scholarly journals Dissection of demethylation and toxicity induced gene expression changes after decitabine treatment

2019 ◽  
Author(s):  
A Turchinovich ◽  
HM Surowy ◽  
AG Tonevitsky ◽  
B Burwinkel
Keyword(s):  
Gene Expression ◽  
Dna Methyltransferase ◽  
Housekeeping Genes ◽  
Dna Demethylation ◽  
Cell Toxicity ◽  
Dna Hypomethylation ◽  
Number Of Genes ◽  
High Doses ◽  
Qpcr Normalization ◽  
The Impact

AbstractThe DNA methyltransferase inhibitor decitabine (DAC) is a widely used drug for both fundamental epigenetics studies and anti-cancer therapy. Besides DNA demethylation, DAC also induces cell toxicity associated with DNA damage. The dual-mode of DAC action on cells provides a significant hurdle to study genes which expression is regulated by CpG methylation. In this work, we performed the analysis of global DNA methylation levels in cultured cancer cells after treatment with increasing doses of DAC and have found the U-shaped curve of the de-methylation efficacy induced by the drug. Specifically, high doses of DAC induced significantly lower DNA hypomethylation as compared to hundred-fold less concentrated doses. At the same time, the impact of DAC on cell viability was dose-dependent. These findings allowed dissecting the demethylation and the cell toxicity impact of DAC on gene expression in subsequent mRNA-seq experiments. Surprisingly, the number of genes that were upregulated due to DNA hypomethylation was comparable to the number of genes induced by DAC toxicity. Furthermore, we show that high DAC concentrations induce downregulation of housekeeping genes which are most widely used for RT-qPCR normalization (including GAPDH, actin and tubulin). The latter suggests that genes unaffected by DAC treatment would manifest themselves as upregulated when their expression is normalized on a downregulated housekeeping reference. Finally, we show that expression of most human oncogenes and tumor-suppressor genes remains unaffected after DAC treatment, and only a few of them were upregulated due to DNA hypomethylation. Our work stresses the importance of closely studying the correlation of the degree of DNA demethylation induced by varying doses of DAC with changes in gene expression levels to avoid erroneous conclusions regarding epigenetic silencing of a gene.

Comprehensive transcriptome analysis reveals link between epigenetic dysregulation, endogenous retrovirus expression and immunogenicity in metastatic colorectal carcinoma.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 3535-3535
Author(s):  
Shehara Ramyalini Mendis ◽  
James Thomas Topham ◽  
Emma Titmuss ◽  
Laura Williamson ◽  
Erin D. Pleasance ◽  
...  
Keyword(s):  
Gene Expression ◽  
Colorectal Carcinoma ◽  
Endogenous Retrovirus ◽  
Dna Demethylation ◽  
Metastatic Colorectal Carcinoma ◽  
Dna Hypomethylation ◽  
Potential Biomarker ◽  
The Mean ◽  
Retroviral Infections ◽  
The Impact

3535 Background: Endogenous retrovirus (ERV) elements represent genomic footprints of ancestral retroviral infections within the human genome. Previous studies have demonstrated increases in ERV mRNA as a result of DNA hypomethylation, and ERV transcription has been associated with increased immunogenicity in metastatic renal cell carcinoma. We performed comprehensive bioinformatics analysis of ERV transcription in metastatic colorectal carcinoma (mCRC), to identify novel links between ERV transcription, epigenetic dysregulation and immunogenicity in metastatic colorectal carcinoma (mCRC). Methods: Tumour samples from 63 patients with mCRC were subjected to RNA sequencing as part of the Personalized OncoGenomics program (POG; NCT02155621) at BC Cancer. Patients were enrolled between 07/2012-07/2017. ERV transcription was quantified across 702,533 distinct loci. Tumors were classified ERV-hi if their total ERV expression (RPKM) was greater than the mean across all samples. High antiviral gene expression tumors (AVG-hi) were designated as having a mean expression of IFIH1, DDX58, TLR3, TANK, TBKBP1, TBK1, IRF3 and IRF7 that was greater than the mean across all samples. All pairwise comparisons of gene expression were subjected to multiple hypothesis correction. Results: Median age was 59 years, with 34 (54%) male and 1 tumor microsatellite unstable. ERV-hi tumors showed increased expression of DNA demethylators TET2 ( q=0.0045) and TET3 ( q<0.0001). Significant overlap existed between ERV-hi and AVG-hi tumors (18/27, p=0.016). Tumors both ERV-hi and AVG-hi trended towards increased PD-L1 expression (p=0.055) and showed a significant increase in survival compared to tumors with high antiviral expression in the absence of high ERV transcription (p=0.0043). Conclusions: Our results suggest DNA demethylation drives increased ERV transcription and ERV-associated immunogenicity in mCRC. Moreover, we provide novel insight into the impact of ERV transcription on the biology of mCRC, highlighting ERV transcription as a potential biomarker and target for precision immunotherapy. Clinical trial information: NCT02155621.

Genome-wide investigation of DNA methylation dynamics reveals a critical role of DNA demethylation during the early somatic embryogenesis of Dimocarpus longan Lour

2020 ◽  
Vol 40 (12) ◽  
pp. 1807-1826
Author(s):  
Xiaohui Chen ◽  
Xiaoping Xu ◽  
Xu Shen ◽  
Hansheng Li ◽  
Chen Zhu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Somatic Embryogenesis ◽  
Dna Methyltransferase ◽  
Expression Patterns ◽  
Critical Role ◽  
Dna Demethylation ◽  
Structure Stability ◽  
Dna Hypomethylation ◽  
Dimocarpus Longan

Abstract DNA methylation plays essential roles in gene regulation, chromatin structure stability, gene imprinting, X chromosome inactivation and embryonic development. However, the dynamics and functions of DNA methylation during the early stage of longan (Dimocarpus longan) somatic embryogenesis (SE) are still unclear. In this study, we carried out whole genome bisulphite sequencing and transcriptome sequencing analyses for embryogenic callus (EC), incomplete compact pro-embryogenic cultures (ICpEC) and globular embryos (GE) in an early SE system. At a global level, the DNA 5-methylcytosine content in EC, ICpEC and GE was 24.59, 19.65 and 19.74%, respectively, suggesting a global decrease in DNA methylation from EC to ICpEC and then a slight increase from ICpEC to GE. Differentially methylated region (DMR) analysis showed that hypomethylation mainly occurred in CHH contexts. Gene ontology and Kyoto encyclopedia of genes and genomes analysis of hypomethylated-CHH-DMR-associated genes revealed that zein biosynthesis, fatty acid biosynthesis, circadian rhythm and mitophagy pathways were involved in longan early SE. Expression patterns of DNA methyltransferase and demethylase genes during longan early SE suggested that the decrease in DNA methylation was probably regulated by DNA methyltransferase genes and the DNA demethylase gene REPRESSOR OF SILENCING 1 (ROS1). The correlation between DNA hypomethylation and gene expression revealed that decreased DNA methylation did not cause extensive changes in gene expression during early longan SE and that gene expression may be affected by methylation changes in gene and downstream regions. Inhibiting DNA methylation with 5-azacytidine treatment in EC promoted the formation of GE and enhanced the capability of longan SE. Our results suggest that DNA demethylation has important roles in longan SE development.

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

2020 ◽  
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.

scholarly journals Is the effect of Decitabine limited to hypomethylation and DNMTs?

2019 ◽  
Author(s):  
Sina Dalvand ◽  
Amin Namdari ◽  
Ashraf Alemi ◽  
Mohammad Hassan Meshkibaf ◽  
Sam Setayesh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Histone Deacetylases ◽  
Dna Methyltransferase ◽  
Chromatin Condensation ◽  
Dna Demethylation ◽  
Histone Tails ◽  
Normal Cells ◽  
Before And After ◽  
Cancerous Cells

Abstract Background: Histone modifications play a crucial role in chromatin structure. Among enzymes, which regulate these processes, histone deacetylases (HDACs) can remove acetyl groups from histone tails, thus increasing their interaction with DNA and leading to chromatin condensation. 5-Aza-2′-deoxycytidine (AZad) or Decitabine is a potent hypomethylating agent that incorporates into DNA and traps DNA methyltransferase in the form of a covalent protein–DNA adduct. Azad, not only change the gene expression through demethylation of the gene's promoter, but it also can change gene expression independently from DNA demethylation. So, the present study was to distinguish whether AZad in addition to inhibitory effects on DNA methyltransferase, can change HDAC3 and HDAC7 mRNA expression in NALM-6, HL-60 cancer cell lines. Methods: HL-60, NALM-6 and normal cells were cultured, and the treatment dose of the AZad was obtained (1µM) by the MTT test. Finally, HDAC3 and HDAC7 mRNA expression were measured by Real Time PCR in HL-60 and NALM-6 cancerous cells before and after treatment. In addition, HDAC3 and HDAC7 mRNA expression in un-treated HL-60 and NALM-6 cancerous cells were compared to the normal cells. Results: Our result revealed that expression of HDAC3 and HDAC7, in HL-60 and NALM-6 cells increases as compared to normal cells. After treatment of HL-60 and NALM-6 cells with AZad, HDAC3 and HDAC7 mRNA expression were decreased significantly. Conclusions: Our data showed, the effects of AZad are not limited to direct hypomethylation of DNMTs but it can indirectly affect other epigenetic factors, such as HDACs activity, through converging pathways. Keywords: HDAC3 ; HDAC7 ; HL-60; NALM-6 ; Decitabine ; AZad

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

2019 ◽  
Vol 31 (3) ◽  
pp. 509 ◽  
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.

Epigenetic understanding of pain mechanisms and modern treatment perspectives

Ból ◽  
2019 ◽  
Vol 20 (1) ◽  
pp. 45-53
Author(s):  
Jadwiga Jośko-Ochojska
Keyword(s):  
Gene Expression ◽  
Chronic Pain ◽  
Dna Methyltransferase ◽  
Histone Deacetylase Inhibitors ◽  
Regulation Of Gene Expression ◽  
Dna Demethylation ◽  
Epigenetic Changes ◽  
Cellular Memory ◽  
Pain Mechanisms ◽  
Long Term Treatment

Human and animal research showed that environmental factors and lifestyle change ‘activate and deactivate’ gene expression due to epigenetic processes, which influences the perception of pain. Chronic and acute pain induce cellular memory of pain. It means that tissue lesion may cause epigenetic changes depending on the scale and kind of injury and conditions related to it. Cellular memory about pain may be transmitted to the cells of offspring through transgenerational transmission, thus inducing in further generations anxiety to neutral stimuli, related to the pain of their ancestors. In the same way, the consequences of excess opioid use may be transmitted to future generations, which is particularly dangerous, as the phenomenon of opioid abuse is becoming a serious threat to public health. It was proved that epigenetic changes in chronic pain occur in concrete genes in various diseases and conditions. Epigenetic changes also occur in genes influencing pain sensitivity. Changes in DNA methylation, histone modifications and non-coding RNA in regions related to pain, cause neuropathic, inflammatory and visceral pains. Epigenetic regulation of gene expression has recently been one of the most important objects in research on pain pathogenesis. The consequences are therapy trials with the use of DNA demethylation or reacetylation of histone lysine residues. In animal and human research, it was proved that the use of histone deacetylase inhibitors and DNA methyltransferase inhibitors decreases pain sensation. The research in the creation of new analgesics is promising. However, current epigenetic medications are highly nonspecific inhibitors that may have unknown side effects. Currently available knowledge does not allow yet to state whether they can be used in a long-term treatment of chronic pain.

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

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Marina Amorim Rocha ◽  
Giovana Maria Breda Veronezi ◽  
Marina Barreto Felisbino ◽  
Maria Silvia Viccari Gatti ◽  
Wirla M. S. C. Tamashiro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Sodium Valproate ◽  
Hela Cells ◽  
Dna Methyltransferase ◽  
Dna Demethylation ◽  
G1 Phase ◽  
Expression Levels ◽  
Demethylation Pathway ◽  
Gene Expression Levels

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.

scholarly journals Regulation of DNA (de)Methylation Positively Impacts Seed Germination during Seed Development under Heat Stress

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 457
Author(s):  
Jaiana Malabarba ◽  
David Windels ◽  
Wenjia Xu ◽  
Jerome Verdier
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
Seed Germination ◽  
Seed Development ◽  
Molecular Mechanisms ◽  
Dna Demethylation ◽  
Mild Stress ◽  
Germination Capacity ◽  
Heat Stress Response ◽  
The Impact

Seed development needs the coordination of multiple molecular mechanisms to promote correct tissue development, seed filling, and the acquisition of germination capacity, desiccation tolerance, longevity, and dormancy. Heat stress can negatively impact these processes and upon the increase of global mean temperatures, global food security is threatened. Here, we explored the impact of heat stress on seed physiology, morphology, gene expression, and methylation on three stages of seed development. Notably, Arabidopsis Col-0 plants under heat stress presented a decrease in germination capacity as well as a decrease in longevity. We observed that upon mild stress, gene expression and DNA methylation were moderately affected. Nevertheless, upon severe heat stress during seed development, gene expression was intensively modified, promoting heat stress response mechanisms including the activation of the ABA pathway. By analyzing candidate epigenetic markers using the mutants’ physiological assays, we observed that the lack of DNA demethylation by the ROS1 gene impaired seed germination by affecting germination-related gene expression. On the other hand, we also observed that upon severe stress, a large proportion of differentially methylated regions (DMRs) were located in the promoters and gene sequences of germination-related genes. To conclude, our results indicate that DNA (de)methylation could be a key regulatory process to ensure proper seed germination of seeds produced under heat stress.

scholarly journals Multi-Omics Data Integration Reveals Link Between Epigenetic Modifications and Gene Expression in Sugar Beet (Beta Vulgaris Subsp. Vulgaris) in Response to Cold

2021 ◽  
Author(s):  
Sindy Gutschker ◽  
José Maria Corral ◽  
Alfred Schmiedl ◽  
Frank Ludewig ◽  
Wolfgang Koch ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Sugar Beet ◽  
Beta Vulgaris ◽  
Environmental Conditions ◽  
Epigenetic Modifications ◽  
Dna Demethylation ◽  
Omics Data ◽  
Dna Hypomethylation ◽  
Transcriptional Responses

Abstract BackgroundDNA methylation is thought to influence the expression of genes, especially in response to changing environmental conditions and developmental changes. Sugar beet (Beta vulgaris ssp. vulgaris), and other biennial or perennial plants are inevitably exposed to fluctuating temperatures throughout their lifecycle and might even require such stimulus to acquire floral competence. Therefore, plants such as beets, need to fine-tune their epigenetic makeup to ensure phenotypic plasticity towards changing environmental conditions while at the same time steering essential developmental processes. Different crop species may show opposing reactions towards the same abiotic stress, or, vice versa, identical species may respond differently depending on the specific kind of stress. ResultsIn this study, we investigated common effects of cold treatment on genome-wide DNA methylation and gene expression of two Beta vulgaris accessions via multi-omics data analysis. Cold exposure resulted in a pronounced reduction of DNA methylation levels, which particularly affected methylation in CHH context (and to a lesser extent CHG) and was accompanied by transcriptional downregulation of the chromomethyltransferase CMT2 and strong upregulation of several genes mediating active DNA demethylation. Conclusion Integration of methylomic and transcriptomic data revealed that, rather than methylation having directly influenced expression, epigenetic modifications correlated with changes in expression of known players involved in DNA (de)methylation. In particular, cold triggered upregulation of genes putatively contributing to DNA demethylation via the ROS1 pathway. Our observations suggest that these transcriptional responses precede the cold-induced global DNA-hypomethylation in non-CpG, preparing beets for additional transcriptional alterations necessary for adapting to upcoming environmental changes.

scholarly journals Recent evolution of a TET-controlled and DPPA3/STELLA-driven pathway of passive demethylation in mammals

2018 ◽  
Author(s):  
Christopher B. Mulholland ◽  
Atsuya Nishiyama ◽  
Joel Ryan ◽  
Ryohei Nakamura ◽  
Merve Yiğit ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Ectopic Expression ◽  
Dna Demethylation ◽  
Mammalian Development ◽  
Dna Hypomethylation ◽  
Tet Proteins ◽  
Genome Wide ◽  
Recent Emergence ◽  
High Conservation ◽  
Early Mammalian Development

AbstractGenome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. So far, it was unclear how mammals specifically achieve global DNA hypomethylation, given the high conservation of the DNA (de-)methylation machinery among vertebrates. We found that DNA demethylation requires TET activity but mostly occurs at sites where TET proteins are not bound suggesting a rather indirect mechanism. Among the few specific genes bound and activated by TET proteins was the naïve pluripotency and germline marker Dppa3 (Pgc7, Stella), which undergoes TDG dependent demethylation. The requirement of TET proteins for genome-wide DNA demethylation could be bypassed by ectopic expression of Dppa3. We show that DPPA3 binds and displaces UHRF1 from chromatin and thereby prevents the recruitment and activation of the maintenance DNA methyltransferase DNMT1. We demonstrate that DPPA3 alone can drive global DNA demethylation when transferred to amphibians (Xenopus) and fish (medaka), both species that naturally do not have a Dppa3 gene and exhibit no post-fertilization DNA demethylation. Our results show that TET proteins are responsible for active and - indirectly also for - passive DNA demethylation; while TET proteins initiate local and gene-specific demethylation in vertebrates, the recent emergence of DPPA3 introduced a unique means of genome-wide passive demethylation in mammals and contributed to the evolution of epigenetic regulation during early mammalian development.

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