The Methylation Inhibitor 5-Aza-2′-Deoxycytidine Induces Genome-Wide Hypomethylation in Rice

DNA methylation is a conserved epigenetic modification which is vital for regulating gene expression and maintaining genome stability in both mammals and plants. Homozygous mutation of rice methyltransferase 1 (met1) gene can cause host death in rice, making it difficult to obtain plant material needed for hypomethylation research. To circumvent this challenge, the methylation inhibitor, 5-Aza-2′-deoxycytidine (AzaD), is used as a cytosine nucleoside analogue to reduce genome wide hypomethylation and is widely used in hypomethylation research. However, how AzaD affects plant methylation profiles at the genome scale is largely unknown. Here, we treated rice seedlings with AzaD and compared the AzaD treatment with osmet1-2 mutants, illustrating that there are similar CG hypomethylation and distribution throughout the whole genome. Along with global methylation loss class I transposable elements (TEs) which are farther from genes compared with class II TEs, were more significantly activated, and the RNA-directed DNA Methylation (RdDM) pathway was activated in specific genomic regions to compensate for severe CG loss. Overall, our results suggest that AzaD is an effective DNA methylation inhibitor that can influence genome wide methylation and cause a series of epigenetic variations.

Integrated Transcriptome Analysis and Single-Base Resolution Methylomes of Watermelon (Citrullus lanatus) Reveal Epigenome Modifications in Response to Osmotic Stress

DNA methylation plays an important role against adverse environment by reshaping transcriptional profile in plants. To better understand the molecular mechanisms of watermelon response to osmotic stress, the suspension cultured watermelon cells were treated with 100mM mannitol, and then a methylated cytosines map was generated by whole genome bisulfite sequencing (WGBS). Combined with transcriptome sequencing, the effects of osmotic stress on differentially methylated expressed genes (DMEGs) were assessed. It was found that genes related to plant hormone synthesis, signal transduction, osmoregulatory substance-related and reactive oxygen species scavenging-related enzyme could rapidly respond to osmotic stress. The overall methylation level of watermelon decreased after osmotic stress treatment, and demethylation occurred in CG, CHG, and CHH contexts. Moreover, differentially methylated expressed genes (DMEGs) were significantly enriched in RNA transport, starch and sucrose metabolism, plant hormone signal transduction and biosynthesis of secondary metabolites, especially in biosynthesis of osmolytes synthase genes. Interestingly, demethylation of a key enzyme gene Cla014489 in biosynthesis of inositol upregulated its expression and promoted accumulation of inositol, which could alleviate the inhibition of cell growth caused by osmotic stress. Meanwhile, a recombinant plasmid pET28a-Cla014489 was constructed and transferred into Escherichia coli BL21 for prokaryotic expression and the expression of ClMIPS protein could improve the tolerance of E. coli to osmotic stress. The effect of methylation level on the expression properties of inositol and its related genes was further confirmed by application of DNA methylation inhibitor 5-azacytidine. These results provide a preliminary insight into the altered methylation levels of watermelon cells in response to osmotic stress and suggest a new mechanism that how watermelon cells adapt to osmotic stress.

Enterovirus 71 structural viral protein 1 promotes autophagy by inducing an m6A-mediated PMP22 overexpression in mouse Schwann cells

Objective Enterovirus 71 (EV71), one of the enteroviruses responsible for the hand, foot and mouth disease (HFMD), can cause severe neurologic diseases such as brainstem encephalitis and demyelination. The molecular mechanism of demyelination is still not fully understood. This study aims to investigate the mechanism of how the EV71 structural viral protein 1, VP1 can act on host cellular pathways in mouse Schwann cells. Methods An EV71 VP1-expressing vector was generated and transfected into mouse Schwann cells (MSCs). Selective mRNA methylation inhibitor (DAA) was employed to identify key members of m6A pathway that are targeted by VP1. To investigate the role of METTL14 and YTHDF1 in PMP22 expression, small interfering RNA against METTL14 and YTHDF1 was employed to knockdown the METTL14 and YTHDF1 expression in MSCs. Real-time PCR and Western blot analysis were performed to determine the expression of PMP22 and m6A modification-associated proteins. Results Our results demonstrated VP1 upregulated m6A pathway by targeting METTL14 and YTHDF1. The expression of PMP22 was decreased by inhibiting the expression of METTL14 and YTHDF1. Conclusions VP1 upregulates m6A modification, which in turn causes the hypermethylation of PMP22 in MSCs, resulting in a higher level of PMP22. Ultimately, this VP1-induced PMP22 overexpression leads to MSC autophagy.

Analysis of Gene Expression Patterns of Epigenetic Enzymes Dnmt3a, Tet1 and Ogt in Murine Chondrogenic Models

We investigated the gene expression pattern of selected enzymes involved in DNA methylation and the effects of the DNA methylation inhibitor 5-azacytidine during in vitro and in vivo cartilage formation. Based on the data of a PCR array performed on chondrifying BMP2-overexpressing C3H10T1/2 cells, the relative expressions of Tet1 (tet methylcytosine dioxygenase 1), Dnmt3a (DNA methyltransferase 3), and Ogt (O-linked N-acetylglucosamine transferase) were further examined with RT-qPCR in murine cell line-based and primary chondrifying micromass cultures. We found very strong but gradually decreasing expression of Tet1 throughout the entire course of in vitro cartilage differentiation along with strong signals in the cartilaginous embryonic skeleton using specific RNA probes for in situ hybridization on frozen sections of 15-day-old mouse embryos. Dnmt3a and Ogt expressions did not show significant changes with RT-qPCR and gave weak in situ hybridization signals. The DNA methylation inhibitor 5-azacytidine reduced cartilage-specific gene expression and cartilage formation when applied during the early stages of chondrogenesis. In contrast, it had a stimulatory effect when added to differentiated chondrocytes, and quantitative methylation-specific PCR proved that the DNA methylation pattern of key chondrogenic marker genes was altered by the treatment. Our results indicate that the DNA demethylation inducing Tet1 plays a significant role during chondrogenesis, and inhibition of DNA methylation exerts distinct effects in different phases of in vitro cartilage formation.

SPOP mutation induces DNA methylation via stabilizing GLP/G9a

Mutations in SPOP E3 ligase gene are reportedly associated with genome-wide DNA hypermethylation in prostate cancer (PCa) although the underlying mechanisms remain elusive. Here, we demonstrate that SPOP binds and promotes polyubiquitination and degradation of histone methyltransferase and DNMT interactor GLP. SPOP mutation induces stabilization of GLP and its partner protein G9a and aberrant upregulation of global DNA hypermethylation in cultured PCa cells and primary PCa specimens. Genome-wide DNA methylome analysis shows that a subset of tumor suppressor genes (TSGs) including FOXO3, GATA5, and NDRG1, are hypermethylated and downregulated in SPOP-mutated PCa cells. DNA methylation inhibitor 5-azacytidine effectively reverses expression of the TSGs examined, inhibits SPOP-mutated PCa cell growth in vitro and in mice, and enhances docetaxel anti-cancer efficacy. Our findings reveal the GLP/G9a-DNMT module as a mediator of DNA hypermethylation in SPOP-mutated PCa. They suggest that SPOP mutation could be a biomarker for effective treatment of PCa with DNA methylation inhibitor alone or in combination with taxane chemotherapeutics.

Potential Therapeutic Options and Perspectives for Alleviation of Endometrial Estrogen Dominance and Progesterone Resistance in Endometriosis

Endometriosis is a chronic disease, influenced by internal and external environment, with long duration from intrauterine life with acme during childbearing, when it is associated to chronic pelvic pains, and infertility/subfertility. DNA hypermethylation of endometrial promoter PRs Hox genes and DNA hypomethylation of promoter ERβ gene is a possible explanation of estrogen dominance, progressive loss of progesterone signaling, followed by progesterone resistance in ectopic, and progesterone attenuance in eutopic endometrium, for failure of hormone therapy (HT), repeated recurrences after surgery, cancers after long time evolution. Animal models, human trials demonstrated progesterone (P4) and progestins influences over progression of disease pathological characteristics, associated to endometrial ER, PR aberrant expressions: ERα loss, and abnormal PRB/PRA ratio. P4 supplementation before mice induced-endometriosis protected from PRs depletion, action that can be translated in women according to the difference of 7 to 12 years between histologic onset and clinical symptoms/signs, parallel to progressive loss of PRs and PR-mediated signaling in ectopic and eutopic endometria. The animal studies have shown that a DNA methylation inhibitor alleviates lesion growth, and induces PRs target gene expression restoration. Continuous/extended contraceptives, dienogest- a new progestin, GnRH agonists/antagonists, aromatase inhibitors, SERM, SPRM, combinated molecules are therapeutic options/perspectives aiming restoration endometrial estrogen-progesterone balance, without disease’s cure. HT may be active alone, or surgery associated.

DNA methylation affects photoperiodic tuberization in potato (Solanum tuberosum L.) by mediating the expression of genes related to the photoperiod and GA pathways

Overcoming short-day-dependent tuberization to adapt to long-day conditions is critical for the widespread geographical success of potato. The genetic pathways of photoperiodic tuberization are similar to those of photoperiodic flowering. DNA methylation plays an important role in photoperiodic flowering. However, little is known about how DNA methylation affects photoperiodic tuberization in potato. Here, we verified the effect of a DNA methylation inhibitor on photoperiodic tuberization and compared the DNA methylation levels and differentially methylated genes (DMGs) in the photoperiodic tuberization process between photoperiod-sensitive and photoperiod-insensitive genotypes, aiming to dissect the role of DNA methylation in the photoperiodic tuberization of potato. We found that a DNA methylation inhibitor could promote tuber initiation in strict short-day genotypes. Whole-genome DNA methylation sequencing showed that the photoperiod-sensitive and photoperiod-insensitive genotypes had distinct DNA methylation modes in which few differentially methylated genes were shared. Transcriptome analysis confirmed that the DNA methylation inhibitor regulated the expression of the key genes involved in the photoperiod and GA pathways to promote tuber initiation in the photoperiod-sensitive genotype. Comparison of the DNA methylation levels and transcriptome levels identified 52 candidate genes regulated by DNA methylation that were predicted to be involved in photoperiodic tuberization. Our findings provide a new perspective for understanding the relationship between photoperiod-dependent and GA-regulated tuberization. Uncovering the epigenomic signatures of these pathways will greatly enhance potato breeding for adaptation to a wide range of environments.

Integrated Methylome and Transcriptome Analyses Reveal the Molecular Mechanism by Which DNA Methylation Regulates Kenaf Flowering

DNA methylation regulates key biological processes in plants. In this study, kenaf seedlings were pretreated with the DNA methylation inhibitor 5-azacytidine (5-azaC) (at concentrations of 0, 100, 200, 400, and 600 μM), and the results showed that pretreatment with 200 μM 5-azaC promoted flowering most effectively. To elucidate the underlying mechanism, phytohormone, adenosine triphosphate (ATP), and starch contents were determined, and genome-wide DNA methylation and transcriptome analyses were performed on anthers pretreated with 200 μM 5-azaC (5-azaC200) or with no 5-azaC (control conditions; 5-azaC0). Biochemical analysis revealed that 5-azaC pretreatment significantly reduced indoleacetic acid (IAA) and gibberellic acid (GA) contents and significantly increased abscisic acid (ABA) and ATP contents. The starch contents significantly increased in response to 200 and 600 μM 5-azaC. Further genome-wide DNA methylation analysis revealed 451 differentially methylated genes (DMGs) with 209 up- and 242 downregulated genes. Transcriptome analysis showed 3,986 differentially expressed genes (DEGs), with 2,171 up- and 1,815 downregulated genes. Integrated genome-wide DNA methylation and transcriptome analyses revealed 72 genes that were both differentially methylated and differentially expressed. These genes, which included ARFs, PP2C, starch synthase, FLC, PIF1, AGL80, and WRKY32, are involved mainly in plant hormone signal transduction, starch and sucrose metabolism, and flowering regulation and may be involved in early flowering. This study serves as a reference and theoretical basis for kenaf production and provides insights into the effects of DNA methylation on plant growth and development.

Hydralazine and Enzalutamide: Synergistic Partners against Prostate Cancer

Advanced prostate cancers frequently develop resistance to androgen-deprivation therapy with serious implications for patient survival. Considering their importance in this type of neoplasia, epigenetic modifications have drawn attention as alternative treatment strategies. The aim of this study was to assess the antitumoral effects of the combination of hydralazine, a DNA methylation inhibitor, with enzalutamide, an antagonist of the androgen receptor, in prostate cancer cell lines. Several biological parameters, such as cell viability, proliferation, DNA damage, and apoptosis, as well as clonogenic and invasive potential, were evaluated. The individual treatments with hydralazine and enzalutamide exerted growth-inhibitory effects in prostate cancer cells and their combined treatment displayed synergistic effects. The combination of these two drugs was very effective in decreasing malignant features of prostate cancer and may become an alternative therapeutic option for prostate cancer patient management.

Epigenetic reprogramming of DCCs into dormancy suppresses metastasis via restored TGFβ–SMAD4 signaling.

Disseminated cancer cells (DCCs) identified in secondary organs, sometimes before the primary tumor becomes detectable and treated, can remain dormant for years to decades before manifesting. Microenvironmental and epigenetic mechanisms may control the onset and escape from dormancy, and here we reveal how a combination of the DNA methylation inhibitor 5-azacytidine (AZA) and retinoic acid receptor ligands all-trans retinoic acid (atRA), orchestrate a novel program of stable dormancy. Treatment of HNSCC tumor cells with AZA+atRA induced a SMAD2/3/4 dependent regulation of downstream transcriptional program that restored the anti-proliferative function of TGFβ signaling. Significantly, AZA+atRA or AZA+AM80, an RARα specific agonist, strongly suppresses lung metastasis formation. The metastatic suppression occurs via the induction and maintenance of phenotypically homogenous dormant SMAD4+/NR2F1+ non-proliferative DCCs. These findings suggest that strategies that maintain or induce dormancy programs may be a viable alternative strategy to improve patient outcomes by preventing or significantly delaying metastasis development.