Passive DNA Demethylation Research Articles

Understanding the role of ten-eleven translocation family proteins in kidney diseases.

Epigenetic mechanisms play a critical role in the pathogenesis of human diseases including kidney disorders. As the erasers of DNA methylation, Ten-eleven translocation (TET) family proteins can oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), thus leading to passive or active DNA demethylation. Similarly, TET family proteins can also catalyze the same reaction on RNA. In addition, TET family proteins can also regulate chromatin structure and gene expression in a catalytic activity-independent manner through recruiting the SIN3A/HDAC co-repressor complex. In 2012, we reported for the first time that the genomic 5-hydroxymethylcytosine level and the mRNA levels of Tet1 and Tet2 were significantly downregulated in murine kidneys upon ischemia and reperfusion injury. Since then, accumulating evidences have eventually established an indispensable role of TET family proteins in not only acute kidney injury but also chronic kidney disease. In this review, we summarize the upstream regulatory mechanisms and the pathophysiological role of TET family proteins in major types of kidney diseases and discuss their potential values in clinical diagnosis and treatment.

In vitro reconstitution of epigenetic reprogramming in the human germ line

Epigenetic reprogramming resets parental epigenetic memories and differentiates primordial germ cells (PGCs) into mitotic pro-spermatogonia or oogonia. This process ensures sexually dimorphic germ cell development for totipotency1. In vitro reconstitution of epigenetic reprogramming in humans remains a fundamental challenge. Here we establish a strategy for inducing epigenetic reprogramming and differentiation of pluripotent stem-cell-derived human PGC-like cells (hPGCLCs) into mitotic pro-spermatogonia or oogonia, coupled with their extensive amplification (about >1010-fold). Bone morphogenetic protein (BMP) signalling is a key driver of these processes. BMP-driven hPGCLC differentiation involves attenuation of the MAPK (ERK) pathway and both de novo and maintenance DNA methyltransferase activities, which probably promote replication-coupled, passive DNA demethylation. hPGCLCs deficient in TET1, an active DNA demethylase abundant in human germ cells2,3, differentiate into extraembryonic cells, including amnion, with de-repression of key genes that bear bivalent promoters. These cells fail to fully activate genes vital for spermatogenesis and oogenesis, and their promoters remain methylated. Our study provides a framework for epigenetic reprogramming in humans and an important advance in human biology. Through the generation of abundant mitotic pro-spermatogonia and oogonia-like cells, our results also represent a milestone for human in vitro gametogenesis research and its potential translation into reproductive medicine.

Epigenetic Reprogramming in Mice and Humans: From Fertilization to Primordial Germ Cell Development.

In this review, advances in the understanding of epigenetic reprogramming from fertilization to the development of primordial germline cells in a mouse and human embryo are discussed. To gain insights into the molecular underpinnings of various diseases, it is essential to comprehend the intricate interplay between genetic, epigenetic, and environmental factors during cellular reprogramming and embryonic differentiation. An increasing range of diseases, including cancer and developmental disorders, have been linked to alterations in DNA methylation and histone modifications. Global epigenetic reprogramming occurs in mammals at two stages: post-fertilization and during the development of primordial germ cells (PGC). Epigenetic reprogramming after fertilization involves rapid demethylation of the paternal genome mediated through active and passive DNA demethylation, and gradual demethylation in the maternal genome through passive DNA demethylation. The de novo DNA methyltransferase enzymes, Dnmt3a and Dnmt3b, restore DNA methylation beginning from the blastocyst stage until the formation of the gastrula, and DNA maintenance methyltransferase, Dnmt1, maintains methylation in the somatic cells. The PGC undergo a second round of global demethylation after allocation during the formative pluripotent stage before gastrulation, where the imprints and the methylation marks on the transposable elements known as retrotransposons, including long interspersed nuclear elements (LINE-1) and intracisternal A-particle (IAP) elements are demethylated as well. Finally, DNA methylation is restored in the PGC at the implantation stage including sex-specific imprints corresponding to the sex of the embryo. This review introduces a novel perspective by uncovering how toxicants and stress stimuli impact the critical period of allocation during formative pluripotency, potentially influencing both the quantity and quality of PGCs. Furthermore, the comprehensive comparison of epigenetic events between mice and humans breaks new ground, empowering researchers to make informed decisions regarding the suitability of mouse models for their experiments.

On the accuracy of the epigenetic copy machine: comprehensive specificity analysis of the DNMT1 DNA methyltransferase.

The specificity of DNMT1 for hemimethylated DNA is a central feature for the inheritance of DNA methylation. We investigated this property in competitive methylation kinetics using hemimethylated (HM), hemihydroxymethylated (OH) and unmethylated (UM) substrates with single CpG sites in a randomized sequence context. DNMT1 shows a strong flanking sequence dependent HM/UM specificity of 80-fold on average, which is slightly enhanced on long hemimethylated DNA substrates. To explain this strong effect of a single methyl group, we propose a novel model in which the presence of the 5mC methyl group changes the conformation of the DNMT1-DNA complex into an active conformation by steric repulsion. The HM/OH preference is flanking sequence dependent and on average only 13-fold, indicating that passive DNA demethylation by 5hmC generation is not efficient in many flanking contexts. The CXXC domain of DNMT1 has a moderate flanking sequence dependent contribution to HM/UM specificity during DNA association to DNMT1, but not if DNMT1 methylates long DNA molecules in processive methylation mode. Comparison of genomic methylation patterns from mouse ES cell lines with various deletions of DNMTs and TETs with our data revealed that the UM specificity profile is most related to cellular methylation patterns, indicating that de novo methylation activity of DNMT1 shapes the DNA methylome in these cells.

Epigenetic Modifications Related to Potato Skin Russeting.

Potato tuber skin is a protective corky tissue consisting of suberized phellem cells. Smooth-skinned varieties are characterized by a clean, shiny appearance compared to the darker hue of russeted potatoes. The rough skin of russeted cultivars is a desired, genetically inherited characteristic; however, unwanted russeting of smooth-skinned cultivars often occurs under suboptimal growth conditions. The involvement of epigenetic modifiers in regulating the smooth skin russeting disorder was tested. We used smooth-skin commercial cultivars with and without the russeting disorder and three lines from a breeding population segregating for russeting. Anatomically, the russet skin showed similar characteristics, whether the cause was environmentally triggered or genetically determined. The old outer layers of the corky phellem remain attached to the newly formed phellem layers instead of being sloughed off. Global DNA methylation analysis indicated a significant reduction in the percentage of 5-methylcytosine in mature vs. immature skin and russet vs. smooth skin. This was true for both the smooth-skin commercial cultivars and the russeted lines. The expression level of selected DNA methyltransferases was reduced in accordance. DNA demethylase expression did not change between the skin types and age. Hence, the reduced DNA methylation in mature and russet skin is more likely to be achieved through passive DNA demethylation and loss of methyltransferase activity.

Structural basis for the unique multifaceted interaction of DPPA3 with the UHRF1 PHD finger.

Ubiquitin-like with PHD and RING finger domain-containing protein 1 (UHRF1)-dependent DNA methylation is essential for maintaining cell fate during cell proliferation. Developmental pluripotency-associated 3 (DPPA3) is an intrinsically disordered protein that specifically interacts with UHRF1 and promotes passive DNA demethylation by inhibiting UHRF1 chromatin localization. However, the molecular basis of how DPPA3 interacts with and inhibits UHRF1 remains unclear. We aimed to determine the structure of the mouse UHRF1 plant homeodomain (PHD) complexed with DPPA3 using nuclear magnetic resonance. Induced α-helices in DPPA3 upon binding of UHRF1 PHD contribute to stable complex formation with multifaceted interactions, unlike canonical ligand proteins of the PHD domain. Mutations in the binding interface and unfolding of the DPPA3 helical structure inhibited binding to UHRF1 and its chromatin localization. Our results provide structural insights into the mechanism and specificity underlying the inhibition of UHRF1 by DPPA3.

Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function.

TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic domain of mammalian TET enzymes favor CGs embedded within basic helix-loop-helix and basic leucine zipper domain transcription factor–binding sites, with up to 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intrasubstrate interactions and CG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germ line. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and octamer-binding transcription factor 4 (OCT4), respectively, illuminating TET function in transcriptional responses and pluripotency support.

NOS2 and S-nitrosothiol signaling induces DNA hypomethylation and LINE-1 retrotransposon expression

Inducible nitric oxide synthase (NOS2) produces high local concentrations of nitric oxide (NO), and its expression is associated with inflammation, cellular stress signals, and cellular transformation. Additionally, NOS2 expression results in aggressive cancer cell phenotypes and is correlated with poor outcomes in patients with breast cancer. DNA hypomethylation, especially of noncoding repeat elements, is an early event in carcinogenesis and is a common feature of cancer cells. In addition to altered gene expression, DNA hypomethylation results in genomic instability via retrotransposon activation. Here, we show that NOS2 expression and associated NO signaling results in substantial DNA hypomethylation in human cell lines by inducing the degradation of DNA (cytosine-5)–methyltransferase 1 (DNMT1) protein. Similarly, NOS2 expression levels were correlated with decreased DNA methylation in human breast tumors. NOS2 expression and NO signaling also resulted in long interspersed noncoding element 1 (LINE-1) retrotransposon hypomethylation, expression, and DNA damage. DNMT1 degradation was mediated by an NO/p38-MAPK/lysine acetyltransferase 5–dependent mechanism. Furthermore, we show that this mechanism is required for NO-mediated epithelial transformation. Therefore, we conclude that NOS2 and NO signaling results in DNA damage and malignant cellular transformation via an epigenetic mechanism.

PPARβ mediates mangiferin-induced neuronal differentiation of neural stem cells through DNA demethylation

Adult hippocampal neurogenesis (AHN) is heavily implicated in the pathogenesis of various neuropsychiatric disorders. The mangiferin (MGF), a bioactive compound of the mango, reportedly produces biological effects on a variety of neuropsychiatric disorders. However, the function and underlying mechanisms of MGF in regulating hippocampal neurogenesis remain unknown. Here we discovered that the transcriptome and methylome of MGF-induced neural stem cells (NSCs) are distinct from the control. RNA-seq analysis revealed that the diferentially expressed genes (DEGs) were signifcantly enriched in the PPARs. Furthermore, we found that MGF enhanced neuronal differentiation and proliferation of neural stem cells (NSCs) via PPARβ but not PPARα and PPARγ. The combination of WGBS and RNA-seq analysis showed that the expression of some neurogenesis genes was negatively correlated with the DNA methylation level generally. We further found that PPARβ increased demethylation of Mash1 promoter by modulating the expressions of active and passive DNA demethylation enzymes in MGF-treated NSCs. Importantly, genetic deficiency of PPARβ decreased hippocampal neurogenesis in the adult mice, whereas the defective neurogenesis was notably rescued by Mash1 overexpression. Our findings uncover a model that PPARβ-mediated DNA demethylation of Mash1 contributes to MGF-induced neuronal genesis, and advance the concept that targeting PPARβ-TET1/DNMT3a-Mash1 axis regulation of neurogenesis might serve as a novel neurotherapeutic strategy.

Gadd45g, A Novel Antidepressant Target, Mediates Metformin-Induced Neuronal Differentiation of Neural Stem Cells Via DNA Demethylation.

Increased neurogenesis elicits antidepressive-like effects. The antidiabetic drug metformin (Met) reportedly promotes hippocampal neurogenesis, which ameliorates spatial memory deficits and depression-like behaviors. However, the precise molecular mechanisms underpinning Met-induced neuronal differentiation of neural stem cells (NSCs) remain unclear. We showed that Met enhanced neuronal differentiation of NSCs via Gadd45g but not Gadd45a and Gadd45b. We further found that Gadd45g increased demethylation of neurogenic differentiation 1 promoter by regulating the activity of passive and active DNA demethylation enzymes through an adenylate-activated protein kinase -independent mechanism in Met-treated NSCs. Importantly, genetic deficiency of Gadd45g decreased hippocampal neurogenesis, which could contribute to spatial memory decline, and depression-like behaviors in the adult mice, whereas forced expression of Gadd45g alleviated the depressive-like behaviors. Our findings provide a model that Gadd45g-mediated DNA demethylation contributes to Met-induced neuronal genesis and its antidepressant-like effects and propose the concept that targeting Gadd45g regulation of neurogenesis might serve as a novel antidepressant strategy.

Anthropometric, biochemical, and haematological indicators associated with hyperhomocysteinemia and their relation to global DNA methylation in a young adult population

ABSTRACT Increased homocysteine (Hcy) levels have been associated with a higher risk of cardiovascular and neurodegenerative diseases. Passive DNA demethylation has been suggested as one of the mechanisms implicated in the development of these conditions, and most studies have investigated this relationship in older adult populations. Therefore, this study aimed to evaluate the relationship between corporal composition and biochemical and haematological indicators with plasma homocysteine levels and genome-wide methylation (Alu, LINE-1, and SAT2) in a population of healthy young adults (median age, 18 years). We showed that the prevalence of hyperhomocysteinemia was significantly higher in men (18.5%) than in women (6.6%) (P = 0.034). Increased Hcy level was substantially associated with higher levels of body mass index and visceral fat in females, whereas in males, it was significantly associated with reduced red cell distribution width and high-density lipoprotein (HDL) cholesterol (HDL-C) levels and increased low-density lipoprotein/HDL ratio. Hypomethylation of Alu was significantly associated with reduced levels of HDL-C (<40.0 mg dL–1), whereas hypomethylation of LINE-1 and SAT2 was significantly associated with higher levels of skeletal muscle (<39.3%) in males. These results highlight the participation of hormonal factors in regulating Hcy metabolism, primarily in the female population, whereas changes in DNA methylation observed in males might be associated with the consumption of a protein diet with high levels of methionine, independent of increased Hcy levels.

Distinguishing Active Versus Passive DNA Demethylation Using Illumina MethylationEPIC BeadChip Microarrays.

The 5-carbon positions on cytosine nucleotides preceding guanines in genomic DNA (CpG) are common targets for DNA methylation (5mC). DNA methylation removal can occur through both active and passive mechanisms. Ten-eleven translocation enzymes (TETs) oxidize 5mC in a stepwise manner to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5mC can also be removed passively through sequential cell divisions in the absence of DNA methylation maintenance. In this chapter, we describe approaches that couple TET-assisted bisulfite (TAB) and oxidative bisulfite (OxBS) conversion to the Illumina MethylationEPIC BeadChIP (EPIC array) and show how these technologies can be used to distinguish active versus passive DNA demethylation. We also describe integrative bioinformatics pipelines to facilitate this analysis.

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

Genome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. Here, we describe a recently evolved pathway in which global hypomethylation is achieved by the coupling of active and passive demethylation. TET activity is required, albeit indirectly, for global demethylation, which mostly occurs at sites devoid of TET binding. Instead, TET-mediated active demethylation is locus-specific and necessary for activating a subset of genes, including the naïve pluripotency and germline marker Dppa3 (Stella, Pgc7). DPPA3 in turn drives large-scale passive demethylation by directly binding and displacing UHRF1 from chromatin, thereby inhibiting maintenance DNA methylation. Although unique to mammals, we show that DPPA3 alone is capable of inducing global DNA demethylation in non-mammalian species (Xenopus and medaka) despite their evolutionary divergence from mammals more than 300 million years ago. Our findings suggest that the evolution of Dppa3 facilitated the emergence of global DNA demethylation in mammals.

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

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.

Estradiol-Induced Epigenetically Mediated Mechanisms and Regulation of Gene Expression.

Gonadal hormone 17β-estradiol (E2) and its receptors are key regulators of gene transcription by binding to estrogen responsive elements in the genome. Besides the classical genomic action, E2 regulates gene transcription via the modification of epigenetic marks on DNA and histone proteins. Depending on the reaction partner, liganded estrogen receptor (ER) promotes DNA methylation at the promoter or enhancer regions. In addition, ERs are important regulators of passive and active DNA demethylation. Furthermore, ERs cooperating with different histone modifying enzymes and chromatin remodeling complexes alter gene transcription. In this review, we survey the basic mechanisms and interactions between estrogen receptors and DNA methylation, demethylation and histone modification processes as well as chromatin remodeling complexes. The particular relevance of these mechanisms to physiological processes in memory formation, embryonic development, spermatogenesis and aging as well as in pathophysiological changes in carcinogenesis is also discussed.

Global DNA Hypomethylation in Epithelial Ovarian Cancer: Passive Demethylation and Association with Genomic Instability.

A hallmark of human cancer is global DNA hypomethylation (GDHO), but the mechanisms accounting for this defect and its pathological consequences have not been investigated in human epithelial ovarian cancer (EOC). In EOC, GDHO was associated with advanced disease and reduced overall and disease-free survival. GDHO (+) EOC tumors displayed a proliferative gene expression signature, including FOXM1 and CCNE1 overexpression. Furthermore, DNA hypomethylation in these tumors was enriched within genomic blocks (hypomethylated blocks) that overlapped late-replicating regions, lamina-associated domains, PRC2 binding sites, and the H3K27me3 histone mark. Increased proliferation coupled with hypomethylated blocks at late-replicating regions suggests a passive hypomethylation mechanism. This hypothesis was further supported by our observation that cytosine DNA methyltransferases (DNMTs) and UHRF1 showed significantly reduced expression in GDHO (+) EOC after normalization to canonical proliferation markers, including MKI67. Finally, GDHO (+) EOC tumors had elevated chromosomal instability (CIN), and copy number alterations (CNA) were enriched at the DNA hypomethylated blocks. Together, these findings implicate a passive DNA demethylation mechanism in ovarian cancer that is associated with genomic instability and poor prognosis.

Phosphorylation of TET2 by AMPK is indispensable in myogenic differentiation

BackgroundTET-mediated oxidation of 5-mC participates in both passive and active DNA demethylation, which exerts a significant influence on diverse biological processes. Mass spectrometry has identified multiple phosphorylation sites of TET2. However, the functions of these phosphosites and their corresponding kinases are mostly unknown.ResultsHere, we showed that AMP-activated protein kinase (AMPK) phosphorylates murine TET2 at the serine residue 97 (S97), and the phosphorylation enhances TET2 stability through promoting its binding to 14-3-3β. AMPK ablation resulted in decreased global 5-hmC levels at the myotube stages, severe differentiation defects of C2C12 cells and significantly, total loss of expression of Pax7. Genome-wide analyses revealed increased DNA methylation at genic and enhancer regions of AMPK-null myoblasts and myotubes. Using CRISPR/Cas9 technology, we showed that a novel enhancer, which is hypermethylated in AMPK-null cells, regulates Pax7 expression. The phospho-mimicking mutant, TET2-S97E, could partly rescue the differentiation defect in AMPK-ablated C2C12 cells.ConclusionsTogether, our data demonstrated that AMPK is a critical regulator of myogenesis, partly through phosphorylating TET2.

Hemi-methylated CpG sites connect Dnmt1-knockdown-induced and Tet1-induced DNA demethylation during somatic cell reprogramming

The relationship between active DNA demethylation induced by overexpressing Tet1 and passive DNA demethylation induced by suppressing Dnmt1 remains unclear. Here, we found that DNMT1 preferentially methylated, but TET1 preferentially demethylated, hemi-methylated CpG sites. These phenomena resulted in a significant overlap in the targets of these two types of DNA demethylation and the counteractions of Dnmt1 and Tet1 during somatic cell reprogramming. Since the hemi-methylated CpG sites generated during cell proliferation were enriched at core pluripotency loci, DNA demethylation induced by Tet1 or sh-RNA against Dnmt1 (sh-Dnmt1) was enriched in these loci, which, in combination with Yamanaka factors, led to the up-regulation of these genes and promoted somatic cell reprogramming. In addition, since sh-Dnmt1 induces DNA demethylation by impairing the further methylation of hemi-methylated CpG sites generated during cell proliferation, while Tet1 induced DNA demethylation by demethylating these hemi-methylated CpG sites, Tet1-induced DNA demethylation, compared with sh-Dnmt1-induced DNA demethylation, exhibited a higher ability to open the chromatin structure and up-regulate gene expression. Thus, Tet1-induced but not sh-Dnmt1-induced DNA demethylation led to the up-regulation of an additional set of genes that can promote the epithelial-mesenchymal transition and impair reprogramming. When vitamin C was used to further increase the demethylation ability of TET1 during reprogramming, Tet1 induced a larger up-regulation of these genes and significantly impaired reprogramming. Therefore, the current studies provide additional information regarding DNA demethylation during somatic cell reprogramming.

Individual retrotransposon integrants are differentially controlled by KZFP/KAP1-dependent histone methylation, DNA methylation and TET-mediated hydroxymethylation in na\xefve embryonic stem cells

BackgroundThe KZFP/KAP1 (KRAB zinc finger proteins/KRAB-associated protein 1) system plays a central role in repressing transposable elements (TEs) and maintaining parent-of-origin DNA methylation at imprinting control regions (ICRs) during the wave of genome-wide reprogramming that precedes implantation. In naïve murine embryonic stem cells (mESCs), the genome is maintained highly hypomethylated by a combination of TET-mediated active demethylation and lack of de novo methylation, yet KAP1 is tethered by sequence-specific KZFPs to ICRs and TEs where it recruits histone and DNA methyltransferases to impose heterochromatin formation and DNA methylation.ResultsHere, upon removing either KAP1 or the cognate KZFP, we observed rapid TET2-dependent accumulation of 5hmC at both ICRs and TEs. In the absence of the KZFP/KAP1 complex, ICRs lost heterochromatic histone marks and underwent both active and passive DNA demethylation. For KAP1-bound TEs, 5mC hydroxylation correlated with transcriptional reactivation. Using RNA-seq, we further compared the expression profiles of TEs upon Kap1 removal in wild-type, Dnmt and Tet triple knockout mESCs. While we found that KAP1 represents the main effector of TEs repression in all three settings, we could additionally identify specific groups of TEs further controlled by DNA methylation. Furthermore, we observed that in the absence of TET proteins, activation upon Kap1 depletion was blunted for some TE integrants and increased for others.ConclusionsOur results indicate that the KZFP/KAP1 complex maintains heterochromatin and DNA methylation at ICRs and TEs in naïve embryonic stem cells partly by protecting these loci from TET-mediated demethylation. Our study further unveils an unsuspected level of complexity in the transcriptional control of the endovirome by demonstrating often integrant-specific differential influences of histone-based heterochromatin modifications, DNA methylation and 5mC oxidation in regulating TEs expression.

Dynamic Methylation Changes of DNA and H3K4 by RG108 Improve Epigenetic Reprogramming of Somatic Cell Nuclear Transfer Embryos in Pigs

Background/Aims: DNA methylation and histone modifications are essential epigenetic marks that can significantly affect the mammalian somatic cell nuclear transfer (SCNT) embryo development. However, the mechanisms by which the DNA methylation affects the epigenetic reprogramming have not been fully elucidated. Methods: In our study, we used quantitative polymerase chain reaction (qPCR), Western blotting, immunofluorescence staining (IF) and sodium bisulfite genomic sequencing to examine the effects of RG108, a DNA methyltransferase inhibitor (DNMTi), on the dynamic pattern of DNA methylation and histone modifications in porcine SCNT embryos and investigate the mechanism by which the epigenome status of donor cells’ affects SCNT embryos development and the crosstalk between epigenetic signals. Results: Our results showed that active DNA demethylation was enhanced by the significantly improving expression levels of TET1, TET2, TET3 and 5hmC, and passive DNA demethylation was promoted by the remarkably inhibitory expression levels of DNMT1, DNMT3A and 5mC in embryos constructed from the fetal fibroblasts (FFs) treated with RG108 (RG-SCNT embryos) compared to the levels in embryos from control FFs (FF-SCNT embryos). The signal intensity of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 9 acetylation (H3K9Ac) was significantly increased and the expression levels of H3K4 methyltransferases were more than 2-fold higher expression in RG-SCNT embryos. RG-SCNT embryos had significantly higher cleavage and blastocyst rates (69.3±1.4%, and 24.72±2.3%, respectively) than FF-SCNT embryos (60.1±2.4% and 18.38±1.9%, respectively). Conclusion: Dynamic changes in DNA methylation caused by RG108 result in dynamic alterations in the patterns of H3K4me3, H3K9Ac and histone H3 lysine 9 trimethylation (H3K9me3), which leads to the activation of embryonic genome and epigenetic modification enzymes associated with H3K4 methylation, and contributes to reconstructing normal epigenetic modifications and improving the developmental efficiency of porcine SCNT embryos.