scholarly journals Genome wide efficiency profiling reveals modulation of maintenance and de novo methylation by Tets

2020 ◽  
Author(s):  
Pascal Giehr ◽  
Charalampos Kyriakopoulos ◽  
Karl Nordström ◽  
Abduhlrahman Salhab ◽  
Fabian Müller ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Epigenetic Modification ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Sequencing Data ◽  
Reduced Representation ◽  
Genome Wide ◽  
Global And Local

AbstractBackgroundDNA methylation is an essential epigenetic modification which is set and maintained by DNA methyl transferases (Dnmts) and removed via active and passive mechanisms involving Tet mediated oxidation. While the molecular mechanisms of these enzymes are well studied, their interplay on shaping cell specific methylomes remains less well understood. In our work we model the activities of Tets and Dnmts at single CpGs across the genome using a novel type of high resolution sequencing data.ResultsTo accurately measure 5mC and 5hmC levels at single CpGs we developed RRHPoxBS, a reduced representation hairpin oxidative bisulfite sequencing approach. Using this method we mapped the methylomes and hydroxymethylomes of wild type and Tet triple knockout mouse embryonic stem cells. These comprehensive datasets were then used to develop an extended Hidden Markov model allowing us i) to determine the symmetrical methylation and hydroxymethylation state at millions of individual CpGs, ii) infer the maintenance and de novo methylation efficiencies of Dnmts and the hydroxylation efficiencies of Tets at individual CpG positions. We find that Tets exhibit their highest activity around unmethylated regulatory elements, i.e. active promoters and enhancers. Furthermore, we find that Tets’ presence has a profound effect on the global and local maintenance and de novo methylation activities by the Dnmts, not only substantially contributing to a universal demethylation of the genome but also shaping the overall methylation landscape.ConclusionsOur analysis demonstrates that a fine tuned and locally controlled interplay between Tets and Dnmts is important to modulate de novo and maintenance activities of Dnmts across the genome. Tet activities contribute to DNA methylation patterning in the following ways: They oxidize 5mC, they locally shield DNA from accidental de novo methylation and at the same time modulate maintenance and de novo methylation efficiencies of Dnmts across the genome.

scholarly journals Transposable element expression in tumors is associated with immune infiltration and increased antigenicity

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Yu Kong ◽  
Christopher M. Rose ◽  
Ashley A. Cass ◽  
Alexander G. Williams ◽  
Martine Darwish ◽  
...  
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Computational Method ◽  
The Cancer Genome Atlas ◽  
Potential Consequence ◽  
Sequencing Data ◽  
Antiviral Responses ◽  
Genome Wide ◽  
Cancer Genome Atlas ◽  
Demethylation Agent

AbstractProfound global loss of DNA methylation is a hallmark of many cancers. One potential consequence of this is the reactivation of transposable elements (TEs) which could stimulate the immune system via cell-intrinsic antiviral responses. Here, we develop REdiscoverTE, a computational method for quantifying genome-wide TE expression in RNA sequencing data. Using The Cancer Genome Atlas database, we observe increased expression of over 400 TE subfamilies, of which 262 appear to result from a proximal loss of DNA methylation. The most recurrent TEs are among the evolutionarily youngest in the genome, predominantly expressed from intergenic loci, and associated with antiviral or DNA damage responses. Treatment of glioblastoma cells with a demethylation agent results in both increased TE expression and de novo presentation of TE-derived peptides on MHC class I molecules. Therapeutic reactivation of tumor-specific TEs may synergize with immunotherapy by inducing inflammation and the display of potentially immunogenic neoantigens.

QSER1 protects DNA methylation valleys from de novo methylation

Science ◽  
2021 ◽  
Vol 372 (6538) ◽  
pp. eabd0875 ◽  
Author(s):  
Gary Dixon ◽  
Heng Pan ◽  
Dapeng Yang ◽  
Bess P. Rosen ◽  
Therande Jashari ◽  
...  
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Embryonic Stem ◽  
Central Question ◽  
Mammalian Development ◽  
Uncharacterized Gene ◽  
Pathological Conditions ◽  
Genome Wide ◽  
A Genome ◽  
De Novo Methylation

DNA methylation is essential to mammalian development, and dysregulation can cause serious pathological conditions. Key enzymes responsible for deposition and removal of DNA methylation are known, but how they cooperate to regulate the methylation landscape remains a central question. Using a knockin DNA methylation reporter, we performed a genome-wide CRISPR-Cas9 screen in human embryonic stem cells to discover DNA methylation regulators. The top screen hit was an uncharacterized gene, QSER1, which proved to be a key guardian of bivalent promoters and poised enhancers of developmental genes, especially those residing in DNA methylation valleys (or canyons). We further demonstrate genetic and biochemical interactions of QSER1 and TET1, supporting their cooperation to safeguard transcriptional and developmental programs from DNMT3-mediated de novo methylation.

scholarly journals A pooling-based approach to mapping genetic variants associated with DNA methylation

2015 ◽  
Author(s):  
Irene Miriam Kaplow ◽  
Julia L MacIsaac ◽  
Sarah M Mah ◽  
Lisa M McEwen ◽  
Michael S Kobor ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Genetic Variants ◽  
Statistical Power ◽  
Epigenetic Modification ◽  
Chromatin Accessibility ◽  
Ctcf Binding ◽  
Sequencing Data ◽  
Individual Level ◽  
Novel Approach ◽  
Genome Wide

DNA methylation is an epigenetic modification that plays a key role in gene regulation. Previous studies have investigated its genetic basis by mapping genetic variants that are associated with DNA methylation at specific sites, but these have been limited to microarrays that cover less than 2% of the genome and cannot account for allele-specific methylation (ASM). Other studies have performed whole-genome bisulfite sequencing on a few individuals, but these lack statistical power to identify variants associated with DNA methylation. We present a novel approach in which bisulfite-treated DNA from many individuals is sequenced together in a single pool, resulting in a truly genome-wide map of DNA methylation. Compared to methods that do not account for ASM, our approach increases statistical power to detect associations while sharply reducing cost, effort, and experimental variability. As a proof of concept, we generated deep sequencing data from a pool of 60 human cell lines; we evaluated almost twice as many CpGs as the largest microarray studies and identified over 2,000 genetic variants associated with DNA methylation. We found that these variants are highly enriched for associations with chromatin accessibility and CTCF binding but are less likely to be associated with traits indirectly linked to DNA, such as gene expression and disease phenotypes. In summary, our approach allows genome-wide mapping of genetic variants associated with DNA methylation in any tissue of any species, without the need for individual-level genotype or methylation data.

scholarly journals YAP contributes to DNA methylation remodeling upon mouse embryonic stem cell differentiation

2020 ◽  
pp. jbc.RA120.015896
Author(s):  
Fabiana Passaro ◽  
Ilaria De Martino ◽  
Federico Zambelli ◽  
Giorgia Di Benedetto ◽  
Matteo Barbato ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Target Genes ◽  
De Novo ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Stem Cell Differentiation ◽  
Embryonic Stem Cell Differentiation ◽  
Nucleosome Remodeling ◽  
Genome Wide ◽  
A Genome

The Yes-associated protein YAP, one of the major effectors of the Hippo pathway together with its related protein TAZ, mediates a range of cellular processes from proliferation and death to morphogenesis. YAP and TAZ regulate a large number of target genes, acting as co-activators of DNA-binding transcription factors or as negative regulators of transcription by interacting with the nucleosome remodeling and histone deacetylase complexes. YAP is expressed in self-renewing embryonic stem cells (ESCs), although it is still debated whether it plays any crucial roles in the control of either stemness or differentiation. Here we show that the transient downregulation of YAP in mouse ESCs perturbs cellular homeostasis, leading to the inability to differentiate properly. Bisulfite genomic sequencing revealed that this transient knockdown caused a genome-wide alteration of the DNA methylation remodeling that takes place during the early steps of differentiation, suggesting that the phenotype we observed might be due to the dysregulation of some of the mechanisms involved in regulation of ESC exit from pluripotency. By gene expression analysis we identified two molecules which could have a role in the altered genome-wide methylation profile: the long non-coding RNA Ephemeron, whose rapid upregulation is crucial for ESCs transition into epiblast, and the methyltransferase-like protein Dnmt3l, which, during the embryo development, cooperates with Dnmt3a and Dnmt3b to contribute to the de novo DNA methylation that governs early steps of ESC differentiation. These data suggest a new role for YAP in the governance of the epigenetic dynamics of exit from pluripotency.

scholarly journals Transposable Element Exprssion in Tumors is Associated with Immune Infiltration and Increased Antigenicity

2018 ◽  
Author(s):  
Yu Kong ◽  
Chris Rose ◽  
Ashley A. Cass ◽  
Martine Darwish ◽  
Steve Lianoglou ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transposable Element ◽  
De Novo ◽  
Computational Method ◽  
The Cancer Genome Atlas ◽  
Sequencing Data ◽  
Genome Wide ◽  
Dna Damage Responses ◽  
Cancer Genome Atlas ◽  
Demethylation Agent

AbstractProfound loss of DNA methylation is a well-recognized hallmark of cancer. Given its role in silencing transposable elements (TEs), we hypothesized that extensive TE expression occurs in tumors with highly demethylated DNA. We developed REdiscoverTE, a computational method for quantifying genome-wide TE expression in RNA sequencing data. Using The Cancer Genome Atlas database, we observed increased expression of over 400 TE subfamilies, of which 262 appeared to result from a proximal loss of DNA methylation. The most recurrent TEs were among the evolutionarily youngest in the genome, predominantly expressed from intergenic loci, and associated with antiviral or DNA damage responses. Treatment of glioblastoma cells with a demethylation agent resulted in both increased TE expression and de novo presentation of TE-derived peptides on MHC class I molecules. Therapeutic reactivation of tumor-specific TEs may synergize with immunotherapy by inducing both inflammation and the display of potentially immunogenic neoantigens.One Sentence SummaryTransposable element expression in tumors is associated with increased immune response and provides tumor-associated antigens

scholarly journals DNA Epigenome Editing Using Crispr-Cas Suntag-Directed DNMT3A

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2707-2707 ◽  
Author(s):  
Yung-Hsin Huang ◽  
Su Jianzhong ◽  
Yong Lei ◽  
Michael C Gundry ◽  
Xiaotian Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Epigenetic Modification ◽  
Poor Correlation ◽  
Reduced Representation ◽  
Peptide Epitopes ◽  
Epigenome Editing ◽  
Multiple Copies

Abstract DNA methylation, an epigenetic modification, has widespread effects on gene expression during development. However, our ability to assign specific function to regions of DNA methylation is limited by the poor correlation between global patterns of DNA methylation and gene expression. To overcome this barrier, we utilized nuclease-deactivated Cas9 protein fused to repetitive peptide epitopes (SunTag) recruiting multiple copies of antibody-fused de novo DNA methyltranferase 3A (DNMT3A) (CRISPR-Cas SunTag-directed DNMT3A) to amplify local DNMT3A concentration and to methylate genomic sites of interest. Here, we demonstrated that CRISPR-Cas SunTag-directed DNMT3A not only dramatically increased CpG methylation but also, to our surprise, CpH (H =A or C or T) methylation at the HOXA5 lociin human embryonic kidney 293T cells (HEK293T). Furthermore, using a single sgRNA, CRISPR-Cas SunTag-directed DNMT3A was capable of methylating 4.5 kb genomic regions, surpassing previous targeted methylation tools whose activity is limited to 200bp. Using reduced representation bisulfite sequencing (RRBS) and RNA-seq, we concluded that CRISPR-Cas SunTag-directed DNMT3A methylated regions of interest without affecting global DNA methylome and transcriptome. This effective and precise tool enables site-specific manipulation of DNA methylation and may be used to address the relationship beteween DNA methylation and gene expression. Disclosures No relevant conflicts of interest to declare.

scholarly journals Differential Recruitment of Methylated CpG Binding Domains by the Orphan Receptor GCNF Initiates the Repression and Silencing of Oct4 Expression

2006 ◽  
Vol 26 (24) ◽  
pp. 9471-9483 ◽  
Author(s):  
Peili Gu ◽  
Damien Le Menuet ◽  
Arthur C.-K. Chung ◽  
Austin J. Cooney
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Epigenetic Modification ◽  
Es Cells ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Orphan Receptor ◽  
Proximal Promoter ◽  
Es Cell ◽  
Oct4 Expression

ABSTRACT The pluripotent factor Oct4 is a key transcription factor that maintains embryonic stem (ES) cell self-renewal and is down-regulated upon the differentiation of ES cells and silenced in somatic cells. A combination of cis elements, transcription factors, and epigenetic modifications, such as DNA methylation, are involved in the regulation of Oct4 gene expression. Here we show that the orphan nuclear receptor GCNF initiates Oct4 repression and DNA methylation by the differential recruitment of MBD (methylated CpG binding domain) factors to the promoter. Compared with wild-type ES cells and gastrulating embryos, Oct4 repression is lost and its proximal promoter is significantly hypomethylated in RA-differentiated GCNF−/− ES cells. The Oct4 gene is reexpressed in some somatic cells of GCNF−/− embryos, showing that it has not been properly silenced coincident with reduced DNA methylation of its promoter. Efforts to characterize mediators of GCNF's repressive function and DNA methylation of the Oct4 promoter identified methyl-DNA binding proteins, MBD3 and MBD2, as GCNF-interacting factors. In P19 and ES cells, upon differentiation, endogenous GCNF binds to the Oct4 proximal promoter and differentially recruits MBD3 and MBD2. In differentiated GCNF−/− ES cells, recruitment of MBD3 and MBD2 to the Oct4 promoter is lost, and repression of Oct4 expression and DNA methylation fails to occur. RNA interference-mediated knockdown of MBD3 and/or MBD2 expression results in reduced Oct4 repression in differentiated P19 and ES cells. Repression of Oct4 expression and recruitment of MBD3 are maintained in de novo DNA methylation-deficient ES cells (Dnmt3A/3B-null cells), while MBD2 recruitment is lost. Thus, recruitment of MBD3 and MBD2 by GCNF links two events, gene-specific repression and DNA methylation, which occur differentially at the Oct4 promoter. GCNF initiates the repression and epigenetic modification of Oct4 gene during ES cell differentiation.

scholarly journals Decoding the function of bivalent chromatin in development and cancer

2021 ◽  
pp. gr.275736.121
Author(s):  
Dhirendra Kumar ◽  
Senthilkumar Cinghu ◽  
Andrew J Oldfield ◽  
Pengyi Yang ◽  
Raja Jothi
Keyword(s):  
Dna Methylation ◽  
Regulatory Mechanism ◽  
De Novo ◽  
Embryonic Stem ◽  
Cell Types ◽  
Gene Promoters ◽  
Genome Wide ◽  
Silent Genes ◽  
Aberrant Dna Methylation ◽  
Rapid Activation

Bivalent chromatin is characterized by the simultaneous presence of H3K4me3 and H3K27me3, histone modifications generally associated with transcriptionally active and repressed chromatin, respectively. Prevalent in embryonic stem cells (ESCs), bivalency is postulated to poise/prime lineage-controlling developmental genes for rapid activation during embryogenesis while maintaining a transcriptionally repressed state in the absence of activation cues; however, this hypothesis remains to be directly tested. Most gene promoters DNA-hypermethylated in adult human cancers are bivalently marked in ESCs, and it was speculated that bivalency predisposes them for aberrant de novo DNA methylation and irreversible silencing in cancer, but evidence supporting this model is largely lacking. Here we show that bivalent chromatin does not poise genes for rapid activation but protects promoters from de novo DNA methylation. Genome-wide studies in differentiating ESCs reveal that activation of bivalent genes is no more rapid than that of other transcriptionally silent genes, challenging the premise that H3K4me3 is instructive for transcription. H3K4me3 at bivalent promoters, a product of the underlying DNA sequence, persists in nearly all cell types irrespective of gene expression and confers protection from de novo DNA methylation. Bivalent genes in ESCs that are frequent targets of aberrant hypermethylation in cancer are particularly strongly associated with loss of H3K4me3/bivalency in cancer. Altogether, our findings suggest that bivalency protects reversibly repressed genes from irreversible silencing and that loss of H3K4me3 may make them more susceptible to aberrant DNA methylation in diseases such as cancer. Bivalency may thus represent a distinct regulatory mechanism for maintaining epigenetic plasticity.

scholarly journals Copy number-dependent DNA methylation of the Pyricularia oryzae MAGGY retrotransposon is triggered by DNA damage

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Ba Van Vu ◽  
Quyet Nguyen ◽  
Yuki Kondo-Takeoka ◽  
Toshiki Murata ◽  
Naoki Kadotani ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Copy Number ◽  
Rna Silencing ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Pyricularia Oryzae ◽  
Transcriptional Gene Silencing ◽  
Physical Interaction ◽  
Uncharacterized Protein ◽  
Form Complex

AbstractTransposable elements are common targets for transcriptional and post-transcriptional gene silencing in eukaryotic genomes. However, the molecular mechanisms responsible for sensing such repeated sequences in the genome remain largely unknown. Here, we show that machinery of homologous recombination (HR) and RNA silencing play cooperative roles in copy number-dependent de novo DNA methylation of the retrotransposon MAGGY in the fungusPyricularia oryzae. Genetic and physical interaction studies revealed thatRecAdomain-containing proteins, includingP. oryzaehomologs ofRad51, Rad55, andRad57, together with an uncharacterized protein, Ddnm1, form complex(es) and mediate either the overall level or the copy number-dependence of de novo MAGGY DNA methylation, likely in conjunction with DNA repair. Interestingly,P. oryzaemutants of specific RNA silencing components (MoDCL1andMoAGO2)were impaired in copy number-dependence of MAGGY methylation. Co-immunoprecipitation of MoAGO2 and HR components suggested a physical interaction between the HR and RNA silencing machinery in the process.

scholarly journals Interplay between Histone and DNA Methylation Seen through Comparative Methylomes in Rare Mendelian Disorders

2021 ◽  
Vol 22 (7) ◽  
pp. 3735
Author(s):  
Guillaume Velasco ◽  
Damien Ulveling ◽  
Sophie Rondeau ◽  
Pauline Marzin ◽  
Motoko Unoki ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Catalytic Domain ◽  
De Novo ◽  
Mutation Screening ◽  
Histone H3 ◽  
Regulatory Elements ◽  
Germline Mutations ◽  
Dna Methyltransferases ◽  
Mendelian Disorders ◽  
Epigenetic Machinery

DNA methylation (DNAme) profiling is used to establish specific biomarkers to improve the diagnosis of patients with inherited neurodevelopmental disorders and to guide mutation screening. In the specific case of mendelian disorders of the epigenetic machinery, it also provides the basis to infer mechanistic aspects with regard to DNAme determinants and interplay between histone and DNAme that apply to humans. Here, we present comparative methylomes from patients with mutations in the de novo DNA methyltransferases DNMT3A and DNMT3B, in their catalytic domain or their N-terminal parts involved in reading histone methylation, or in histone H3 lysine (K) methylases NSD1 or SETD2 (H3 K36) or KMT2D/MLL2 (H3 K4). We provide disease-specific DNAme signatures and document the distinct consequences of mutations in enzymes with very similar or intertwined functions, including at repeated sequences and imprinted loci. We found that KMT2D and SETD2 germline mutations have little impact on DNAme profiles. In contrast, the overlapping DNAme alterations downstream of NSD1 or DNMT3 mutations underlines functional links, more specifically between NSD1 and DNMT3B at heterochromatin regions or DNMT3A at regulatory elements. Together, these data indicate certain discrepancy with the mechanisms described in animal models or the existence of redundant or complementary functions unforeseen in humans.

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