scholarly journals Loss of Fam60a, a Sin3a subunit, results in embryonic lethality and is associated with aberrant methylation at a subset of gene promoters

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Ryo Nabeshima ◽  
Osamu Nishimura ◽  
Takako Maeda ◽  
Natsumi Shimizu ◽  
Takahiro Ide ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Embryonic Stem ◽  
Dna Demethylation ◽  
Specific Gene ◽  
Aberrant Methylation ◽  
Gene Promoters ◽  
Mouse Development ◽  
Promoter Regions ◽  
Corepressor Complex ◽  
Genome Wide

We have examined the role of Fam60a, a gene highly expressed in embryonic stem cells, in mouse development. Fam60a interacts with components of the Sin3a-Hdac transcriptional corepressor complex, and most Fam60a–/– embryos manifest hypoplasia of visceral organs and die in utero. Fam60a is recruited to the promoter regions of a subset of genes, with the expression of these genes being either up- or down-regulated in Fam60a–/– embryos. The DNA methylation level of the Fam60a target gene Adhfe1 is maintained at embryonic day (E) 7.5 but markedly reduced at E9.5 in Fam60a–/– embryos, suggesting that DNA demethylation is enhanced in the mutant. Examination of genome-wide DNA methylation identified several differentially methylated regions, which were preferentially hypomethylated, in Fam60a–/– embryos. Our data suggest that Fam60a is required for proper embryogenesis, at least in part as a result of its regulation of DNA methylation at specific gene promoters.

scholarly journals Dnmt1 binds and represses genomic retroelements via DNA methylation in mouse early embryos

2020 ◽  
Vol 48 (15) ◽  
pp. 8431-8444 ◽  
Author(s):  
Byungkuk Min ◽  
Jung Sun Park ◽  
Young Sun Jeong ◽  
Kyuheum Jeon ◽  
Yong-Kook Kang
Keyword(s):  
Dna Methylation ◽  
Cpg Islands ◽  
Endogenous Retrovirus ◽  
Dna Demethylation ◽  
Mouse Development ◽  
Genome Wide ◽  
Early Embryos ◽  
Dna Adenine Methylase ◽  
Early Mouse ◽  
Early Developmental

Abstract Genome-wide passive DNA demethylation in cleavage-stage mouse embryos is related to the cytoplasmic localization of the maintenance methyltransferase DNMT1. However, recent studies provided evidences of the nuclear localization of DNMT1 and its contribution to the maintenance of methylation levels of imprinted regions and other genomic loci in early embryos. Using the DNA adenine methylase identification method, we identified Dnmt1-binding regions in four- and eight-cell embryos. The unbiased distribution of Dnmt1 peaks in the genic regions (promoters and CpG islands) as well as the absence of a correlation between the Dnmt1 peaks and the expression levels of the peak-associated genes refutes the active participation of Dnmt1 in the transcriptional regulation of genes in the early developmental period. Instead, Dnmt1 was found to associate with genomic retroelements in a greatly biased fashion, particularly with the LINE1 (long interspersed nuclear elements) and ERVK (endogenous retrovirus type K) sequences. Transcriptomic analysis revealed that the transcripts of the Dnmt1-enriched retroelements were overrepresented in Dnmt1 knockdown embryos. Finally, methyl-CpG-binding domain sequencing proved that the Dnmt1-enriched retroelements, which were densely methylated in wild-type embryos, became demethylated in the Dnmt1-depleted embryos. Our results indicate that Dnmt1 is involved in the repression of retroelements through DNA methylation in early mouse development.

scholarly journals Deletion of Tet proteins results in quantitative disparities during ESC differentiation partially attributable to alterations in gene expression

2019 ◽  
Author(s):  
Michael J Reimer ◽  
Kirthi Pulakanti ◽  
Linzheng Shi ◽  
Alex Abel ◽  
Mingyu Liang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Critical Role ◽  
Embryonic Stem ◽  
Dna Demethylation ◽  
Intermediate Phenotype ◽  
Gene Promoters ◽  
Dna Hypermethylation ◽  
Tet Proteins ◽  
Tet Protein

Abstract Background: The Tet protein family (Tet1, Tet2, and Tet3) regulate DNA methylation through conversion of 5-methylcytosine to 5-hydroxymethylcytosine which can ultimately result in DNA demethylation and play a critical role during early mammalian development and pluripotency¬. While multiple groups have generated knockouts combining loss of different Tet proteins in murine embryonic stem cells (ESCs), differences in genetic background and approaches has made it difficult to directly compare results and discern the direct mechanism by which Tet proteins regulate the transcriptome. To address this concern, we utilized genomic editing in an isogenic pluripotent background which permitted a quantitative, flow-cytometry based measurement of pluripotency in combination with genome-wide assessment of gene expression and DNA methylation changes. Our ultimate goal was to generate a resource of large-scale datasets to permit hypothesis-generating experiments. Results: We demonstrate a quantitative disparity in the differentiation ability among Tet protein deletions, with Tet2 single knockout exhibiting the most severe defect, while loss of Tet1 ¬alone or combinations of Tet genes showed a quantitatively intermediate phenotype. Using a combination of transcriptomic and epigenomic approaches we demonstrate an increase in DNA hypermethylation and a divergence of transcriptional profiles in pluripotency among Tet deletions, with loss of Tet2 having the most profound effect in undifferentiated ESCs. Conclusions: We conclude that loss of Tet2 has the most dramatic effect both on the phenotype of ESCs and the transcriptome compared to other genotypes. While loss of Tet proteins increased DNA hypermethylation, especially in gene promoters, these changes in DNA methylation did not correlate with gene expression changes. Thus, while loss of different Tet proteins alters DNA methylation, this change does not appear to be directly responsible for transcriptome changes. Thus, loss of Tet proteins likely regulates the transcriptome epigenetically both through altering 5mC but also through additional mechanisms. Nonetheless, the transcriptome changes in pluripotent Tet2-/- ESCs compared to wild-type implies that the disparities in differentiation can be partially attributed to baseline alterations in gene expression.

scholarly journals Deletion of Tet proteins results in quantitative disparities during ESC differentiation partially attributable to differences in gene expression

2019 ◽  
Author(s):  
Michael J Reimer ◽  
Kirthi Pulakanti ◽  
Linzheng Shi ◽  
Alex Abel ◽  
Mingyu Liang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Critical Role ◽  
Embryonic Stem ◽  
Dna Demethylation ◽  
Intermediate Phenotype ◽  
Gene Promoters ◽  
Dna Hypermethylation ◽  
Tet Proteins ◽  
Tet Protein

Abstract Background: The Tet protein family (Tet1, Tet2, and Tet3) regulate DNA methylation through conversion of 5-methylcytosine to 5-hydroxymethylcytosine which can ultimately result in DNA demethylation and play a critical role during early mammalian development and pluripotency¬. While multiple groups have generated knockouts combining loss of different Tet proteins in murine embryonic stem cells (ESCs), differences in genetic background and approaches has made it difficult to directly compare results and discern the direct mechanism by which Tet proteins regulate the transcriptome. To address this concern, we utilized genomic editing in an isogenic pluripotent background which permitted a quantitative, flow-cytometry based measurement of pluripotency in combination with genome-wide assessment of gene expression and DNA methylation changes. Our ultimate goal was to generate a resource of large-scale datasets to permit hypothesis-generating experiments. Results: We demonstrate a quantitative disparity in the differentiation ability among Tet protein deletions, with Tet2 single knockout exhibiting the most severe defect, while loss of Tet1 ¬alone or combinations of Tet genes showed a quantitatively intermediate phenotype. Using a combination of transcriptomic and epigenomic approaches we demonstrate an increase in DNA hypermethylation and a divergence of transcriptional profiles in pluripotency among Tet deletions, with loss of Tet2 having the most profound effect in undifferentiated ESCs. Conclusions: We conclude that loss of Tet2 has the most dramatic effect both on the phenotype of ESCs and the transcriptome compared to other genotypes. While loss of Tet proteins increased DNA hypermethylation, especially in gene promoters, these changes in DNA methylation did not correlate with gene expression changes. Thus, while loss of different Tet proteins alters DNA methylation, this change does not appear to be directly responsible for transcriptome changes. Thus, loss of Tet proteins likely regulates the transcriptome epigenetically both through altering 5mC but also through additional mechanisms. Nonetheless, the transcriptome changes in pluripotent Tet2-/- ESCs compared to wild-type implies that the disparities in differentiation can be partially attributed to baseline alterations in gene expression.

scholarly journals Deletion of Tet proteins results in quantitative disparities during ESC differentiation partially attributable to alterations in gene expression

2019 ◽  
Author(s):  
Michael J Reimer ◽  
Kirthi Pulakanti ◽  
Linzheng Shi ◽  
Alex Abel ◽  
Mingyu Liang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Critical Role ◽  
Embryonic Stem ◽  
Dna Demethylation ◽  
Intermediate Phenotype ◽  
Gene Promoters ◽  
Dna Hypermethylation ◽  
Tet Proteins ◽  
Tet Protein

Abstract Background: The Tet protein family (Tet1, Tet2, and Tet3) regulate DNA methylation through conversion of 5-methylcytosine to 5-hydroxymethylcytosine which can ultimately result in DNA demethylation and play a critical role during early mammalian development and pluripotency¬. While multiple groups have generated knockouts combining loss of different Tet proteins in murine embryonic stem cells (ESCs), differences in genetic background and approaches has made it difficult to directly compare results and discern the direct mechanism by which Tet proteins regulate the transcriptome. To address this concern, we utilized genomic editing in an isogenic pluripotent background which permitted a quantitative, flow-cytometry based measurement of pluripotency in combination with genome-wide assessment of gene expression and DNA methylation changes. Our ultimate goal was to generate a resource of large-scale datasets to permit hypothesis-generating experiments. Results: We demonstrate a quantitative disparity in the differentiation ability among Tet protein deletions, with Tet2 single knockout exhibiting the most severe defect, while loss of Tet1 ¬alone or combinations of Tet genes showed a quantitatively intermediate phenotype. Using a combination of transcriptomic and epigenomic approaches we demonstrate an increase in DNA hypermethylation and a divergence of transcriptional profiles in pluripotency among Tet deletions, with loss of Tet2 having the most profound effect in undifferentiated ESCs. Conclusions: We conclude that loss of Tet2 has the most dramatic effect both on the phenotype of ESCs and the transcriptome compared to other genotypes. While loss of Tet proteins increased DNA hypermethylation, especially in gene promoters, these changes in DNA methylation did not correlate with gene expression changes. Thus, while loss of different Tet proteins alters DNA methylation, this change does not appear to be directly responsible for transcriptome changes. Thus, loss of Tet proteins likely regulates the transcriptome epigenetically both through altering 5mC but also through additional mechanisms. Nonetheless, the transcriptome changes in pluripotent Tet2-/- ESCs compared to wild-type implies that the disparities in differentiation can be partially attributed to baseline alterations in gene expression.

scholarly journals Frequent lack of repressive capacity of promoter DNA methylation identified through genome-wide epigenomic manipulation

2017 ◽  
Author(s):  
Ethan Ford ◽  
Matthew R. Grimmer ◽  
Sabine Stolzenburg ◽  
Ozren Bogdanovic ◽  
Alex de Mendoza ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Fusion Protein ◽  
Rna Polymerase Ii ◽  
Zinc Finger ◽  
Gene Promoters ◽  
Natural Conditions ◽  
Promoter Regions ◽  
Genome Wide ◽  
Promoter Dna Methylation ◽  
Promoter Dna

AbstractIt is widely assumed that the addition of DNA methylation at CpG rich gene promoters silences gene transcription. However, this conclusion is largely drawn from the observation that promoter DNA methylation inversely correlates with gene expression in natural conditions. The effect of induced DNA methylation on endogenous promoters has yet to be comprehensively assessed. Here, we induced the simultaneous methylation of thousands of promoters in the genome of human cells using an engineered zinc finger-DNMT3A fusion protein, enabling assessment of the effect of forced DNA methylation upon transcription, histone modifications, and DNA methylation persistence after the removal of the fusion protein. We find that DNA methylation is frequently insufficient to transcriptionally repress promoters. Furthermore, DNA methylation deposited at promoter regions associated with H3K4me3 is rapidly erased after removal of the zinc finger-DNMT3A fusion protein. Finally, we demonstrate that induced DNA methylation can exist simultaneously on promoter nucleosomes that possess the active histone modification H3K4me3, or DNA bound by the initiated form of RNA polymerase II. These findings suggest that promoter DNA methylation is not generally sufficient for transcriptional inactivation, with implications for the emerging field of epigenome engineering.One Sentence SummaryGenome-wide epigenomic manipulation of thousands of human promoters reveals that induced promoter DNA methylation is unstable and frequently does not function as a primary instructive biochemical signal for gene silencing and chromatin reconfiguration.

scholarly journals DNA Methylation of Enhancer Elements in Myeloid Neoplasms: Think Outside the Promoters?

Cancers ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 1424 ◽  
Author(s):  
Ordoñez ◽  
Martínez-Calle ◽  
Agirre ◽  
Prosper
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Epigenetic Modification ◽  
Myeloproliferative Neoplasms ◽  
Regulation Of Gene Expression ◽  
Fine Tuning ◽  
Specific Gene ◽  
Gene Promoters ◽  
Regulatory Regions ◽  
Genome Wide

Gene regulation through DNA methylation is a well described phenomenon that has a prominent role in physiological and pathological cell-states. This epigenetic modification is usually grouped in regions denominated CpG islands, which frequently co-localize with gene promoters, silencing the transcription of those genes. Recent genome-wide DNA methylation studies have challenged this paradigm, demonstrating that DNA methylation of regulatory regions outside promoters is able to influence cell-type specific gene expression programs under physiologic or pathologic conditions. Coupling genome-wide DNA methylation assays with histone mark annotation has allowed for the identification of specific epigenomic changes that affect enhancer regulatory regions, revealing an additional layer of complexity to the epigenetic regulation of gene expression. In this review, we summarize the novel evidence for the molecular and biological regulation of DNA methylation in enhancer regions and the dynamism of these changes contributing to the fine-tuning of gene expression. We also analyze the contribution of enhancer DNA methylation on the expression of relevant genes in acute myeloid leukemia and chronic myeloproliferative neoplasms. The characterization of the aberrant enhancer DNA methylation provides not only a novel pathogenic mechanism for different tumors but also highlights novel potential therapeutic targets for myeloid derived neoplasms.

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.

Abstract 40: Disturbed Flow Alters Genomewide DNA Methylation Patterns, Regulating Endothelial Gene Expression and Atherosclerosis

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Jessilyn Dunn ◽  
Haiwei Qiu ◽  
Soyeon Kim ◽  
Daudi Jjingo ◽  
Ryan Hoffman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Methylation Status ◽  
Western Diet ◽  
Dependent Manner ◽  
Gene Promoters ◽  
Genome Wide ◽  
Methylation Patterns ◽  
Endothelial Gene Expression

Atherosclerosis preferentially occurs in arterial regions of disturbed blood flow (d-flow), which alters gene expression, endothelial function, and atherosclerosis. Here, we show that d-flow regulates genome-wide DNA methylation patterns in a DNA methyltransferase (DNMT)-dependent manner. We found that d-flow induced expression of DNMT1, but not DNMT3a or DNMT3b, in mouse arterial endothelium in vivo and in cultured endothelial cells by oscillatory shear (OS) compared to unidirectional laminar shear in vitro. The DNMT inhibitor 5-Aza-2’deoxycytidine (5Aza) or DNMT1 siRNA significantly reduced OS-induced endothelial inflammation. Moreover, 5Aza reduced lesion formation in two atherosclerosis models using ApoE-/- mice (western diet for 3 months and the partial carotid ligation model with western diet for 3 weeks). To identify the 5Aza mechanisms, we conducted two genome-wide studies: reduced representation bisulfite sequencing (RRBS) and transcript microarray using endothelial-enriched gDNA and RNA, respectively, obtained from the partially-ligated left common carotid artery (LCA exposed to d-flow) and the right contralateral control (RCA exposed to s-flow) of mice treated with 5Aza or vehicle. D-flow induced DNA hypermethylation in 421 gene promoters, which was significantly prevented by 5Aza in 335 genes. Systems biological analyses using the RRBS and the transcriptome data revealed 11 mechanosensitive genes whose promoters were hypermethylated by d-flow but rescued by 5Aza treatment. Of those, five genes contain hypermethylated cAMP-response-elements in their promoters, including the transcription factors HoxA5 and Klf3. Their methylation status could serve as a mechanosensitive master switch in endothelial gene expression. Our results demonstrate that d-flow controls epigenomic DNA methylation patterns in a DNMT-dependent manner, which in turn alters endothelial gene expression and induces atherosclerosis.

Lung cancer risk stratification using methylation profile in the oral epithelium

2018 ◽  
Vol 27 (2) ◽  
pp. 87-92 ◽  
Author(s):  
Hiroaki Harada ◽  
Kazuaki Miyamaoto ◽  
Masami Kimura ◽  
Tetsuro Ishigami ◽  
Kiyomi Taniyama ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Dna Methylation ◽  
Cancer Risk ◽  
Risk Stratification ◽  
Lung Cancer Risk ◽  
Cancer Risk Assessment ◽  
Oral Epithelium ◽  
Specific Gene ◽  
Aberrant Methylation ◽  
Cancer Genes

Background Assuming that the entire airway is affected by the same inhaled carcinogen, similar molecular alterations may occur in the lung and oral cavity. Thus, we hypothesized that DNA methylation profiles in the oral epithelium may be a promising biomarker for lung cancer risk stratification. Methods A methylation-specific polymerase chain reaction was performed on oral epithelium from 16 patients with lung cancer and 32 controls without lung cancer. Genes showing aberrant methylation profiles in the oral epithelium were compared between patients and controls. Results The analysis revealed that HOXD11 and PCDHGB6 were methylated more frequently in patients than in controls ( p = 0.0055 and p = 0.0247, respectively). Combined analyses indicated that 8 of 16 (50%) patients and 3 of 32 (9.4%) controls showed DNA methylation in both genes ( p = 0.0016). Among the population limited to current and former smokers, 6 of 11 (54.5%) patients showed methylation in both genes, compared to 1 of 17 (5.9%) controls ( p = 0.0037). In a subgroup analysis limited to the population above 50-years old, 8 of 16 (50%) patients and 2 of 16 (12.5%) controls showed methylation in both genes ( p = 0.0221). Conclusions The results of this study indicate that specific gene methylation in the oral epithelium might be a promising biomarker for lung cancer risk assessment, especially among smokers. Risk stratification through the analysis of DNA methylation profiles in the oral epithelium may be a useful and less invasive first-step approach in an efficient two-step lung cancer screening strategy.

scholarly journals DAXX safeguards pericentromeric heterochromatin formation in embryonic stem cells

2021 ◽  
Author(s):  
Antoine Canat ◽  
Adeline Veillet ◽  
Robert Illingworth ◽  
Emmanuelle Fabre ◽  
Pierre Therizols
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Dna Damage ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Pericentric Heterochromatin ◽  
Dna Demethylation ◽  
Pericentromeric Heterochromatin ◽  
Major Satellite ◽  
Heterochromatin Formation

AbstractDNA methylation is essential for heterochromatin formation and repression of DNA repeat transcription, both of which are essential for genome integrity. Loss of DNA methylation is associated with disease, including cancer, but is also required for development. Alternative pathways to maintain heterochromatin are thus needed to limit DNA damage accumulation. Here, we find that DAXX, an H3.3 chaperone, protects pericentromeric heterochromatin and is essential for embryonic stem cells (ESCs) maintenance in the ground-state of pluripotency. Upon DNA demethylation-mediated damage, DAXX relocalizes to pericentromeric regions, and recruits PML and SETDB1, thereby promoting heterochromatin formation. In the absence of DAXX, the 3D-architecture and physical properties of pericentric heterochromatin are disrupted, resulting in derepression of major satellite DNA. Using epigenome editing tools, we demonstrate that H3.3, and specifically H3.3K9 modification, directly contribute to maintaining pericentromeric chromatin conformation. Altogether, our data reveal that DAXX and H3.3 unite DNA damage response and heterochromatin maintenance in ESCs.

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