scholarly journals DNA methylation modulates transcription factor occupancy chiefly at sites of high intrinsic cell-type variability

2014 ◽  
Author(s):  
Matthew Maurano ◽  
Hao Wang ◽  
Sam John ◽  
Anthony Shafer ◽  
Theresa Canfield ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcription Factor ◽  
Dna Methyltransferase ◽  
Nuclear Genome ◽  
Cell Types ◽  
Intrinsic Variability ◽  
Occupancy Patterns ◽  
Target Sites ◽  
Transcription Factor Occupancy

The nuclear genome of every cell harbors millions of unoccupied transcription factor (TF) recognition sequences that harbor methylated cytosines. Although DNA methylation is commonly invoked as a repressive mechanism, the extent to which it actively silences specific TF occupancy sites is unknown. To define the role of DNA methylation in modulating TF binding, we quantified the effect of DNA methyltransferase abrogation on the occupancy patterns of a ubiquitous TF capable of autonomous binding to its target sites in chromatin (CTCF). Here we show that the vast majority of unoccupied, methylated CTCF recognition sequences remain unbound upon depletion of DNA methylation. Rather, methylation-regulated binding is restricted to a small fraction of elements that exhibit high intrinsic variability in CTCF occupancy across cell types. Our results suggest that DNA methylation is not a major groundskeeper of genomic transcription factor occupancy landscapes, but rather a specialized mechanism for stabilizing epigenetically labile sites.

scholarly journals Role of DNA Methylation in Modulating Transcription Factor Occupancy

Cell Reports ◽  
2015 ◽  
Vol 12 (7) ◽  
pp. 1184-1195 ◽  
Author(s):  
Matthew T. Maurano ◽  
Hao Wang ◽  
Sam John ◽  
Anthony Shafer ◽  
Theresa Canfield ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcription Factor ◽  
Transcription Factor Occupancy

scholarly journals Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Krystyna Ślaska-Kiss ◽  
Nikolett Zsibrita ◽  
Mihály Koncz ◽  
Pál Albert ◽  
Ákos Csábrádi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Binding ◽  
Binding Affinity ◽  
Dna Methyltransferase ◽  
Restriction Enzymes ◽  
Dna Binding Protein ◽  
E Coli ◽  
Dna Binding Affinity ◽  
Higher Eukaryotes

AbstractTargeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.

scholarly journals DNA Methyltransferase 3A Is Involved in the Sustained Effects of Chronic Stress on Synaptic Functions and Behaviors

2020 ◽  
Author(s):  
Jing Wei ◽  
Jia Cheng ◽  
Nicholas J Waddell ◽  
Zi-Jun Wang ◽  
Xiaodong Pang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Epigenetic Modification ◽  
Substantial Reduction ◽  
Dna Methyltransferases ◽  
Male Rats ◽  
Neural Pathways ◽  
Dnmt Inhibitors ◽  
Synaptic Functions

Abstract Emerging evidence suggests that epigenetic mechanisms regulate aberrant gene transcription in stress-associated mental disorders. However, it remains to be elucidated about the role of DNA methylation and its catalyzing enzymes, DNA methyltransferases (DNMTs), in this process. Here, we found that male rats exposed to chronic (2-week) unpredictable stress exhibited a substantial reduction of Dnmt3a after stress cessation in the prefrontal cortex (PFC), a key target region of stress. Treatment of unstressed control rats with DNMT inhibitors recapitulated the effect of chronic unpredictable stress on decreased AMPAR expression and function in PFC. In contrast, overexpression of Dnmt3a in PFC of stressed animals prevented the loss of glutamatergic responses. Moreover, the stress-induced behavioral abnormalities, including the impaired recognition memory, heightened aggression, and hyperlocomotion, were partially attenuated by Dnmt3a expression in PFC of stressed animals. Finally, we found that there were genome-wide DNA methylation changes and transcriptome alterations in PFC of stressed rats, both of which were enriched at several neural pathways, including glutamatergic synapse and microtubule-associated protein kinase signaling. These results have therefore recognized the potential role of DNA epigenetic modification in stress-induced disturbance of synaptic functions and cognitive and emotional processes.

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 Brown Fat Dnmt3b Deficiency Ameliorates Obesity in Female Mice

Life ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1325
Author(s):  
Fenfen Li ◽  
Xin Cui ◽  
Jia Jing ◽  
Shirong Wang ◽  
Huidong Shi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Energy Expenditure ◽  
Knockout Mice ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Brown Fat ◽  
Chow Diet ◽  
Female Mice ◽  
Diet Induced Obesity

Obesity results from a chronic energy imbalance due to energy intake exceeding energy expenditure. Activation of brown fat thermogenesis has been shown to combat obesity. Epigenetic regulation, including DNA methylation, has emerged as a key regulator of brown fat thermogenic function. Here we aimed to study the role of Dnmt3b, a DNA methyltransferase involved in de novo DNA methylation, in the regulation of brown fat thermogenesis and obesity. We found that the specific deletion of Dnmt3b in brown fat promotes the thermogenic and mitochondrial program in brown fat, enhances energy expenditure, and decreases adiposity in female mice fed a regular chow diet. With a lean phenotype, the female knockout mice also exhibit increased insulin sensitivity. In addition, Dnmt3b deficiency in brown fat also prevents diet-induced obesity and insulin resistance in female mice. Interestingly, our RNA-seq analysis revealed an upregulation of the PI3K-Akt pathway in the brown fat of female Dnmt3b knockout mice. However, male Dnmt3b knockout mice have no change in their body weight, suggesting the existence of sexual dimorphism in the brown fat Dnmt3b knockout model. Our data demonstrate that Dnmt3b plays an important role in the regulation of brown fat function, energy metabolism and obesity in female mice.

scholarly journals DNA methylomes and transcriptomes analysis reveal implication of host DNA methylation machinery in BmNPV proliferation in Bombyx mori

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Haoling Huang ◽  
Ping Wu ◽  
Shaolun Zhang ◽  
Qi Shang ◽  
Haotong Yin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bombyx Mori ◽  
Dna Methyltransferase ◽  
Transcript Level ◽  
Research Direction ◽  
Methylation Pattern ◽  
Integrated Analysis ◽  
Infected Cells ◽  
Structural Maintenance Of Chromosomes

Abstract Background Bombyx mori nucleopolyhedrosis virus (BmNPV) is a major pathogen that threatens the sustainability of the sericultural industry. DNA methylation is a widespread gene regulation mode in epigenetics, which plays an important role in host immune response. Until now, little has been known about epigenetic regulation on virus diseases in insects. This study aims to explore the role of DNA methylation in BmNPV proliferation. Results Inhibiting DNA methyltransferase (DNMT) activity of silkworm can suppress BmNPV replication. The integrated analysis of transcriptomes and DNA methylomes in silkworm midguts infected with or without BmNPV showed that both the expression pattern of transcriptome and DNA methylation pattern are changed significantly upon BmNPV infection. A total of 241 differentially methylated regions (DMRs) were observed in BmNPV infected midguts, among which, 126 DMRs were hyper-methylated and 115 DMRs were hypo-methylated. Significant differences in both mRNA transcript level and DNA methylated levels were found in 26 genes. BS-PCR validated the hypermethylation of BGIBMGA014008, a structural maintenance of chromosomes protein gene in the BmNPV-infected midgut. In addition, DNMT inhibition reduced the expression of inhibitor of apoptosis family genes, iap1 from BmNPV, Bmiap2, BmSurvivin1 and BmSurvivin2. Conclusion Our results indicate that DNA methylation plays positive roles in BmNPV proliferation and loss of DNMT activity could induce the apoptosis of infected cells to suppress BmNPV proliferation. Our results may provide a new idea and research direction for the molecular mechanism on insect-virus interaction.

scholarly journals Complex DNA sequence readout mechanisms of the DNMT3B DNA methyltransferase

2020 ◽  
Vol 48 (20) ◽  
pp. 11495-11509
Author(s):  
Michael Dukatz ◽  
Sabrina Adam ◽  
Mahamaya Biswal ◽  
Jikui Song ◽  
Pavel Bashtrykov ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Catalytic Mechanism ◽  
Catalytic Domain ◽  
Dna Methyltransferases ◽  
Flanking Sequence ◽  
Cpg Sites ◽  
Binding Interface ◽  
Target Sites ◽  
Flanking Sequences

Abstract DNA methyltransferases interact with their CpG target sites in the context of variable flanking sequences. We investigated DNA methylation by the human DNMT3B catalytic domain using substrate pools containing CpX target sites in randomized flanking context and identified combined effects of CpG recognition and flanking sequence interaction together with complex contact networks involved in balancing the interaction with different flanking sites. DNA methylation rates were more affected by flanking sequences at non-CpG than at CpG sites. We show that T775 has an essential dynamic role in the catalytic mechanism of DNMT3B. Moreover, we identify six amino acid residues in the DNA-binding interface of DNMT3B (N652, N656, N658, K777, N779, and R823), which are involved in the equalization of methylation rates of CpG sites in favored and disfavored sequence contexts by forming compensatory interactions to the flanking residues including a CpG specific contact to an A at the +1 flanking site. Non-CpG flanking preferences of DNMT3B are highly correlated with non-CpG methylation patterns in human cells. Comparison of the flanking sequence preferences of human and mouse DNMT3B revealed subtle differences suggesting a co-evolution of flanking sequence preferences and cellular DNMT targets.

scholarly journals Thrombomodulin Is Silenced in Malignant Mesothelioma by a Poly(ADP-ribose) Polymerase-1-mediated Epigenetic Mechanism

2011 ◽  
Vol 286 (22) ◽  
pp. 19478-19488 ◽  
Author(s):  
Linda Nocchi ◽  
Marco Tomasetti ◽  
Monica Amati ◽  
Jiri Neuzil ◽  
Lory Santarelli ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Malignant Mesothelioma ◽  
Promoter Methylation ◽  
Dna Methyltransferase ◽  
Critical Role ◽  
Gene Promoter ◽  
Epigenetic Mechanism ◽  
Heterogeneous Expression ◽  
High Level

Malignant mesothelioma (MM) is often complicated by thromboembolic episodes, with thrombomodulin (TM) playing a critical role in the anticoagulant process. Heterogeneous expression of TM has been observed in cancer, and low or no TM expression in cancer cells is associated with poor prognosis. In this study, we analyzed TM expression in biopsies of MM patients and compared them with normal mesothelial tissue. The role of DNA methylation-associated gene silencing in TM expression was investigated. To evaluate poly(ADP-ribose) polymerase-1 (PARP1) as responsible for gene promoter epigenetic modifications, nonmalignant mesothelial cells (Met-5A) and MM cells (H28) were silenced for PARP1 and the DNA methylation/acetylation-associated TM expression evaluated. A correlation between low TM expression and high level of TM promoter methylation was found in MM biopsies. Low expression of TM was restored in MM cells by their treatment with 5-aza-2′-deoxycytidine and, to a lesser extent, with trichostatin, whereas the epigenetic agents did not affect TM expression in Met-5A cells. Silencing of PARP1 resulted in a strong down-regulation of TM expression in Met-5A cells, while restoring TM expression in H28 cells. PARP1 silencing induced TM promoter methylation in Met-5A cells and demethylation in MM cells, and this was paralleled by corresponding changes in the DNA methyltransferase activity. We propose that methylation of the TM promoter is responsible for silencing of TM expression in MM tissue, a process that is regulated by PARP1.

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.

scholarly journals Architectural Epigenetics: Mitotic Retention of Mammalian Transcriptional Regulatory Information

2010 ◽  
Vol 30 (20) ◽  
pp. 4758-4766 ◽  
Author(s):  
Sayyed K. Zaidi ◽  
Daniel W. Young ◽  
Martin Montecino ◽  
Jane B. Lian ◽  
Janet L. Stein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Transcription Factor ◽  
Epigenetic Mechanisms ◽  
Cellular Phenotype ◽  
Transcriptional Regulatory ◽  
Regulatory Information ◽  
Cellular Identity ◽  
Epigenetic Signatures ◽  
Transcription Factor Occupancy

ABSTRACT Epigenetic regulatory information must be retained during mammalian cell division to sustain phenotype-specific and physiologically responsive gene expression in the progeny cells. Histone modifications, DNA methylation, and RNA-mediated silencing are well-defined epigenetic mechanisms that control the cellular phenotype by regulating gene expression. Recent results suggest that the mitotic retention of nuclease hypersensitivity, selective histone marks, as well as the lineage-specific transcription factor occupancy of promoter elements contribute to the epigenetic control of sustained cellular identity in progeny cells. We propose that these mitotic epigenetic signatures collectively constitute architectural epigenetics, a novel and essential mechanism that conveys regulatory information to sustain the control of phenotype and proliferation in progeny cells by bookmarking genes for activation or suppression.

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