Epigenetic Mechanisms Research Articles

Epigenetic Regulation of Anthocyanin Biosynthesis in Betula pendula ‘Purple Rain’

Betula pendula ‘Purple Rain’ is characterized by its purple leaves and has ornamental applications. A green mutant line NL, which was mutated by line NZ of B. pendula ‘Purple Rain’ during tissue culture, shows green leaves instead of the typical purple color of B. pendula ‘Purple Rain’. This study quantified the leaf color traits of NL and a normal B. pendula ‘Purple Rain’ line NZ, and uncovered differentially expressed genes involved in flavonoid biosynthesis pathway genes in NL through RNA-Seq analysis. Compared to NZ, reduced levels of six anthocyanins contained in NL were revealed via flavonoids-targeted metabolomics. Sequence mutations in transcription factors that could explain NL’s phenotype failed to be screened via whole-genome resequencing, suggesting an epigenetic basis for this variant. Therefore, a key gene, BpMYB113, was identified in NL via the combined analysis of small RNA sequencing, whole-genome methylation sequencing, and transcriptomics. In NL, this gene features a hyper CHH context methylation site and a lower transcription level compared to NZ, disrupting the expression of downstream genes in the phenylalanine metabolism pathway, and thereby reducing flavonoid biosynthesis. Our study elucidates an epigenetic mechanism underlying color variation in variegated trees, providing pivotal insights for the breeding and propagation of colored-leaf tree species.

Substrate specificity and protein stability drive the divergence of plant-specific DNA methyltransferases.

DNA methylation is an important epigenetic mechanism essential for transposon silencing and genome integrity. Across evolution, the substrates of DNA methylation have diversified between kingdoms. In plants, chromomethylase3 (CMT3) and CMT2 mediate CHG and CHH methylation, respectively. However, how these two methyltransferases diverge on substrate specificities during evolution remains unknown. Here, we reveal that CMT2 originates from a duplication of an evolutionarily ancient CMT3 in flowering plants. Lacking a key arginine residue recognizing CHG in CMT2 impairs its CHG methylation activity in most flowering plants. An engineered V1200R mutation empowers CMT2 to restore CHG and CHH methylations in Arabidopsis cmt2cmt3 mutant, testifying a loss-of-function effect for CMT2 during evolution. CMT2 has evolved a long and unstructured amino terminus critical for protein stability, especially under heat stress, and is plastic to tolerate various natural mutations. Together, this study reveals the mechanism of chromomethylase divergence for context-specific DNA methylation in plants and sheds important lights on DNA methylation evolution and function.

Integration of epigenomic and transcriptomic profiling uncovers EZH2 target genes linked to cysteine metabolism in hepatocellular carcinoma

Enhancer of zeste homolog 2 (EZH2), a key protein implicated in various cancers including hepatocellular carcinoma (HCC), is recognized for its association with epigenetic dysregulation and pathogenesis. Despite clinical explorations into EZH2-targeting therapies, the mechanisms underlying its role in gene suppression in HCC have remained largely unexplored. Here, we integrate epigenomic and transcriptomic analyses to uncover the transcriptional landscape modulated by selective EZH2 inhibition in HCC. By reanalyzing transcriptomic data of HCC patients, we demonstrate that EZH2 overexpression correlates with poor patient survival. Treatment with the EZH2 inhibitor tazemetostat restored expression of genes involved in cysteine-methionine metabolism and lipid homeostasis, while suppressing angiogenesis and oxidative stress-related genes. Mechanistically, we demonstrate EZH2-mediated H3K27me3 enrichment at cis-regulatory elements of transsulfuration pathway genes, which is reversed upon inhibition, leading to increased chromatin accessibility. Among 16 EZH2-targeted candidate genes, BHMT and CDO1 were notably correlated with poor HCC prognosis. Tazemetostat treatment of HCC cells increased BHMT and CDO1 expression while reducing levels of ferroptosis markers FSP1, NFS1, and SLC7A11. Functionally, EZH2 inhibition dose-dependently reduced cell viability and increased lipid peroxidation in HCC cells. Our findings reveal a novel epigenetic mechanism controlling lipid peroxidation and ferroptosis susceptibility in HCC, providing a rationale for exploring EZH2-targeted therapies in this malignancy.

Epigenetic mechanisms of alveolar macrophage activation in chemical-induced acute lung injury

Airways, alveoli and the pulmonary tissues are the most vulnerable to the external environment including occasional deliberate or accidental exposure to highly toxic chemical gases. However, there are many effective protective mechanisms that maintain the integrity of the pulmonary tissues and preserve lung function. Alveolar macrophages form the first line of defense against any pathogen or chemical/reactant that crosses the airway mucociliary barrier and reaches the alveolar region. Resident alveolar macrophages are activated or circulating monocytes infiltrate the airspace to contribute towards inflammatory or reparative responses. Studies on response of alveolar macrophages to noxious stimuli are rapidly emerging and alveolar macrophage are also being sought as therapeutic target. Here such studies have been reviewed and put together for a better understanding of the role pulmonary macrophages in general and alveolar macrophage in particular play in the pathogenesis of disease caused by chemical induced acute lung injury.

Intercontinental Insights into Autism Spectrum Disorder: A Synthesis of Environmental Influences and DNA Methylation

Abstract Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder characterized by a broad range of symptoms. The etiology of ASD is thought to involve complex gene-environment interactions, which are crucial to understanding its various causes and symptoms. DNA methylation is an epigenetic mechanism that potentially links genetic predispositions to environmental factors in the development of ASD. This review provides a global perspective on ASD, focusing on how DNA methylation studies may reveal gene-environment interactions characteristic of specific geographical regions. It delves into the role of DNA methylation in influencing the causes and prevalence of ASD in regions where environmental influences vary significantly. We also address potential explanations for the high ASD prevalence in North America, considering lifestyle factors, environmental toxins, and diagnostic considerations. Asian and European studies offer insights into endocrine-disrupting compounds (EDC), persistent organic pollutants (POP), maternal smoking, and their associations with DNA methylation alterations in ASD. In areas with limited data on DNA methylation and ASD, such as Africa, Oceania, and South America, we discuss prevalent environmental factors based on epidemiological studies. Additionally, the review integrates global and country-specific prevalence data from various studies, providing a comprehensive picture of the variables influencing ASD diagnoses over region and year of assessment. This prevalence data, coupled with regional environmental variables and DNA methylation studies, provides a perspective on the complexities of ASD research. Integrating global prevalence data, we underscore the need for a comprehensive global understanding of ASD’s complex etiology. Expanded research into epigenetic mechanisms of ASD is needed, particularly in underrepresented populations and locations, to enhance biomarker development for diagnosis and intervention strategies for ASD that reflect the varied environmental and genetic landscapes worldwide.

ARGONAUTE4 and the DNA demethylase ROS1c mediate dehydroascorbate-induced intergenerational nematode resistance in rice.

Plants can transmit information to the next generation and modulate the phenotype of their offspring through epigenetic mechanisms. In this study, we demonstrate the activation of 'intergenerational acquired resistance' (IAR) in the progeny of rice (Oryza sativa) plants exogenously treated with dehydroascorbate (DHA). The offspring of lifelong DHA-treated plants (DHA-IAR) were significantly less susceptible to the root-knot nematode Meloidogyne graminicola and partially inherited the DHA-induced transcriptional response found in the parental plants. Phytohormone analyses on the DHA-IAR plants unveiled higher basal abscisic acid (ABA) levels and a primed induction of the jasmonic acid (JA) pathway. RNA-seq analysis on the embryonic tissues of immature seeds of DHA-treated plants revealed major shifts in the expression of genes associated with epigenetic pathways. We confirmed that DHA treatment leads to a significant but transient pattern of global DNA hypomethylation in the parental plants at 12 to 24 hours after treatment. The induction of resistance in the parental plants requires the DNA demethylase REPRESSOR OF SILENCING 1C (ROS1c) and ARGONAUTE 4 (AGO), suggesting a role for DNA demethylation and subsequent re-methylation in establishment of this phenotype. Confirming the transience of global hypomethylation upon DHA treatment, no significant change in global DNA methylation levels was observed in DHA-IAR versus naïve plants. Finally, DHA could not induce IAR in the Ros1c mutant line and in the ARGONAUTE 4 (ago4ab)-RNAi line. These data indicate that a controlled collaboration between transient DNA demethylation and remethylation underlies the induced resistance and IAR phenotypes upon DHA treatment.

Investigating the differential structural organization and gene expression regulatory networks of lamin A Ig fold domain mutants of muscular dystrophy.

Lamins form a proteinaceous meshwork as a major structural component of the nucleus. Lamins, along with their interactors, act as determinants for chromatin organization throughout the nucleus. The major dominant missense mutations responsible for autosomal dominant forms of muscular dystrophies reside in the Ig fold domain of lamin A. However, how lamin A contributes to the distribution of heterochromatin and balances euchromatin, and how it relocates epigenetic marks to shape chromatin states, remains poorly defined, making it difficult to draw conclusions about the prognosis of lamin A-mediated muscular dystrophies.In the first part of this report, we identified the in-vitro organization of full-length lamin A proteins due to two well-documented Ig LMNA mutations, R453W and W514R. We further demonstrated that both lamin A/C mutant cells predominantly expressed nucleoplasmic aggregates. Labelling specific markers of epigenetics allowed correlation of lamin A mutations with epigenetic mechanisms. In addition to manipulating epigenetic mechanisms, our proteomic studies traced diverse expressions of transcription regulators, RNA synthesis and processing proteins, protein translation components, and posttranslational modifications. These data suggest severe perturbations in targeting other proteins to the nucleus.

Renal endowment in men and women: start from the beginning.

The development of the human kidney leads to the establishment of nephron endowment through a process influenced by both genetic and environmental factors. There is individual variability regarding nephron endowment and factors including aging and pathological conditions contribute to the decline in the number of nephrons, impacting renal function. Genetic determinants, such as mutations in crucial developmental genes like Pax2, and epigenetic mechanisms mediated by key enzymes including sirtuin 3, play critical roles in the regulation of the number of nephrons, with implications for kidney disease susceptibility. Sexual dimorphism significantly influences kidney development and function, with the number of nephrons being significantly lower in females, consistent with lower female birth weight, which is considered a surrogate for nephron endowment. Also, although females have fewer nephrons, they experience a slower decline in GFR compared to males. Gender disparity in chronic kidney disease progression has been attributed to factors such as metabolism, oxidative stress, renal hemodynamics, and sex hormones. Understanding the complexities of nephron endowment variability, genetic determinants, and sexual dimorphism in kidney development and function is crucial for elucidating the mechanisms underlying individual kidney disease susceptibility and progression. Further research in this field holds promise for the development of personalized approaches to kidney disease prevention, management, and treatment.

Epigenetic mechanisms linking pregnancy complications to cardiovascular disease in offspring

Epigenetic mechanisms linking pregnancy complications to cardiovascular disease in offspring

Simultaneous single-nucleus RNA sequencing and single-nucleus ATAC sequencing of neuroblastoma cell lines

Neuroblastoma is the most common extracranial solid tumor in children, and a leading cause of childhood cancer deaths. All neuroblastomas arise from neural crest-derived sympathetic neuronal progenitors, but numerous mutations, the most common of which is MYCN amplification, give rise to these lesions. Epigenetic aberrations also play a role in oncogenesis and tumor progression. To better understand biologic diversity of neuroblastomas, we performed joint single-nucleus ATAC sequencing and single-nucleus RNA sequencing on six neuroblastoma cell lines, three of which are MYCN amplified. After standard filtering for high-quality nuclei, we obtained chromatin accessibility and transcript abundance data from 41,733 neuroblastoma tumor cells. Preliminary analysis reveals significant diversity in chromatin landscape and gene expression across neuroblastoma cell lines. This dataset is a valuable resource for studying the transcriptional and epigenetic mechanisms of this deadly childhood disease.

Epigenetic Regulation of Immune Cells in Health and Disease: A Comprehensive Review

Epigenetic regulation plays a crucial role in the development, differentiation, and function of immune cells. Mechanisms such as DNA methylation, histone modifications, chromatin remodeling, and non-coding RNA interactions dynamically shape gene expression, enabling immune cells to adapt to environmental stimuli and mediate appropriate immune responses. Aberrant epigenetic regulation is implicated in a range of diseases, including autoimmune disorders, cancer, and chronic inflammatory conditions. In autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis, altered DNA methylation and histone modifications contribute to immune dysregulation and tissue damage. In cancer, immune cells in the tumor microenvironment often exhibit epigenetic changes that facilitate immune evasion. Emerging therapeutic strategies targeting epigenetic pathways, including DNA methyltransferase inhibitors, histone deacetylase inhibitors, and CRISPR-based epigenome editing, offer promising approaches for treating immune-mediated diseases. This review provides a comprehensive overview of the epigenetic mechanisms governing immune cell behavior, their dysregulation in disease, and the potential of epigenetic therapies to restore immune homeostasis and improve patient outcomes. Keywords: Epigenetics, immune cells, DNA methylation, histone modifications, non-coding RNAs, autoimmune diseases, immunotherapy

Global DNA methylation level in spermatozoa is not associated with ICSI fertilization outcome and embryo quality in donor oocyte programme

Epigenetic mechanisms, including DNA methylation and histone modifications, govern chromatin arrangement in sperm, enhancing motility and safeguarding DNA integrity for accurate epigenetic inheritance. Abnormal methylation is linked to poor sperm quality and fertility issues, underscoring the need to study sperm DNA methylation and its impact on sperm function and embryo development in assisted reproductive technology. In this study, processed spermatozoa from 75 normozoospermic and 15 abnormal ejaculates were examined for sperm global DNA methylation levels using a colourimetric absorbance method. Although semen characteristics were poor in abnormal ejaculates, no significant correlation was found between sperm global DNA methylation levels and sperm characteristics in either normozoospermic or abnormal cohorts. However, mean global DNA methylation levels were significantly lowered in abnormal sperm samples compared to normozoospermic samples (p < 0.05). Furthermore, injecting spermatozoa from these patients (N = 50) into donor oocytes did not show a significant relationship between sperm global DNA methylation and embryo developmental competence. These findings highlight the limitation of sperm global DNA methylation as a biomarker for embryo development and quality.

Epigenetic Mechanisms of the Anti-inflammatory Action of the Opioid Peptide Leutragin: Role of Sirtuin 1

Sirtuin 1 (SIRT1) is a class III histone deacetylase that plays a key role in resolving inflammation through known epigenetic mechanisms involving histone and nuclear factor κB (NF-κB) deacetylation. Deacetylation reduces the transcriptional activity of NF-κB and the associated production of pro-inflammatory cytokines such as interleukin 1β (IL-1β), tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). In the present study, we show for the first time that biomodeling of acute lung inflammation by a single injection of lipopolysaccharide (LPS) induces synchronous oscillations of mRNA levels of cytokines IL-1β, TNF-α, IL-6 and SIRT1 deacetylase in the lungs, the maximum amplitudes of cytokine mRNA oscillations are observed between 1.5 and 5 hours, whereas high levels of SIRT1 mRNA are observed up to 24 hours, when cytokine mRNA oscillations have already faded, which is consistent with the hypothesis about the role of SIRT1 as a factor acting in the phase of inflammation resolution. The study shows that the mechanism of anti-inflammatory action of inhaled hexapeptide Leutragin, a δ-opioid receptor agonist, is related to its ability to increase SIRT1 mRNA expression and decrease the amplitudes of IL-1β, TNF-α, and IL-6 mRNA oscillations in the lungs, which generally leads to the resolution of inflammation in the conditions of biomodeling of acute lung inflammation.

Normative implications of postgenomic deterministic narratives: the case study of epigenetic harm.

What do we mean when we talk about epigenetic harm? This paper presents a multidimensional view of epigenetic harm. It is a plea to take a step back from discussions of epigenetic responsibility distributions prevalent in ELSA literature on epigenetics. Instead, it urges researchers to take a closer look at the normative role played by the concept of epigenetic harm. It starts out by showing that the ways in which the object of epigenetic responsibility has already been conceptualized are all related to 'epigenetic harm': something negative that happens in which epigenetic mechanisms play a role, or rather something that needs to be avoided. Epigenetic harm is then characterized as a bridging concept between relatively neutral findings on epigenetics on the one side, and potential ethical and societal implications of those findings, primarily in terms of responsibility ascriptions and distributions, on the other. The paper proposes that a sufficiently nuanced account of epigenetic harm should include at least three dimensions. The dimension of causation alone leads to an overly narrow understanding of harm, and a wrong understanding of this dimension might prompt researchers to support an excessively simplistic epigenetic determinism. It is argued that a multidimensional analysis of epigenetic harm is less vulnerable to this threat and more reflective of the various kinds of harm that may be experienced by the subjects of epigenetic alterations. The paper applies insights from disability studies and feminist philosophy to draw attention to two other dimensions of epigenetic harm, namely lived experiences and relationality. The paper concludes by exploring what a shift towards a multidimensional approach to epigenetic harm might mean for epigenetic research and responsibility ascriptions by formulating some concrete implications.

NAT10-mediated mRNA N4-acetylcytidine reprograms serine metabolism to drive leukaemogenesis and stemness in acute myeloid leukaemia.

RNA modification has emerged as an important epigenetic mechanism that controls abnormal metabolism and growth in acute myeloid leukaemia (AML). However, the roles of RNA N4-acetylcytidine (ac4C) modification in AML remain elusive. Here, we report that ac4C and its catalytic enzyme NAT10 drive leukaemogenesis and sustain self-renewal of leukaemic stem cells/leukaemia-initiating cells through reprogramming serine metabolism. Mechanistically, NAT10 facilitates exogenous serine uptake and de novo biosynthesis through ac4C-mediated translation enhancement of the serine transporter SLC1A4 and the transcription regulators HOXA9 and MENIN that activate transcription of serine synthesis pathway genes. We further characterize fludarabine as an inhibitor of NAT10 and demonstrate that pharmacological inhibition of NAT10 targets serine metabolic vulnerability, triggering substantial anti-leukaemia effects both in vitro and in vivo. Collectively, our study demonstrates the functional importance of ac4C and NAT10 in metabolism control and leukaemogenesis, providing insights into the potential of targeting NAT10 for AML therapy.

17α-Ethynylestradiol alters testicular epigenetic profiles and histone-to-protamine exchange in mice

Spermatogenesis starts with the onset of puberty within the seminiferous epithelium of the testes. It is a complex process under intricate control of the endocrine system. Physiological regulations by steroid hormones in general and by estrogens in particular are due to their chemical nature prone to be disrupted by exogenous factors acting as endocrine disruptors (EDs). 17α-Ethynylestradiol (EE2) is an environmental pollutant with a confirmed ED activity and a well-known effect on spermatogenesis and chromatin remodeling in haploid germ cells. The aim of our study was to assess possible effects of two doses (2.5ng/ml; 2.5 μg/ml) of EE2 on both histone-to-protamine exchange and epigenetic profiles during spermatogenesis performing a multi/transgenerational study in mice. Our results demonstrated an impaired histone-to-protamine exchange with a significantly higher histone retention in sperm nuclei of exposed animals, when this process was accompanied by the changes of histone post-translational modifications (PTMs) abundancies with a prominent effect on H3K9Ac and partial changes in protamine 1 promoter methylation status. Furthermore, individual changes in molecular phenotypes were partially transmitted to subsequent generations, when no direct trans-generational effect was observed. Finally, the uncovered specific localization of the histone retention in sperm nuclei and their specific PTMs profile after EE2 exposure may indicate an estrogenic effect on sperm motility and early embryonic development via epigenetic mechanisms.

Super-Enhancer Reprograming Driven by SOX9 and TCF7L2 Represents Transcription-Targeted Therapeutic Vulnerability for Treating Gallbladder Cancer.

Gallbladder cancer (GBC) is a highly aggressive malignancy lacking clinically available targeted therapeutic agents. Super-enhancers (SEs) are crucial epigenetic cis-regulatory elements whose extensive reprogramming drives aberrant transcription in cancers. To study SE in GBC, the genomic distribution of H3K27ac is profiled in multiple GBC tissue and cell line samples to establish the SE landscape and its associated core regulatory circuitry (CRC). The biliary lineage factor SOX9 and Wnt pathway effector TCF7L2, two master transcription factor (TF) candidates identified by CRC analysis, are verified to co-occupy each other's SE region, forming a mutually autoregulatory loop to drive oncogenic SE reprogramming in a subset of GBC. The SOX9/TCF7L2 double-high GBC cells are highly dependent on the two TFs and enriched of SE-associated gene signatures related to stemness, ErbB and Wnt pathways. Patients with more such GBC cells exhibited significantly worse prognosis. Furthermore, SOX9/TCF7L2 double-high GBC preclinical models are found to be susceptible to SE-targeted CDK7 inhibition therapy in vitro and in vivo. Together, this study provides novel insights into the epigenetic mechanisms underlying the oncogenesis of a subset of GBCs with poorer prognosis and illustrates promising prognostic stratification and therapeutic strategies for treating those GBC patients in future clinical trials.

Genomic Insights into Drug Abuse: The Role of DNA and Protein-Coding Regions in Addiction and Genetic Mutation

Abstract This paper explores the genetic and epigenetic impacts of drug abuse, focusing on how chronic drug use can induce mutations in DNA, particularly within protein-coding regions, and alter gene expression through mechanisms such as DNA methylation. The study highlights the significant role of these genetic changes in the brain's reward pathways, which contribute to the development and persistence of addiction. Through a comprehensive literature review, the paper identifies key genetic factors associated with addiction susceptibility and the long-term effects of drug-induced epigenetic modifications. A proposed research framework is presented, emphasizing the need for longitudinal studies to track epigenetic changes over time, targeted genomic analysis to identify critical mutations, and the integration of multi-omics approaches to provide a holistic understanding of drug abuse's impact on the genome. The paper also addresses ethical considerations, including the importance of protecting genetic privacy and preventing stigmatization based on genetic findings. The study concludes that advancing our understanding of the genetic and epigenetic mechanisms underlying addiction can lead to more effective, personalized treatment strategies and inform public health policies aimed at mitigating the effects of drug abuse.

Epigenetic Mechanisms Induced by Mycobacterium tuberculosis to Promote Its Survival in the Host.

Tuberculosis caused by the obligate intracellular pathogen, Mycobacterium tuberculosis, is one among the prime causes of death worldwide. An urgent remedy against tuberculosis is of paramount importance in the current scenario. However, the complex nature of this appalling disease contributes to the limitations of existing medications. The quest for better treatment approaches is driving the research in the field of host epigenomics forward in context with tuberculosis. The interplay between various host epigenetic factors and the pathogen is under investigation. A comprehensive understanding of how Mycobacterium tuberculosis orchestrates such epigenetic factors and favors its survival within the host is in increasing demand. The modifications beneficial to the pathogen are reversible and possess the potential to be better targets for various therapeutic approaches. The mechanisms, including histone modifications, DNA methylation, and miRNA modification, are being explored for their impact on pathogenesis. In this article, we are deciphering the role of mycobacterial epigenetic regulators on various strategies like cytokine expression, macrophage polarization, autophagy, and apoptosis, along with a glimpse of the potential of host-directed therapies.

Whole blood global DNA methylation [5-methylcytosine (5-mC)] levels in psoriatic patients before and after methotrexate treatment

BackgroundPsoriasis is a chronic inflammatory skin disease characterised by hyperproliferation of keratinocytes and abnormal immune response. It manifests in genetically predisposed individuals exposed to environmental triggers like infections, drugs, stress, and trauma. Epigenetic mechanisms are a critical component of the complex etiopathogenesis of psoriasis, as they modulate gene expression and increase disease risk. There are many conflicting reports regarding DNA methylation in patients with psoriasis. This could well reflect the choice of tissue and technique used. Data on whole blood DNA methylation in psoriasis is sparse. ObjectiveThe present study aims to measure the levels of DNA methylation [5-methylcytosine (5-mC)] in psoriatic patients from whole blood pre and post-treatment with methotrexate and its correlation with disease severity. MethodsWe recruited thirty cases of psoriasis vulgaris and 30 healthy controls. Thirty psoriatic patients were on methotrexate treatment and followed up for three months. 5-methylcytosine was measured using a global DNA methylation ELISA kit. ResultsThe 5-methylcytosine levels from whole blood DNA were 8.13±1.89 % in cases and 5.37±2.33 % in controls, which was statistically significant (p = 0.0001). Post-treatment with methotrexate, there was a significant reduction (p = 0.0001) in the 5-methylcytosine levels in cases (4.54±1.74 %). Pre and post-treatment 5-methylcytosine levels were not significantly associated with the Psoriasis Area Severity Index (PASI) score. ConclusionPatients with psoriasis have whole-blood DNA global hypermethylation. Treatment with methotrexate leads to a significant reduction in DNA hypermethylation.