Assessment of Changes in Global DNA Methylation Levels by Pyrosequencing® of Repetitive Elements

Tabish, Ali Mustafa; Baccarelli, Andrea; Godderis, Lode; Barrow, Timothy M.; Hoet, Peter; Byun, Hyang‐Min

doi:10.1007/978-1-4939-2715-9_15

Cited by 14 publications

(11 citation statements)

References 15 publications

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“…DNMT1 is classically described as the "maintenance" methyltransferase, whereas DNMT3a and DNMT3b are responsible for de novo DNA methylation, but increasing evidence over the past several years has challenged this distinction and subverted this simplistic model (53). We did not observe a difference in LINE-1 methylation, which is often used to approximate global methylation (43), after TGF-␤1 treatment. DNA methylation of LINE-1 and other retrotransposon elements, however, do not encapsulate the methylation changes that might occur in other regions of the genome.…”

Section: Discussioncontrasting

confidence: 57%

“…TGF-␤1 has also been shown to increase the expression of collagen type I by inducing DNA methylation changes in cardiac fibroblasts (27). To determine whether global DNA methylation levels were affected by TGF-␤1 treatment, we assayed the DNA methylation of the retrotransposon LINE-1, which is frequently used to estimate global methylation (43). Bisulfite sequencing revealed no change in LINE-1 methylation after TGF-␤1 treatment (Fig.…”

Section: Tgf-␤1 Inhibited Dnmt1 Ubiquitination By Phosphorylating Andmentioning

confidence: 99%

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Transforming Growth Factor-β1 Increases DNA Methyltransferase 1 and 3a Expression through Distinct Post-transcriptional Mechanisms in Lung Fibroblasts

Koh

Scruggs

Huang

2016

Journal of Biological Chemistry

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DNA methylation is a fundamental epigenetic mark that plays a critical role in differentiation and is mediated by the actions of DNA methyltransferases (DNMTs). TGF-β1 is one of the most potent inducers of fibroblast differentiation, and although many of its actions on fibroblasts are well described, the ability of TGF-β1 to modulate DNA methylation in mesenchymal cells is less clear. Here, we examine the ability of TGF-β1 to modulate the expression of various DNMTs in primary lung fibroblasts (CCL210). TGF-β1 increased the protein expression, but not RNA levels, of both DNMT1 and DNMT3a. The increases in DNMT1 and DNMT3a were dependent on TGF-β1 activation of focal adhesion kinase and PI3K/Akt. Activation of mammalian target of rapamycin complex 1 by Akt resulted in increased protein translation of DNMT3a. In contrast, the increase in DNMT1 by TGF-β1 was not dependent on new protein synthesis and instead was due to decreased protein degradation. TGF-β1 treatment led to the phosphorylation and inactivation of glycogen synthase kinase-3β, which resulted in inhibition of DNMT1 ubiquitination and proteosomal degradation. The phosphorylation and inactivation of glycogen synthase kinase-3β was dependent on mammalian target of rapamycin complex 1. These results demonstrate that TGF-β1 increases expression of DNMT1 and DNMT3a through different post-transcriptional mechanisms. Because DNA methylation is critical to many processes including development and differentiation, for which TGF-β1 is known to be crucial, the ability of TGF-β1 to increase expression of both DNMT1 and DNMT3a demonstrates a novel means by which TGF-β1 may regulate DNA methylation in these cells.

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Section: Discussioncontrasting

confidence: 57%

Section: Tgf-␤1 Inhibited Dnmt1 Ubiquitination By Phosphorylating Andmentioning

confidence: 99%

Transforming Growth Factor-β1 Increases DNA Methyltransferase 1 and 3a Expression through Distinct Post-transcriptional Mechanisms in Lung Fibroblasts

Koh

Scruggs

Huang

2016

Journal of Biological Chemistry

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“…For example, LINE-1 retrotransposons retain ~80–100 copies throughout the human genome that remain capable of retrotransposition, but are epigenetically silenced in normal cells. LINE-1 demethylation has thus been used as a control measure for the induction of global hypomethylation by HMAs in a given experimental setting [72–74]. …”

Section: Introductionmentioning

confidence: 99%

The double-edged sword of (re)expression of genes by hypomethylating agents: from viral mimicry to exploitation as priming agents for targeted immune checkpoint modulation

et al. 2017

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Hypomethylating agents (HMAs) have been widely used over the last decade, approved for use in myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML) and acute myeloid leukemia (AML). The proposed central mechanism of action of HMAs, is the reversal of aberrant methylation in tumor cells, thus reactivating CpG-island promoters and leading to (re)expression of tumor suppressor genes. Recent investigations into the mode of action of azacitidine (AZA) and decitabine (DAC) have revealed new molecular mechanisms that impinge on tumor immunity via induction of an interferon response, through activation of endogenous retroviral elements (ERVs) that are normally epigenetically silenced. Although the global demethylation of DNA by HMAs can induce anti-tumor effects, it can also upregulate the expression of inhibitory immune checkpoint receptors and their ligands, resulting in secondary resistance to HMAs. Recent studies have, however, suggested that this could be exploited to prime or (re)sensitize tumors to immune checkpoint inhibitor therapies. In recent years, immune checkpoints have been targeted by novel therapies, with the aim of (re)activating the host immune system to specifically eliminate malignant cells. Antibodies blocking checkpoint receptors have been FDA-approved for some solid tumors and a plethora of clinical trials testing these and other checkpoint inhibitors are under way. This review will discuss AZA and DAC novel mechanisms of action resulting from the re-expression of pathologically hypermethylated promoters of gene sets that are related to interferon signaling, antigen presentation and inflammation. We also review new insights into the molecular mechanisms of action of transient, low-dose HMAs on various tumor types and discuss the potential of new treatment options and combinations.

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“…No significant changes in the 5-mC levels were observed either. We subsequently studied the methylation levels of LINE-1 , which is considered a good indicator of global DNA methylation [ 34 ]. Sequence specific analysis of LINE-1 did not reveal significant changes in methylation, except for a single concentration of SWCNT (25 µg/ml) at a CpG position 3.…”

Section: Discussionmentioning

confidence: 99%

“…Sequence specific methylation of repetitive element ( LINE-1 ) and gene promoters ( SKI, DNMT 1, HDAC 4, NPAT/ATM, BCL2L11, MAP3K10, PIK3R2, MYO1C, TCF3, FGFR 1, AGRN ) were analyzed using bisulfite pyrosequencing, as described previously [ 34 ]. In addition to RNA sequencing results, we obtained the present set of genes based on our preliminary experiments in vitro (in THP-1 cells, 16 HBE) [ 28 , 29 ], in vivo [ 27 ] and human [ 17 ].…”

Section: Methodsmentioning

confidence: 99%

Single-walled and multi-walled carbon nanotubes induce sequence-specific epigenetic alterations in 16 HBE cells

et al. 2018

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Recent studies have identified carbon nanotube (CNT)-induced epigenetic changes as one of the key players in patho-physiological response. In the present study, we investigated whether CNT exposure is associated with epigenetic changes in human bronchial epithelial cells (16 HBE), in vitro. We focused on global DNA methylation, methylation of LINE-1 elements and promoter sequence of twelve functionally important genes (SKI, DNMT1, HDAC4, NPAT, ATM, BCL2L11, MAP3K10, PIK3R2, MYO1C, TCF3, FGFR 1 and AGRN). Additionally, we studied the influence of CNT exposure on miRNA expression. Using a LC-MS/MS method and pyrosequencing for LINE-1, we observed no significant changes in global DNA methylation (%) between the concentrations of multi-walled and single-walled CNT (MWCNT and SWCNT, respectively). Significant changes in sequence-specific methylation was observed in at least one CpG site for DNMT1 (SWCNT), HDAC4 (MWCNT), NPAT/ATM (MWCNT and SWCNT), MAP3K10 (MWCNT), PIK3R2 (MWCNT and SWCNT) and MYO1C (SWCNT). While changes in DNA methylation of the genes were relatively small, these changes were associated with changes in RNA expression, especially for MWCNT. However, the study did not reveal any significant alteration in the miRNA expression, associated with MWCNT and SWCNT exposure. Based on our results, mainly MWCNT influence DNA methylation and expression of the studied genes and could have significant impact on several critical cellular processes.

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Assessment of Changes in Global DNA Methylation Levels by Pyrosequencing® of Repetitive Elements

Cited by 14 publications

References 15 publications

Transforming Growth Factor-β1 Increases DNA Methyltransferase 1 and 3a Expression through Distinct Post-transcriptional Mechanisms in Lung Fibroblasts

Transforming Growth Factor-β1 Increases DNA Methyltransferase 1 and 3a Expression through Distinct Post-transcriptional Mechanisms in Lung Fibroblasts

The double-edged sword of (re)expression of genes by hypomethylating agents: from viral mimicry to exploitation as priming agents for targeted immune checkpoint modulation

Single-walled and multi-walled carbon nanotubes induce sequence-specific epigenetic alterations in 16 HBE cells

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