our knowledge, this is the first study to investigate, demonstrate, and independently validate a prognostic biomarker potential for MEIS2 at the transcriptional expression level and at the DNA methylation level in PC.

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Epigenetic modification of the human CCR6 gene is associated with stable CCR6 expression in T cells

Blood ◽

10.1182/blood-2010-06-293027 ◽

2011 ◽

Vol 117 (10) ◽

pp. 2839-2846 ◽

Cited By ~ 39

Author(s):

Svenja Steinfelder ◽

Stefan Floess ◽

Dirk Engelbert ◽

Barbara Haeringer ◽

Udo Baron ◽

...

Keyword(s):

Dna Methylation ◽

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Cd4 T Cells ◽

Transcriptional Activity ◽

Epigenetic Modification ◽

Cell Receptor ◽

Noncoding Region ◽

Differentially Methylated Region

Abstract CCR6 is a chemokine receptor expressed on Th17 cells and regulatory T cells that is induced by T-cell priming with certain cytokines, but how its expression and stability are regulated at the molecular level is largely unknown. Here, we identified and characterized a noncoding region of the human CCR6 locus that displayed unmethylated CpG motifs (differentially methylated region [DMR]) selectively in CCR6+ lymphocytes. CCR6 expression on circulating CD4+ T cells was stable on cytokine-induced proliferation but partially down-regulated on T-cell receptor stimulation. However, CCR6 down-regulation was mostly transient, and the DMR within the CCR6 locus remained demethylated. Notably, in vitro induction of CCR6 expression with cytokines in T-cell receptor-activated naive CD4+ T cells was not associated with a demethylated DMR and resulted in unstable CCR6 expression. Conversely, treatment with the DNA methylation inhibitor 5′-azacytidine induced demethylation of the DMR and led to increased and stable CCR6 expression. Finally, when cloned into a reporter gene plasmid, the DMR displayed transcriptional activity in memory T cells that was suppressed by DNA methylation. In summary, we have identified a noncoding region of the human CCR6 gene with methylation-sensitive transcriptional activity in CCR6+ T cells that controls stable CCR6 expression via epigenetic mechanisms.

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IL-10 Production By CLL Cells Is Enhanced in the Anergic IGHV Mutated Subset and Associates with Reduced DNA Methylation of the IL-10 Locus

Blood ◽

10.1182/blood.v126.23.2917.2917 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2917-2917

Author(s):

Samantha Drennan ◽

Annalisa D'Avola ◽

Yifang Gao ◽

Eleni Chrysostomou ◽

Andrew J Steele ◽

...

Keyword(s):

Dna Methylation ◽

Gene Locus ◽

Conflicts Of Interest ◽

Methylation Status ◽

Differentially Methylated Region ◽

Variable Degree ◽

Immunoglobulin Gene ◽

Transcript Levels ◽

Low Levels ◽

B10 Cells

Abstract The B-cell receptor (BCR) of chronic lymphocytic leukemia (CLL) cells with unmutated (U-CLL) or mutated (M-CLL) immunoglobulin gene heavy-chain variable (IGHV) regions shows a variable degree of anergy, associated with low surface IgM (sIgM) levels and signaling capacity, more evident in M-CLL. DNA methylation is also different between M-CLL and U-CLL. Patients from both subsets are immunosuppressed from the early stages, possibly influenced by the ability of activated CLL cells to produce IL-10, a property of B10 cells. The aim of this study was to investigate associations between capacity of circulating CLL cells to differentiate into B10 cells and features of anergy. In parallel, the methylation status of the IL-10 gene locus was probed in order to investigate associations between IL-10 gene methylation and production. CLL cells were isolated and cultured +/- TLR9 activation with CpG (ODN-2006). Cytokine secretion was measured in supernatants (Luminex) and IL10 transcript was measured (RT-qPCR) from cell pellets. IL-10 production by CLL cells was measured by intracellular flow cytometry following stimulation with CpG for 24 hours. Epigenetic profiling was performed using MassARRAY and the 450K Array. We found that activation of CLL cells by CpG consistently induced production and secretion of IL-10 protein, while no or low amounts of pro-inflammatory cytokines were detected. Production of IL-10 was significantly higher in M-CLL than in U-CLL. Levels also correlated with lower sIgM levels and signaling capacity, both features of anergy. A linear correlation was present between secretion, intracellular production and transcript levels of IL-10, suggesting no aberrant post-transcriptional controls. In the absence of CpG activation, IL-10 transcript levels were also detected at low levels in M-CLL, while they were even lower in U-CLL. To identify a potential basis for the differential expression, DNA methylation analysis of IL-10 gene locus was performed. While the promoter region displayed similarly low levels of methylation in both U-CLL and M-CLL, methylation differences were detected immediately downstream of the promoter within the first intron (approximately +200 to +500 bp, differentially methylated region 1, DMR1) and in the gene body (approximately +1300 to +1800 bp, DMR2). Both DMR1 and DMR2 were markedly more hypomethylated in M-CLL than U-CLL, with the greatest difference detected at cg17067005 within DMR1. Each of these regions display histone H3 lysine 27 acetylation (H3K27ac) in B cells (GM12878 cells), thus are likely to represent functional DNA elements. Analysis of IL-10 transcript levels with methylation demonstrated a mutually exclusive pattern between expression and methylation of both DMR1 and DMR2. These data document a strong link between capacity to differentiate into B10-like cells and anergy, and suggest an epigenetic component in the regulation of IL-10 production in CLL cells. This capacity may contribute to immunosuppression. While U-CLL appears less able to produce IL-10 on a per cell basis, higher tumor load may compensate, accounting for clinical immunosuppression in both subsets. Disclosures No relevant conflicts of interest to declare.

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DNA methylation profiling identifies epigenetic differences between early versus late stages of diabetic chronic kidney disease

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa226 ◽

2020 ◽

Author(s):

Ashani Lecamwasam ◽

Boris Novakovic ◽

Braydon Meyer ◽

Elif I Ekinci ◽

Karen M Dwyer ◽

...

Keyword(s):

Chronic Kidney Disease ◽

Dna Methylation ◽

Kidney Disease ◽

Secretory Protein ◽

Differentially Methylated Region ◽

P Value ◽

Operating Characteristics ◽

Cross Sectional ◽

Cpg Sites ◽

Methylation Patterns

Abstract Background We investigated a cross-sectional epigenome-wide association study of patients with early and late diabetes-associated chronic kidney disease (CKD) to identify possible epigenetic differences between the two groups as well as changes in methylation across all stages of diabetic CKD. We also evaluated the potential of using a panel of identified 5′-C-phosphate-G-3′ (CpG) sites from this cohort to predict the progression of diabetic CKD. Methods This cross-sectional study recruited 119 adults. DNA was extracted from blood using the Qiagen QIAampDNA Mini Spin Kit. Genome-wide methylation analysis was performed using Illumina Infinium MethylationEPIC BeadChips (HM850K). Intensity data files were processed and analysed using the minfi and MissMethyl packages for R. We examined the degree of methylation of CpG sites in early versus late diabetic CKD patients for CpG sites with an unadjusted P-value <0.01 and an absolute change in methylation of 5% (n = 239 CpG sites). Results Hierarchical clustering of the 239 CpG sites largely separated the two groups. A heat map for all 239 CpG sites demonstrated distinct methylation patterns in the early versus late groups, with CpG sites showing evidence of progressive change. Based on our differentially methylated region (DMR) analysis of the 239 CpG sites, we highlighted two DMRs, namely the cysteine-rich secretory protein 2 (CRISP2) and piwi-like RNA-mediated gene silencing 1 (PIWIL1) genes. The best predictability for the two groups involved a receiver operating characteristics curve of eight CpG sites alone and achieved an area under the curve of 0.976. Conclusions We have identified distinct DNA methylation patterns between early and late diabetic CKD patients as well as demonstrated novel findings of potential progressive methylation changes across all stages (1–5) of diabetic CKD at specific CpG sites. We have also identified associated genes CRISP2 and PIWIL1, which may have the potential to act as stage-specific diabetes-associated CKD markers, and showed that the use of a panel of eight identified CpG sites alone helps to increase the predictability for the two groups.

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Genome-Wide DNA Methylation Comparison between Brassica napus Genic Male Sterile Line and Restorer Line

International Journal of Molecular Sciences ◽

10.3390/ijms19092689 ◽

2018 ◽

Vol 19 (9) ◽

pp. 2689 ◽

Cited By ~ 6

Author(s):

Zhixin Wang ◽

Xiangping Wu ◽

Zengxiang Wu ◽

Hong An ◽

Bin Yi ◽

...

Keyword(s):

Dna Methylation ◽

Male Sterility ◽

Bisulfite Sequencing ◽

Epigenetic Modification ◽

Differentially Methylated Region ◽

Male Sterile ◽

Male Sterile Line ◽

Floral Buds ◽

Global Comparison ◽

Genome Wide

DNA methylation is an essential epigenetic modification that dynamically regulates gene expression during plant development. However, few studies have determined the DNA methylation profiles of male-sterile rapeseed. Here, we conducted a global comparison of DNA methylation patterns between the rapeseed genic male sterile line 7365A and its near-isogenic fertile line 7365B by whole-genome bisulfite sequencing (WGBS). Profiling of the genome-wide DNA methylation showed that the methylation level in floral buds was lower than that in leaves and roots. Besides, a total of 410 differentially methylated region-associated genes (DMGs) were identified in 7365A relative to 7365B. Traditional bisulfite sequencing polymerase chain reaction (PCR) was performed to validate the WGBS data. Eleven DMGs were found to be involved in anther and pollen development, which were analyzed by quantitative PCR. In particular, Bnams4 was hypo-methylated in 7365A, and its expression was up-regulated, which might affect other DMGs and thus control the male sterility. This study provided genome-wide DNA methylation profiles of floral buds and important clues for revealing the molecular mechanism of genic male sterility in rapeseed.

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Prenatal substance exposure and offspring development: Does DNA methylation play a role?

Neurotoxicology and Teratology ◽

10.1016/j.ntt.2018.01.009 ◽

2019 ◽

Vol 71 ◽

pp. 50-63 ◽

Cited By ~ 8

Author(s):

Valerie S. Knopik ◽

Kristine Marceau ◽

L. Cinnamon Bidwell ◽

Emily Rolan

Keyword(s):

Dna Methylation ◽

Prenatal Substance Exposure ◽

Offspring Development ◽

Substance Exposure

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Dietary supplementation with polyunsaturated fatty acid during pregnancy modulates DNA methylation at IGF2/H19 imprinted genes and growth of infants

Physiological Genomics ◽

10.1152/physiolgenomics.00061.2014 ◽

2014 ◽

Vol 46 (23) ◽

pp. 851-857 ◽

Cited By ~ 63

Author(s):

Ho-Sun Lee ◽

Albino Barraza-Villarreal ◽

Carine Biessy ◽

Talita Duarte-Salles ◽

Peter D. Sly ◽

...

Keyword(s):

Dna Methylation ◽

Mononuclear Cells ◽

Dietary Supplementation ◽

Positive Association ◽

Child Growth ◽

Normal Weight ◽

Differentially Methylated Region ◽

Control Group ◽

Imprinted Genes ◽

Maternal Body Mass Index

Epigenetic regulation of imprinted genes is regarded as a highly plausible explanation for linking dietary exposures in early life with the onset of diseases during childhood and adulthood. We sought to test whether prenatal dietary supplementation with docosahexaenoic acid (DHA) during pregnancy may modulate epigenetic states at birth. This study was based on a randomized intervention trial conducted in Mexican pregnant women supplemented daily with 400 mg of DHA or a placebo from gestation week 18–22 to parturition. We applied quantitative profiling of DNA methylation states at IGF2 promoter 3 ( IGF2 P3), IGF2 differentially methylated region (DMR), and H19 DMR in cord blood mononuclear cells of the DHA-supplemented group ( n = 131) and the control group ( n = 130). In stratified analyses, DNA methylation levels in IGF2 P3 were significantly higher in the DHA group than the control group in preterm infants ( P = 0.04). We also observed a positive association between DNA methylation levels and maternal body mass index; IGF2 DMR methylation was higher in the DHA group than the control group in infants of overweight mothers ( P = 0.03). In addition, at H19 DMR, methylation levels were significantly lower in the DHA group than the control group in infants of normal weight mothers ( P = 0.01). Finally, methylation levels at IGF2/H19 imprinted regions were associated with maternal BMI. These findings suggest that epigenetic mechanisms may be modulated by DHA, with potential impacts on child growth and development.

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DNA methylation changes in CD4+ T cells isolated from multiple sclerosis patients on dimethyl fumarate

Multiple Sclerosis Journal - Experimental Translational and Clinical ◽

10.1177/2055217318787826 ◽

2018 ◽

Vol 4 (3) ◽

pp. 205521731878782 ◽

Cited By ~ 1

Author(s):

Vicki E Maltby ◽

Rodney A Lea ◽

Karen A Ribbons ◽

Katherine A Sanders ◽

Daniel Kennedy ◽

...

Keyword(s):

Multiple Sclerosis ◽

Dna Methylation ◽

T Cells ◽

Transcriptional Start Site ◽

Oral Treatment ◽

Dimethyl Fumarate ◽

Differentially Methylated Region ◽

Start Site ◽

Before And After ◽

Multiple Sclerosis Patients

Background Dimethyl fumarate is an oral treatment for multiple sclerosis, whose mechanism of action is not fully understood. Objective To investigate the effects of dimethyl fumarate on DNA methylation in the CD4+ T cells of multiple sclerosis patients. Methods We performed Illumina EPIC arrays to investigate the DNA methylation profiles of CD4+ T cells derived from multiple sclerosis patients before and after dimethyl fumarate treatment. Results Treatment with dimethyl fumarate resulted in 97% of differentially methylated positions showing hypermethylation. Four genes, SNORD1A, SHTN1, MZB1 and TNF had a differentially methylated region located within the transcriptional start site. Conclusion This study investigates the effect of dimethyl fumarate on DNA methylation in multiple sclerosis patients.

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Bisphenol A Exposure Disrupts Metabolic Health Across Multiple Generations in the Mouse

Endocrinology ◽

10.1210/en.2014-2027 ◽

2015 ◽

Vol 156 (6) ◽

pp. 2049-2058 ◽

Cited By ~ 78

Author(s):

Martha Susiarjo ◽

Frances Xin ◽

Amita Bansal ◽

Martha Stefaniak ◽

Changhong Li ◽

...

Keyword(s):

Dna Methylation ◽

Bisphenol A ◽

Glucose Homeostasis ◽

Early Life ◽

Metabolic Health ◽

Male Offspring ◽

Differentially Methylated Region ◽

Developmental Defects ◽

Metabolic Phenotypes ◽

Multiple Generations

Abstract Accumulating evidence has suggested that a suboptimal early life environment produces multigenerational developmental defects. A proposed mechanism is stable inheritance of DNA methylation. Here we show that maternal bisphenol A (BPA) exposure in C57BL/6 mice produces multigenerational metabolic phenotypes in their offspring. Using various methods including dual-energy X-ray absorptiometry analyses, glucose tolerance tests, and perifusion islet studies, we showed that exposure to 10 μg/kg/d and 10 mg/kg/d BPA in pregnant F0 mice was associated with higher body fat and perturbed glucose homeostasis in F1 and F2 male offspring but not female offspring. To provide insight into the mechanism of the multigenerational metabolic abnormalities, we investigated the maternal metabolic milieu and inheritance of DNA methylation across generations. We showed that maternal glucose homeostasis during pregnancy was altered in the F0 but not F1 female mice. The results suggested that a compromised maternal metabolic milieu may play a role in the health of the F1 offspring but cannot account for all of the observed multigenerational phenotypes. We further demonstrated that the metabolic phenotypes in the F1 and F2 BPA male offspring were linked to fetal overexpression of the imprinted Igf2 gene and increased DNA methylation at the Igf2 differentially methylated region 1. Studies in H19Δ3.8/+ mouse mutants supported the role of fetal Igf2 overexpression in altered adult glucose homeostasis. We conclude that early life BPA exposure at representative human exposure levels can perturb metabolic health across multiple generations in the mouse through stable inheritance of DNA methylation changes at the Igf2 locus.

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