Methylome inheritance and enhancer dememorization reset an epigenetic gate safeguarding embryonic programs

2021 ◽  
Author(s):  
Xiaotong Wu ◽  
Hongmei Zhang ◽  
Bingjie Zhang ◽  
Yu Zhang ◽  
Qiuyan Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Early Development ◽  
Epigenetic Reprogramming ◽  
Adult Tissue ◽  
Dna Hypermethylation ◽  
Tissue Specific ◽  
Early Embryos

Drastic epigenetic reprogramming is essential to convert terminally-differentiated gametes to totipotent embryos. However, it remains puzzling why post-fertilization global DNA reprogramming occurs only in mammals but not in non-mammalian vertebrates. In zebrafish, global methylome inheritance is however accompanied by sweeping enhancer "dememorization" as they become fully methylated. By depleting maternal dnmt1 using oocyte microinjection in situ, we eliminated DNA methylation in zebrafish early embryos, which died around gastrulation with severe differentiation defects. Strikingly, methylation deficiency leads to extensive derepression of adult tissue-specific genes and CG-rich enhancers, which acquire ectopic TF binding and, unexpectedly, H3K4me3. By contrast, embryonic enhancers are generally CG-poor and evade DNA methylation repression. Hence, global DNA hypermethylation inheritance coupled with enhancer dememorization installs an epigenetic gate that safeguards embryonic programs and ensures temporally ordered gene expression. We propose that "enhancer dememorization" underlies and unifies distinct epigenetic reprogramming modes in early development between mammals and non-mammals.

Single-cell multiomics sequencing reveals the functional regulatory landscape of early embryos

2019 ◽  
Author(s):  
Yang Wang ◽  
Peng Yuan ◽  
Zhiqiang Yan ◽  
Ming Yang ◽  
Ying Huo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Single Cell ◽  
Regulatory Network ◽  
Chromatin Accessibility ◽  
Epigenetic Reprogramming ◽  
Preimplantation Development ◽  
Rna Expression ◽  
Early Embryos ◽  
Allele Specific

AbstractExtensive epigenetic reprogramming occurs during preimplantation embryo development and is accompanied by zygotic genome activation (ZGA) and first cell fate specification. Recent studies using single-cell epigenome sequencing techniques have provided global views of the dynamics of different epigenetic layers during this period. However, it remains largely unclear how the drastic epigenetic reprogramming contributes to transcriptional regulatory network. Here, we developed a single-cell multiomics sequencing technology (scNOMeRe-seq) that enables profiling of genome-wide chromatin accessibility, DNA methylation and RNA expression in the same individual cell with improved performance compared to that of earlier techniques. We applied this method to analyze the global dynamics of different molecular layers and their associations in mouse preimplantation embryos. We found that global DNA methylation remodeling facilitates the reconstruction of genetic lineages in early embryos and revealed that the gradual increases in heterogeneity among blastomeres are driven by asymmetric cleavage. Allele-specific DNA methylation pattern is maintained throughout preimplantation development and is accompanied by allele-specific associations between DNA methylation and gene expression in the gene body that are inherited from oocytes and sperm. Through integrated analyses of the collective dynamics between gene expression and chromatin accessibility, we constructed a ZGA-associated regulatory network and revealed coordination among multiple epigenetic layers, transcription factors (TFs) and repeat elements that instruct the proper ZGA process. Moreover, we found that inner cell mass (ICM)/trophectoderm (TE) lineage-associated cis-regulatory elements are stepwise activated in blastomeres during post-ZGA embryo stages. TE lineage-specific TFs play dual roles in promoting the TE program while repressing the ICM program, thereby separating the TE lineage from the ICM lineage. Taken together, our findings not only depict the first single-cell triple-omics map of chromatin accessibility, DNA methylation and RNA expression during mouse preimplantation development but also enhance the fundamental understanding of epigenetic regulation in early embryos.

scholarly journals Reversible DNA Hypermethylation of the Interleukin-15 (IL-15) Promoter Induces IL-15 Expression, Drives the Pathogenesis of T-Cell Large Granular Lymphocytic Leukemia and Provides a Potential Therapeutic Approach Using 5-Azacitidine

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3776-3776
Author(s):  
Jonathan E Brammer ◽  
Amy E Boles ◽  
Anthony Mansour ◽  
Aharon G. Freud ◽  
Monique Mathé-Allainmat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
T Cell ◽  
Research Funding ◽  
Marked Decrease ◽  
Lymphocytic Leukemia ◽  
Dna Hypermethylation ◽  
Reduced Representation ◽  
Primary Driver ◽  
Interleukin 15

Background and Rationale: T-cell large granular lymphocytic leukemia (T-LGLL) is an incurable clonal proliferation of CD8+ memory T-cells that leads to profound neutropenia and anemia with limited treatment options. The primary driver of T-LGLL is overexpression of interleukin-15 (IL-15), a gamma-chain cytokine. Previously, we have demonstrated that mice overexpressing IL-15 develop DNA hypermethylation and chromosomal instability that leads to the spontaneous development of LGLL (Mishra et al. Cancer Cell 2012). Further, the IL-15 promoter is known to be hypermethylated in cutaneous T-cell lymphoma (CTCL), another IL-15 driven malignancy (Mishra et al. Cancer Discovery 2016). In CTCL patients, the counterintuitive increase in IL-15 mRNA was due to hypermethylation of its promoter at the repressor binding sequences in the IL-15 gene. However, the methylation status of the IL-15 promoter in T-LGLL patients remains unknown. Concept: We hypothesize that the IL-15 promoter is hypermethylated in patients with T-LGLL, leading to aberrant overexpression of IL-15 and that this hypermethylation is a critical event in the leukemogenesis of T-LGLL. If true, demethylation of the IL-15 promoter with a resultant decrease in IL-15 transcripts should lead to apoptosis of T-LGLL cells. Hypomethylation of the IL-15 promoter, therefore, may provide a novel therapeutic approach to inhibiting IL-15, the primary driver of T-LGLL. Results: CD3+/CD8+/CD5-/dim T-cells were purified from peripheral blood of LGLL patient (n=3) and normal donor (ND) (n=3) by flow cytometry sorting. We analyzed DNA methylation and gene expression profiling using reduced representation bisulfite and RNA sequencing. With bioinformatics analysis, we determined differential methylation (1-way ANOVA P= 0.0178) and expression (1-way ANOVA P =0.0059). These data sets revealed significant differential hypermethylation of gene promoters in leukemic samples, compared to controls (Figure 1A). Reduced representation bisulfite sequencing that can identify differentially methylated regions at single base-pair resolutions demonstrated an increase in DNA methylation of the IL-15 promoter in patient samples over controls. To determine the functional significance of this finding, we treated the MOTN-1 T-LGLL cell line in vitro with the hypomethylating agent, 5-azacytidine (5-aza) at concentrations of 0.5 uM, 1 uM, 2.5 uM, and 5 uM. At 24 and 48 hours, a marked decrease in the viability of T-LGLL cells was observed, from 100% to 49.50%, p=0.037; particularly at higher concentrations of 5-aza (100% to 27% +11.30%, p=0.0030). Next, we sought to determine whether 5-aza induced hypomethylation of the IL-15 promoter. IL-15 gene expression in MOTN-1 T-LGLL cells treated with 5-aza was measured in comparison to control treated MOTN-1 cells. A marked decrease in IL-15 expression was observed at all concentrations of 5-aza compared to control (Figure 1B, p=0.0001). These results confirm that 5-aza leads to decreased transcription of the IL-15 gene, possibly due to hypomethylation of the IL-15 promoter. Finally, to determine whether a decrease in IL-15 alone was the cause of increased apoptosis of T-LGLL cells, we exposed MOTN-1 cells to a novel IL-15 inhibitor, IBI-15, and compared cell viability against MOTN-1 cells exposed to an inactive control, IBI-40. Even more profound decrease in cell viability was observed utilizing IBI-15 that targets the binding of IL-15 to its receptor (Figure 1C). Together, these data suggest that hypermethylation of the IL-15 promoter is critical to the pathogenesis of T-LGLL, and that treatment with 5-aza is sufficient to induce hypomethylation of the IL-15 promoter, decrease IL-15 transcription, and induce apoptosis in T-LGLL cells. Conclusions: Hypermethylation of the IL-15 promoter, with subsequent increase in IL-15, is critical to the pathogenesis of T-LGLL. Inhibition of the IL-15 promoter hypermethylation by 5-aza leads to down-regulation of the IL-15 gene transcript, which is sufficient to induce apoptosis of T-LGLL cells. These data suggest that 5-aza induced hypomethylation may be a novel method to induce IL-15 inhibition and a potentially efficacious clinical strategy against T-LGLL. Disclosures Brammer: Bioniz Therapeutics, Inc.: Research Funding; Viracta Therapeutics, Inc.: Research Funding; Verastem, Inc: Research Funding. Porcu:Daiichi: Research Funding; BeiGene: Other: Scientific Board, Research Funding; Spectrum: Consultancy; Viracta: Honoraria, Other: Scientific Board, Research Funding; Innate Pharma: Honoraria, Other: Scientific Board, Research Funding; Kyowa: Honoraria, Other: Scientific Board, Research Funding; ADCT: Research Funding; Incyte: Research Funding. OffLabel Disclosure: IBI-15 IBI-40 IL-15 inhibitor

scholarly journals DNA hypermethylation associated with upregulated gene expression in prostate cancer demonstrates the diversity of epigenetic regulation

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Ieva Rauluseviciute ◽  
Finn Drabløs ◽  
Morten Beck Rye
Keyword(s):  
Gene Expression ◽  
Prostate Cancer ◽  
Dna Methylation ◽  
Epigenetic Regulation ◽  
Normal Tissue ◽  
Expression Profiles ◽  
Expression Data ◽  
Dna Hypermethylation ◽  
Genome Wide ◽  
A Genome

Abstract Background Prostate cancer (PCa) has the highest incidence rates of cancers in men in western countries. Unlike several other types of cancer, PCa has few genetic drivers, which has led researchers to look for additional epigenetic and transcriptomic contributors to PCa development and progression. Especially datasets on DNA methylation, the most commonly studied epigenetic marker, have recently been measured and analysed in several PCa patient cohorts. DNA methylation is most commonly associated with downregulation of gene expression. However, positive associations of DNA methylation to gene expression have also been reported, suggesting a more diverse mechanism of epigenetic regulation. Such additional complexity could have important implications for understanding prostate cancer development but has not been studied at a genome-wide scale. Results In this study, we have compared three sets of genome-wide single-site DNA methylation data from 870 PCa and normal tissue samples with multi-cohort gene expression data from 1117 samples, including 532 samples where DNA methylation and gene expression have been measured on the exact same samples. Genes were classified according to their corresponding methylation and expression profiles. A large group of hypermethylated genes was robustly associated with increased gene expression (UPUP group) in all three methylation datasets. These genes demonstrated distinct patterns of correlation between DNA methylation and gene expression compared to the genes showing the canonical negative association between methylation and expression (UPDOWN group). This indicates a more diversified role of DNA methylation in regulating gene expression than previously appreciated. Moreover, UPUP and UPDOWN genes were associated with different compartments — UPUP genes were related to the structures in nucleus, while UPDOWN genes were linked to extracellular features. Conclusion We identified a robust association between hypermethylation and upregulation of gene expression when comparing samples from prostate cancer and normal tissue. These results challenge the classical view where DNA methylation is always associated with suppression of gene expression, which underlines the importance of considering corresponding expression data when assessing the downstream regulatory effect of DNA methylation.

scholarly journals Microarray analysis of gene expression during early development: a cautionary overview

Reproduction ◽  
2010 ◽  
Vol 140 (6) ◽  
pp. 787-801 ◽  
Author(s):  
Claude Robert
Keyword(s):  
Gene Expression ◽  
Early Development ◽  
Microarray Analysis ◽  
Developmental Stages ◽  
Sample Handling ◽  
Early Embryos ◽  
High Throughput Method ◽  
Full Benefit ◽  
Key Steps ◽  
Early Developmental

The rise of the ‘omics’ technologies started nearly a decade ago and, among them, transcriptomics has been used successfully to contrast gene expression in mammalian oocytes and early embryos. The scarcity of biological material that early developmental stages provide is the prime reason why the field of transcriptomics is becoming more and more popular with reproductive biologists. The potential to amplify scarce mRNA samples and generate the necessary amounts of starting material enables the relative measurement of RNA abundance of thousands of candidates simultaneously. So far, microarrays have been the most commonly used high-throughput method in this field. Microarray platforms can be found in a wide variety of formats, from cDNA collections to long or short oligo probe sets. These platforms generate large amounts of data that require the integration of comparative RNA abundance values in the physiological context of early development for their full benefit to be appreciated. Unfortunately, significant discrepancies between datasets suggest that direct comparison between studies is difficult and often not possible. We have investigated the sample-handling steps leading to the generation of microarray data produced from prehatching embryo samples and have identified key steps that significantly impact the downstream results. This review provides a discussion on the best methods for the preparation of samples from early embryos for microarray analysis and focuses on the challenges that impede dataset comparisons from different platforms and the reasons why methodological benchmarking performed using somatic cells may not apply to the atypical nature of prehatching 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 Gene expression levels of DNA methyltransferase enzymes in Shank3-deficient mouse model of autism during early development

2021 ◽  
Vol 55 (4) ◽  
pp. 234-237
Author(s):  
Annamaria Srancikova ◽  
Alexandra Reichova ◽  
Zuzana Bacova ◽  
Jan Bakos
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Brain Development ◽  
Early Development ◽  
Molecular Mechanisms ◽  
Altered Expression ◽  
Expression Levels ◽  
The Brain ◽  
Deficient Mice ◽  
Gene Expression Levels

Abstract Objectives. The balance between DNA methylation and demethylation is crucial for the brain development. Therefore, alterations in the expression of enzymes controlling DNA methylation patterns may contribute to the etiology of neurodevelopmental disorders, including autism. SH3 and multiple ankyrin repeat domains 3 (Shank3)-deficient mice are commonly used as a well-characterized transgenic model to investigate the molecular mechanisms of autistic symptoms. DNA methyltransferases (DNMTs), which modulate several cellular processes in neurodevelopment, are implicated in the pathophysiology of autism. In this study, we aimed to describe the gene expression changes of major Dnmts in the brain of Shank3-deficient mice during early development. Methods and Results. The Dnmts gene expression was analyzed by qPCR in 5-day-old homo-zygous Shank3-deficient mice. We found significantly lower Dnmt1 and Dnmt3b gene expression levels in the frontal cortex. However, no such changes were observed in the hippocampus. However, significant increase was observed in the expression of Dnmt3a and Dnmt3b genes in the hypothalamus of Shank3-deficient mice. Conclusions. The present data indicate that abnormalities in the Shank3 gene are accompanied by an altered expression of DNA methylation enzymes in the early brain development stages, therefore, specific epigenetic control mechanisms in autism-relevant models should be more extensively investigated.

5 Gene expression analysis and DNA methylation patterns of porcine somatic cell nuclear transfer blastocysts with high and low incidence of apoptosis

2019 ◽  
Vol 31 (1) ◽  
pp. 128
Author(s):  
L. Moley ◽  
R. Jones ◽  
R. Kaundal ◽  
A. Thomas ◽  
A. Benninghoff ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Enrichment Analysis ◽  
Epigenetic Reprogramming ◽  
Reduced Representation ◽  
False Discovery ◽  
Reduced Representation Bisulfite Sequencing ◽  
Methylation Patterns ◽  
Dna Methylation Analysis ◽  
Go Terms

Somatic cell NT (SCNT) efficiency remains poor, preventing the technology from being regularly used in the agricultural industry. It is believed that faulty epigenetic reprogramming of SCNT embryos leads to the low overall success. A clear apoptotic signature is associated with inappropriate gene expression and epigenomic aberrancies in many experimental cell culture systems, and we hypothesised that an apoptosis biomarker could be used to effectively separate properly reprogrammed porcine SCNT embryos from those that are destined to fail due to incomplete reprogramming. Therefore, our objective was to evaluate global gene expression and DNA methylation patterns in high- and low-apoptosis individual embryos in an effort to characterise the extent of genomic reprogramming that had taken place. Porcine SCNT blastocysts on Day 6 of development were stained with a nontoxic, noninvasive caspase activity reporter, and the top and bottom 20% of detected caspase activity were classified as high and low apoptosis, respectively (3 replicate cloning sessions; n=13 embryos per group). Genomic DNA and total RNA were isolated from each individual blastocyst. The RNA sequencing libraries were prepared using the Ovation SoLo RNA-Seq system (NuGen, San Carlos, CA, USA). Reduced representation bisulfite sequencing libraries were prepared for DNA methylation analysis using a modification of the single-cell reduced representation bisulfite sequencing global DNA methylation analysis approach detailed by Guo et al. (2015 Nat. Protoc. 10, 645-59). The RNA sequencing analysis using EdgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html) revealed 175 total differentially expressed genes (fold change ≥1.5; false discovery rate ≤0.05) between the high- and low-apoptosis SCNT embryos. This list of differentially expressed genes was used to perform enrichment analysis to identify overrepresented Gene Ontology (GO) terms or Kyoto Encyclopedia of Genes and Genomes pathways (DAVID Ease version 6.8 (https://david.ncifcrf.gov/) against the Sus scrofa background genome). However, no significantly enriched GO terms or pathways were identified (false discovery rate P>0.05). Analysis of global DNA methylation patterns between high- and low-apoptosis SCNT embryos using MethylKit (Akalin et al. 2012Genome Biol. 13, R87) revealed 335 differentially methylated 100-bp regions with at least 25% difference in methylation (adjusted P ≤ 0.01). Gene transcription start sites associated with these regions were used for enrichment analysis; again, no significant enrichment of GO terms or Kyoto Encyclopedia of Genes and Genomes pathways was identified. Principal component analysis of CpG methylation showed the low-apoptosis embryos clustering more tightly than the high-apoptosis embryos, which were highly scattered. Ongoing comparisons of high- and low-apoptosis cloned embryos with naturally fertilized embryos produced invivo may provide more information about which embryos were properly reprogrammed. Although we are still pursuing a link between reprogramming and gene expression in high- and low-apoptosis embryos, we conclude that these data support a model of stochastic epigenetic reprogramming following SCNT and reinforce the necessity of identifying embryos most likely to be successful due to proper epigenetic reprogramming in order to increase SCNT efficiency.

Sign in / Sign up

Export Citation Format

Share Document