Sodium valproate and 5-aza-2′-deoxycytidine differentially modulate DNA demethylation in G1 phase-arrested and proliferative HeLa cells

AbstractSodium valproate/valproic acid (VPA), a histone deacetylase inhibitor, and 5-aza-2-deoxycytidine (5-aza-CdR), a DNA methyltransferase 1 (DNMT1) inhibitor, induce DNA demethylation in several cell types. In HeLa cells, although VPA leads to decreased DNA 5-methylcytosine (5mC) levels, the demethylation pathway involved in this effect is not fully understood. We investigated this process using flow cytometry, ELISA, immunocytochemistry, Western blotting and RT-qPCR in G1 phase-arrested and proliferative HeLa cells compared to the presumably passive demethylation promoted by 5-aza-CdR. The results revealed that VPA acts predominantly on active DNA demethylation because it induced TET2 gene and protein overexpression, decreased 5mC abundance, and increased 5-hydroxy-methylcytosine (5hmC) abundance, in both G1-arrested and proliferative cells. However, because VPA caused decreased DNMT1 gene expression levels, it may also act on the passive demethylation pathway. 5-aza-CdR attenuated DNMT1 gene expression levels but increased TET2 and 5hmC abundance in replicating cells, although it did not affect the gene expression of TETs at any stage of the cell cycle. Therefore, 5-aza-CdR may also function in the active pathway. Because VPA reduces DNA methylation levels in non-replicating HeLa cells, it could be tested as a candidate for the therapeutic reversal of DNA methylation in cells in which cell division is arrested.

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A Sparse and Low-Rank Regression Model for Identifying the Relationships Between DNA Methylation and Gene Expression Levels in Gastric Cancer and the Prediction of Prognosis

Genes ◽

10.3390/genes12060854 ◽

2021 ◽

Vol 12 (6) ◽

pp. 854

Author(s):

Yishu Wang ◽

Lingyun Xu ◽

Dongmei Ai

Keyword(s):

Gene Expression ◽

Gastric Cancer ◽

Dna Methylation ◽

Regression Model ◽

The Cancer Genome Atlas ◽

Low Rank ◽

Expression Levels ◽

Rank Regression ◽

Prediction Of Prognosis ◽

Gene Expression Levels

DNA methylation is an important regulator of gene expression that can influence tumor heterogeneity and shows weak and varying expression levels among different genes. Gastric cancer (GC) is a highly heterogeneous cancer of the digestive system with a high mortality rate worldwide. The heterogeneous subtypes of GC lead to different prognoses. In this study, we explored the relationships between DNA methylation and gene expression levels by introducing a sparse low-rank regression model based on a GC dataset with 375 tumor samples and 32 normal samples from The Cancer Genome Atlas database. Differences in the DNA methylation levels and sites were found to be associated with differences in the expressed genes related to GC development. Overall, 29 methylation-driven genes were found to be related to the GC subtypes, and in the prognostic model, we explored five prognoses related to the methylation sites. Finally, based on a low-rank matrix, seven subgroups were identified with different methylation statuses. These specific classifications based on DNA methylation levels may help to account for heterogeneity and aid in personalized treatments.

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Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

10.1101/2020.03.28.012518 ◽

2020 ◽

Cited By ~ 1

Author(s):

Daniel M. Sapozhnikov ◽

Moshe Szyf

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Promoter Region ◽

Dna Methyltransferase ◽

Dna Demethylation ◽

New Method ◽

Inducible Promoters ◽

Specific Targeting ◽

Main Effect

AbstractAlthough associations between DNA methylation and gene expression were established four decades ago, the causal role of DNA methylation in gene expression remains unresolved. Different strategies to address this question were developed; however, all are confounded and fail to disentangle cause and effect. We developed here a highly effective new method using only deltaCas9(dCas9):gRNA site-specific targeting to physically block DNA methylation at specific targets in the absence of a confounding flexibly-tethered enzymatic activity, enabling examination of the role of DNA methylation per se in living cells. We show that the extensive induction of gene expression achieved by TET/dCas9-based targeting vectors is confounded by DNA methylation-independent activities, inflating the role of DNA methylation in the promoter region. Using our new method, we show that in several inducible promoters, the main effect of DNA methylation is silencing basal promoter activity. Thus, the effect of demethylation of the promoter region in these genes is small, while induction of gene expression by different inducers is large and DNA methylation independent. In contrast, targeting demethylation to the pathologically silenced FMR1 gene targets robust induction of gene expression. We also found that standard CRISPR/Cas9 knockout generates a broad unmethylated region around the deletion, which might confound interpretation of CRISPR/Cas9 gene depletion studies. In summary, this new method could be used to reveal the true extent, nature, and diverse contribution to gene regulation of DNA methylation at different regions.

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Occupational exposure to gases/fumes and mineral dust affect DNA methylation levels of genes regulating expression

Human Molecular Genetics ◽

10.1093/hmg/ddz067 ◽

2019 ◽

Vol 28 (15) ◽

pp. 2477-2485 ◽

Cited By ~ 1

Author(s):

Diana A van der Plaat ◽

Judith M Vonk ◽

Natalie Terzikhan ◽

Kim de Jong ◽

Maaike de Vries ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Occupational Exposure ◽

Health Effects ◽

Mineral Dust ◽

Occupational Exposures ◽

Expression Levels ◽

Adverse Health Effects ◽

Never Smokers ◽

Gene Expression Levels

Abstract Many workers are daily exposed to occupational agents like gases/fumes, mineral dust or biological dust, which could induce adverse health effects. Epigenetic mechanisms, such as DNA methylation, have been suggested to play a role. We therefore aimed to identify differentially methylated regions (DMRs) upon occupational exposures in never-smokers and investigated if these DMRs associated with gene expression levels. To determine the effects of occupational exposures independent of smoking, 903 never-smokers of the LifeLines cohort study were included. We performed three genome-wide methylation analyses (Illumina 450 K), one per occupational exposure being gases/fumes, mineral dust and biological dust, using robust linear regression adjusted for appropriate confounders. DMRs were identified using comb-p in Python. Results were validated in the Rotterdam Study (233 never-smokers) and methylation-expression associations were assessed using Biobank-based Integrative Omics Study data (n = 2802). Of the total 21 significant DMRs, 14 DMRs were associated with gases/fumes and 7 with mineral dust. Three of these DMRs were associated with both exposures (RPLP1 and LINC02169 (2×)) and 11 DMRs were located within transcript start sites of gene expression regulating genes. We replicated two DMRs with gases/fumes (VTRNA2-1 and GNAS) and one with mineral dust (CCDC144NL). In addition, nine gases/fumes DMRs and six mineral dust DMRs significantly associated with gene expression levels. Our data suggest that occupational exposures may induce differential methylation of gene expression regulating genes and thereby may induce adverse health effects. Given the millions of workers that are exposed daily to occupational exposures, further studies on this epigenetic mechanism and health outcomes are warranted.

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Effects of ten–eleven translocation 1 (Tet1) on DNA methylation and gene expression in chicken primordial germ cells

Reproduction Fertility and Development ◽

10.1071/rd18145 ◽

2019 ◽

Vol 31 (3) ◽

pp. 509 ◽

Cited By ~ 1

Author(s):

Minli Yu ◽

Dongfeng Li ◽

Wanyan Cao ◽

Xiaolu Chen ◽

Wenxing Du

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Germ Cells ◽

Dna Methyltransferase ◽

Primordial Germ Cells ◽

Dna Demethylation ◽

Caveolin 1 ◽

Expression Of Genes ◽

Deleted In Azoospermia

Ten–eleven translocation 1 (Tet1) is involved in DNA demethylation in primordial germ cells (PGCs); however, the precise regulatory mechanism remains unclear. In the present study the dynamics of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing PGCs and the role of Tet1 in PGC demethylation were analysed. Results show that 5mC levels dropped significantly after embryonic Day 4 (E4) and 5hmC levels increased reaching a peak at E5–E5.5. Interestingly, TET1 protein was highly expressed during E5 to E5.5, which showed a consistent trend with 5hmC. The expression of pluripotency-associated genes (Nanog, PouV and SRY-box 2 (Sox2)) and germ cell-specific genes (caveolin 1 (Cav1), piwi-like RNA-mediated gene silencing 1 (Piwi1) and deleted in azoospermia-like (Dazl)) was upregulated after E5, whereas the expression of genes from the DNA methyltransferase family was decreased. Moreover, the Dazl gene was highly methylated in early PGCs and then gradually hypomethylated. Knockdown of Tet1 showed impaired survival and proliferation of PGCs, as well as increased 5mC levels and reduced 5hmC levels. Further analysis showed that knockdown of Tet1 led to elevated DNA methylation levels of Dazl and downregulated gene expression including Dazl. Thus, this study reveals the dynamic epigenetic reprogramming of chicken PGCs invivo and the molecular mechanism of Tet1 in regulating genomic DNA demethylation and hypomethylation of Dazl during PGC development.

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A Genome-Wide Study of DNA Methylation Patterns and Gene Expression Levels in Multiple Human and Chimpanzee Tissues

PLoS Genetics ◽

10.1371/journal.pgen.1001316 ◽

2011 ◽

Vol 7 (2) ◽

pp. e1001316 ◽

Cited By ~ 142

Author(s):

Athma A. Pai ◽

Jordana T. Bell ◽

John C. Marioni ◽

Jonathan K. Pritchard ◽

Yoav Gilad

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Expression Levels ◽

Genome Wide ◽

A Genome ◽

Methylation Patterns ◽

Genome Wide Study ◽

Gene Expression Levels

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Gene expression levels of DNA methyltransferase enzymes in Shank3-deficient mouse model of autism during early development

Endocrine Regulations ◽

10.2478/enr-2021-0025 ◽

2021 ◽

Vol 55 (4) ◽

pp. 234-237

Author(s):

Annamaria Srancikova ◽

Alexandra Reichova ◽

Zuzana Bacova ◽

Jan Bakos

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Brain Development ◽

Early Development ◽

Molecular Mechanisms ◽

Altered Expression ◽

Expression Levels ◽

The Brain ◽

Deficient Mice ◽

Gene Expression Levels

Abstract Objectives. The balance between DNA methylation and demethylation is crucial for the brain development. Therefore, alterations in the expression of enzymes controlling DNA methylation patterns may contribute to the etiology of neurodevelopmental disorders, including autism. SH3 and multiple ankyrin repeat domains 3 (Shank3)-deficient mice are commonly used as a well-characterized transgenic model to investigate the molecular mechanisms of autistic symptoms. DNA methyltransferases (DNMTs), which modulate several cellular processes in neurodevelopment, are implicated in the pathophysiology of autism. In this study, we aimed to describe the gene expression changes of major Dnmts in the brain of Shank3-deficient mice during early development. Methods and Results. The Dnmts gene expression was analyzed by qPCR in 5-day-old homo-zygous Shank3-deficient mice. We found significantly lower Dnmt1 and Dnmt3b gene expression levels in the frontal cortex. However, no such changes were observed in the hippocampus. However, significant increase was observed in the expression of Dnmt3a and Dnmt3b genes in the hypothalamus of Shank3-deficient mice. Conclusions. The present data indicate that abnormalities in the Shank3 gene are accompanied by an altered expression of DNA methylation enzymes in the early brain development stages, therefore, specific epigenetic control mechanisms in autism-relevant models should be more extensively investigated.

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Integrative genomic strategies applied to a lymphoblast cell line model reveal specific transcriptomic signatures associated with clozapine response

10.1101/2020.09.22.308262 ◽

2020 ◽

Author(s):

SAJ de With ◽

APS Ori ◽

T Wang ◽

SL Pulit ◽

E Strengman ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Adverse Effects ◽

Drug Targets ◽

Molecular Mechanisms ◽

Disease Risk ◽

Density Lipoprotein ◽

Expression Levels ◽

Genome Wide ◽

Gene Expression Levels

AbstractClozapine is an important antipsychotic drug. However, its use is often accompanied by metabolic adverse effects and, in rare instances, agranulocytosis. The molecular mechanisms underlying these adverse events are unclear. To gain more insights into the response to clozapine at the molecular level, we exposed lymphoblastoid cell lines (LCLs) to increasing concentrations of clozapine and measured genome-wide gene expression and DNA methylation profiles. We observed robust and significant changes in gene expression levels due to clozapine (n = 463 genes at FDR < 0.05) affecting cholesterol and cell cycle pathways. At the level of DNA methylation, we find significant changes upstream of the LDL receptor, in addition to global enrichments of regulatory, immune and developmental pathways. By integrating these data with human tissue gene expression levels obtained from the Genotype-Tissue Expression project (GTEx), we identified specific tissues, including liver and several tissues involved in immune, endocrine and metabolic functions, that clozapine treatment may disproportionately affect. Notably, differentially expressed genes were not enriched for genome-wide disease risk of schizophrenia or for known psychotropic drug targets. However, we did observe a nominally significant association of genetic signals related to total cholesterol and low-density lipoprotein levels. Together, these results shed light on the biological mechanisms through which clozapine functions. The observed associations with cholesterol pathways, its genetic architecture and specific tissue effects may be indicative of the metabolic adverse effects observed in clozapine users. LCLs may thus serve as a useful tool to study these molecular mechanisms further.

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Genetic analysis of DNA methylation and gene expression levels in whole blood of healthy human subjects

BMC Genomics ◽

10.1186/1471-2164-13-636 ◽

2012 ◽

Vol 13 (1) ◽

pp. 636 ◽

Cited By ~ 138

Author(s):

Kristel R van Eijk ◽

Simone de Jong ◽

Marco PM Boks ◽

Terry Langeveld ◽

Fabrice Colas ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Genetic Analysis ◽

Whole Blood ◽

Human Subjects ◽

Healthy Human ◽

Expression Levels ◽

Healthy Human Subjects ◽

Gene Expression Levels

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DUSP1Gene Polymorphisms Are Associated with Obesity-Related Metabolic Complications among Severely Obese Patients and Impact on Gene Methylation and Expression

International Journal of Genomics ◽

10.1155/2013/609748 ◽

2013 ◽

Vol 2013 ◽

pp. 1-10 ◽

Cited By ~ 7

Author(s):

F. Guénard ◽

L. Bouchard ◽

A. Tchernof ◽

Y. Deshaies ◽

F. S. Hould ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Obese Patients ◽

Gene Methylation ◽

Metabolic Complications ◽

Expression Levels ◽

Cpg Sites ◽

Severely Obese ◽

The Impact ◽

Gene Expression Levels

TheDUSP1gene encodes a member of the dual-specificity phosphatase family previously identified as being differentially expressed in visceral adipose tissue (VAT) of severely obese men with versus without the metabolic syndrome.Objective.To test the association betweenDUSP1polymorphisms, obesity-related metabolic complications, gene methylation, and expression levels in VAT.Methods.TheDUSP1locus and promoter region were sequenced in 25 individuals. SNPs were tested for association with obesity-related complications in a cohort of more than 1900 severely obese individuals. The impact of SNPs on methylation levels of 36 CpG sites and correlations between DNA methylation and gene expression levels in VAT were computed in a subset of 14 samples.Results.Heterozygotes for rs881150 had lower HDL-cholesterol levels (HDL-C;P=0.01), and homozygotes for the minor allele of rs13184134 and rs7702178 had increased fasting glucose levels (P=0.04and 0.01, resp.). rs881150 was associated with methylation levels of CpG sites located ~1250 bp upstream the transcription start site. Methylation levels of 4 CpG sites were inversely correlated withDUSP1gene expression.Conclusion.These results suggest thatDUSP1polymorphisms modulate plasma glucose and HDL-C levels in obese patients possibly through alterations of DNA methylation and gene expression levels.

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Rare Genetic Variants Underlie Outlying levels of DNA Methylation and Gene-Expression

10.1101/2020.02.19.950659 ◽

2020 ◽

Author(s):

V. Kartik Chundru ◽

Riccardo E. Marioni ◽

James G. D. Pendergast ◽

Tian Lin ◽

Allan J. Beveridge ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Phenotypic Variation ◽

Genetic Variants ◽

Rare Variants ◽

Effect Sizes ◽

Expression Levels ◽

Low Level ◽

Rare Genetic Variants ◽

Gene Expression Levels

AbstractTesting the effect of rare variants on phenotypic variation is difficult due to the need for extremely large cohorts to identify associated variants given expected effect sizes. An alternative approach is to investigate the effect of rare genetic variants on low-level genomic traits, such as gene expression or DNA methylation (DNAm), as effect sizes are expected to be larger for low-level compared to higher-order complex traits. Here, we investigate DNAm in healthy ageing populations - the Lothian Birth cohorts of 1921 and 1936 and identify both transient and stable outlying DNAm levels across the genome. We find an enrichment of rare genetic variants within 1kb of DNAm sites in individuals with stable outlying DNAm, implying genetic control of this extreme variation. Using a family-based cohort, the Brisbane Systems Genetics Study, we observed increased sharing of DNAm outliers among more closely related individuals, consistent with these outliers being driven by rare genetic variation. We demonstrated that outlying DNAm levels have a functional consequence on gene expression levels, with extreme levels of DNAm being associated with gene expression levels towards the tails of the population distribution. Overall, this study demonstrates the role of rare variants in the phenotypic variation of low-level genomic traits, and the effect of extreme levels of DNAm on gene expression.

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