DNA Demethylation Research Articles

Exogenous Methylation Inhibitor 5‐Azacytidine Accelerates Postharvest Leaf Senescence in Pak Choi by Modulating Transcriptomic and Metabolomic Profiles

ABSTRACTThe senescence of leafy vegetables is inherent and general after harvest. DNA methylation affects the senescence process of plant leaves. Limited studies have explored the impact of exogenous methylation inhibitors on postharvest vegetables and the mechanisms of their effects on the transcriptome and metabolome during storage. In this study, pak choi was immersed in 100 mg L−1 of 5‐azacytidine (AZ) (an inhibitor of methyltransferase) solutions, indicated that AZ yielded significant acceleration in leaf senescence. Compared to the control group, the AZ‐treated pak choi exhibited faster weight loss, higher malondialdehyde content, greater color change, and higher chlorophyllase activity. Transcriptomic and widely targeted metabolomic analyses were then performed on pak choi samples. Transcriptomic investigation disclosed that the chlorophyll degradation genes BraSGR2 and BraPPH were upregulated by AZ, leading to the degradation of more chlorophyll content. AZ effectively stimulated upregulation of the senescence‐associated genes (BraSAG20, BraSAG21, BraSRG1, and BraSRG2), thereby accelerating the pace of the senescence process. The widely targeted metabolomic analyses demonstrated that AZ downregulated the flavonoid synthase genes BraFLS1 and BraF3H, causing a relative decline in flavonoid levels. The study also unveiled a diminution in carbohydrate content during storage, further exacerbated by AZ. To encapsulate, our preliminary findings suggest that the application of AZ effectively accelerates the process of leaf senescence in stored pak choi, which helps us understand the relationship between DNA demethylation and vegetable senescence.

DNA methylation profiling of CpG islands in trigeminal ganglion of rats with orofacial pain induced by experimental tooth movement

BackgroundTooth movement induced orofacial pain is the most cited negative effect during orthodontic treatment, while treatment options without side effects are limited. The differential expression of pain-related genes due to DNA methylation and demethylation is instrumental in pain. The purpose of the study was to evaluate the DNA methylation profiling of CpG islands (CGI) and CGI shores in promoter regions in trigeminal ganglions (TG) of tooth movement induced orofacial pain rats, thus to further insight the DNA methylation regulation in orofacial pain.Materials and methodsAn orofacial pain rat model was constructed by ligating coil springs between the incisor and first maxillary molar with 40 g of force. The Rat Grimace Score (RGS) was used for pain evaluation. The genome methylation status was analyzed by the reduced representation bisulfite sequencing (RRBS) technique. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analyses were conducted in the differentially methylated regions (DMRs). Moreover, a protein-protein interaction (PPI) network was established to detect annotated genes associated with pain.ResultsRGS was significantly higher in orofacial pain rats than in sham rats. RRBS showed widespread methylation changes in CGI and CGI shores in TG promoter regions. Both 902 hypermethylated DMRs and 862 hypomethylated DMRs were found in the CGIs of promoter regions. KEGG analysis revealed that annotated genes are participated in endocrine, nervous, immune, and sensory systems. Moreover, the “Calcium signaling pathway”, “Wnt signaling pathway” and “Neuroactive ligand-receptor interaction” were significantly enriched pathways. Furthermore, PPI network showed several genes (Ctnnb1, Dlg4, Creb1, Camk2g, Bmp2, etc.) with different methylation statuses were reported to be associated with pain.ConclusionsThis study demonstrated methylation changes were existed in CGI and CGI shores in TG promoter regions when pain occurs, thus providing a basis for further study on the mechanism of DNA methylation in orofacial pain.

Concomitant loss of TET2 and TET3 results in T cell expansion and genomic instability in mice

Ten eleven translocation (TET) proteins are tumor suppressors that through their catalytic activity oxidize 5-methylcytosine to 5-hydroxymethylcytosine, to promote DNA demethylation and to regulate gene expression. Notably, TET2 is one of the most frequently mutated genes in hematological malignancies, including T cell lymphomas. However, murine models with deletion of TET2 do not exhibit T cell expansion, presumably due to redundancy with other members of the TET family of proteins. In order to gain insight on the TET mediated molecular events that safeguard T cells from aberrant proliferation we performed serial adoptive transfers of murine CD4 T cells that lack concomitantly TET2 and TET3 to fully immunocompetent congenic mice. Here we show a progressive acquisition of malignant traits upon loss of TET2 and TET3 that is characterized by loss of genomic integrity, acquisition of aneuploidy and upregulation of the protooncogene Myc.

CpG Traffic Lights Are Involved in Active DNA Demethylation

CpG Traffic Lights Are Involved in Active DNA Demethylation

Longikaurin A induces ferroptosis and inhibits glioblastoma progression through DNA methylation - Mediated GPX4 suppression.

Glioblastoma (GBM) is the most common primary intracranial tumor highly resistant to conventional clinical chemotherapy. Recently, the induction of ferroptosis is emerging as a putative strategy to treat various tumors. However, the identification of the effective and applicable tumor ferroptosis-inducing agents remains challenging. In this study, we showed that longikaurin A (LK-A), a natural diterpenoid isolated from the medicinal plant Isodon ternifolius with strong anti-GBM capacities, induced remarkable GBM cell ferroptosis along with suppressing the key anti-ferroptosis factor glutathione peroxidase 4 (GPX4). GPX4 promoter contains conserved CpG islands. The LK-A-induced GPX4 suppression coincided with the inhibition of ten-eleven translocation 2 (TET2), a key DNA demethylation enzyme and an increase in the hypermethylation of the GPX4 promoter. Further, LK-A promoted the GBM ferroptotic alterations and inhibited GBM progression in both subcutaneous and orthotopic xenograft mouse models, whereas GPX4 overexpression largely abrogated its anti-GBM effects both in vitro and in vivo, suggesting that LK-A inductions of the DNA methylation-incurred GPX4 suppression and ferroptosis are crucial for its anti-GBM functions. Together, our study has elaborated an important epigenetic pathway of GBM ferroptosis and uncovered a critical pharmacological property of LK-A for treating GBM patients.

In silico screening of phytochemicals against chromatin modifier, SETD7 for remodeling of the immunosuppressive tumor microenvironment in renal cancer.

The tumor microenvironment and immune evasion function in a complex cellular network profoundly challenge the clinical outcome of promising therapies. Our recently published study reported that the subset of genes upregulated in ccRCC due to H3K4me1 and DNA demethylation potentially leads to an immunosuppressive environment. Thus, modulating H3K4me1 chromatin modifier SETD7 with a natural inhibitor in combination with immunotherapy might improve the immune landscape for a better therapeutic outcome.The present study was conducted via virtual screening and MD simulation using compounds from the literature, IMPPAT, and SuperNatural database. The phytochemical IMPHY002979 showed better binding affinity and lower energy than the reported R-PFI-2 and cyproheptadine inhibitors. The phytochemicals interact with the SET domain through H-bonding, as confirmed by MD simulation and molecular interaction analysis. Further, the compound was assessed using ADME parameters and free energy estimation, showing better pharmacokinetic properties. Therefore, the non-accessibility of the histone methyltransferase activity domain of SET7 with IMPHY002979 can downregulate H3K4me1 and, thereby, the expression of genes potentially responsible for immunosuppressive TME. Thus, patient stratification based on molecular markers for immunotherapy and combining epigenetic modulators with therapeutic drugs will improve the efficacy of immunotherapy in ccRCC.

Ventral Hippocampal CA1 GADD45B Regulates Susceptibility to Social Stress by Influencing NMDA Receptor-Mediated Synaptic Plasticity.

Growth arrest DNA damage-inducible protein 45 β (GADD45B) has been reported to be a regulatory factor for active DNA demethylation and is implicated in the modulation of synaptic plasticity and chronic stress-related psychopathological processes. However, its precise role and mechanism of action in stress susceptibility remain elusive. In this study, we found a significant reduction in GADD45B expression specifically in the ventral, but not the dorsal hippocampal CA1 (dCA1) of stress-susceptible mice. Furthermore, we demonstrated that GADD45B negatively regulates susceptibility to social stress and NMDA receptor-dependent long-term potentiation (LTP) in the ventral hippocampal CA1 (vCA1). Importantly, through pharmacological inhibition using the NMDA receptor antagonist MK801, we provided further evidence supporting the hypothesis that GADD45B potentially modulates susceptibility to social stress by influencing NMDA receptor-mediated LTP. Collectively, these results suggested that modulation of NMDA receptor-mediated synaptic plasticity is a pivotal mechanism underlying the regulation of susceptibility to social stress by GADD45B.

CXXC5 stabilizes DNA methylation patterns in mouse embryonic stem cells.

Mammalian genomes encode 12 proteins that contain a CXXC zinc finger domain. Most members of this family are large multi-domain proteins that function in the control of DNA methylation and histone methylation patterns. CXXC5 is a smaller member of the family, along with its closest homologue CXXC4. These two proteins lack known catalytic domains. Here, we have characterized CXXC5 in mouse embryonic stem (ES) cells. We used gene knockouts, RNA sequencing, and DNA methylation analysis by whole-genome bisulfite sequencing. We show that CXXC5 is a nuclear protein that interacts with 5-methylcytosine oxidases (TET proteins). Removal of CXXC5 from ES cells leads to very few changes in gene expression. CXXC5 extensively colocalizes with TET1 and TET2 at CpG islands. CXXC5 inactivation leads to a substantial reduction of DNA methylation levels that affects all genomic compartments including genic and intergenic regions and CpG island shores. We propose a model in which CXXC5 serves as an anchor for TET proteins at CpG islands. In the absence of CXXC5, the 5-methylcytosine oxidases become dislodged from CpG islands and are enabled to induce genome-scale DNA demethylation. Thus, CXXC5 serves as a stabilizer of DNA methylation patterns.

Human HDAC6 senses valine abundancy to regulate DNA damage.

As an essential branched amino acid, valine is pivotal for protein synthesis, neurological behaviour, haematopoiesis and leukaemia progression1-3. However, the mechanism by which cellular valine abundancy is sensed for subsequent cellular functions remains undefined. Here we identify that human histone deacetylase 6 (HDAC6) serves as a valine sensor by directly binding valine through a primate-specific SE14 repeat domain. The nucleus and cytoplasm shuttling of human, but not mouse, HDAC6 is tightly controlled by the intracellular levels of valine. Valine deprivation leads to HDAC6 retention in the nucleus and induces DNA damage. Mechanistically, nuclear-localized HDAC6 binds and deacetylates ten-eleven translocation 2 (TET2) to initiate active DNA demethylation, which promotes DNA damage through thymine DNA glycosylase-driven excision. Dietary valine restriction inhibits tumour growth in xenograft and patient-derived xenograft models, and enhances the therapeutic efficacy of PARP inhibitors. Collectively, our study identifies human HDAC6 as a valine sensor that mediates active DNA demethylation and DNA damage in response to valine deprivation, and highlights the potential of dietary valine restriction for cancer treatment.

PROSER1 modulates DNA demethylation through dual mechanisms to prevent syndromic developmental malformations.

The link between DNA methylation and neurodevelopmental disorders is well established. However, how DNA methylation is fine-tuned-ensuring precise gene expression and developmental fidelity-remains poorly understood. PROSER1, a known TET2 interactor, was recently linked to a severe neurodevelopmental disorder. Here, we demonstrate that PROSER1 interacts with all TET enzymes and stabilizes chromatin-bound TET-OGT-PROSER1-DBHS (TOPD) complexes, which regulate DNA demethylation and developmental gene expression. Surprisingly, we found that PROSER1 also sequesters TET enzymes, preventing widespread demethylation and transposable element derepression. Our findings identify PROSER1 as a key factor that both positively and negatively regulates DNA demethylation essential for mammalian neurodevelopment.

Enhancing Radiofrequency Ablation for Hepatocellular Carcinoma: Nano-Epidrug Effects on Immune Modulation and Antigenicity Restoration.

Radiofrequency ablation (RFA), a critical therapy for hepatocellular carcinoma (HCC), carries a significant risk of recurrence and metastasis, particularly owing to mechanisms involving immune evasion and antigen downregulation via epigenetic modifications. This study introduces a "nano-epidrug" named MFMP. MFMP, which is composed of hollow mesoporous manganese dioxide (MnO2) nanoparticles, FIDAS-5 as an MAT2A inhibitor, macrophage membrane, and anti-PD-L1 (aPD-L1), targets HCC cells. By selectively binding to these cells, MFMP initially reverses immune suppression via PD-L1 inhibition. After endocytosis, MFMP disassembles in the tumor microenvironment, releasing FIDAS-5 and Mn2+. FIDAS-5 prevents cGAS methylation, whereas Mn2+ aids STING pathway restoration. In addition, FIDAS-5 reduces m6A RNA modification, suppressing EGFR expression. These changes enhance HCC antigenicity to promote cytotoxic T cell recognition and cytotoxic killing. Furthermore, MFMP mediates immunogenic cell death in HCC by synergizing with RFA through cGAS DNA demethylation, EGFR mRNA demethylation, and TBK1 protein phosphorylation, thereby inhibiting recurrence and metastasis and enhancing immune memory. Thus, MFMP is a potential adjunctive therapy requiring clinical validation.

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.

Epigenetic dynamics of partially methylated domains in human placenta and trophoblast stem cells

BackgroundThe placenta is essential for nutrient exchange and hormone production between the mother and the developing fetus and serves as an invaluable model for epigenetic research. Most epigenetic studies of the human placenta have used whole placentas from term pregnancies and have identified the presence of partially methylated domains (PMDs). However, the origin of these domains, which are typically absent in most somatic cells, remains unclear in the placental context.ResultsUsing whole-genome bisulfite sequencing and analysis of histone H3 modifications, we generated epigenetic profiles of human cytotrophoblasts during the first trimester and at term, as well as human trophoblast stem cells. Our study focused specifically on PMDs. We found that genomic regions likely to form PMDs are resistant to global DNA demethylation during trophectoderm reprogramming, and that PMDs arise through a slow methylation process within condensed chromatin near the nuclear lamina. In addition, we found significant differences in histone H3 modifications between PMDs in cytotrophoblasts and trophoblast stem cells.ConclusionsOur findings suggest that spatiotemporal genomic features shape megabase-scale DNA methylation patterns, including PMDs, in the human placenta and highlight distinct differences in PMDs between human cytotrophoblasts and trophoblast stem cells. These findings advance our understanding of placental biology and provide a basis for further research into human development and related diseases.

FoxA1/2-dependent epigenomic reprogramming drives lineage switching in lung adenocarcinoma.

The ability of cancer cells to undergo identity changes (i.e., lineage plasticity) plays a key role in tumor progression and response to therapy. Loss of the pulmonary lineage specifier NKX2-1 in KRAS-driven lung adenocarcinoma (LUAD) enhances tumor progression and causes a FoxA1/2-dependent pulmonary-to-gastric lineage switch. However, the mechanisms by which FoxA1/2 activate a latent gastric identity in the lung remain largely unknown. Here, we show that FoxA1/2 reprogram the epigenetic landscape of gastric-specific genes after NKX2-1 loss in mouse models by facilitating ten-eleven translocation (TET)2/3 recruitment, DNA demethylation, histone 3 lysine 27 acetylation (H3K27ac) deposition, and three-dimensional (3D) chromatin interactions. FoxA1/2-mediated DNA methylation changes are highly conserved in human endodermal development and in progression of human lung and pancreatic neoplasia. Furthermore, oncogenic signaling is required for specific elements of FoxA1/2-dependent epigenetic reprogramming. This work demonstrates the role of FoxA1/2 in rewiring the DNA methylation and 3D chromatin landscape of NKX2-1-negative LUAD to drive cancer cell lineage switching.

Hypermethylation of CDKN2A CpG island drives resistance to PRC2 inhibitors in SWI/SNF loss-of-function tumors.

Polycomb repressive complex 2 (PRC2) catalyzes the writing of the tri-methylated histone H3 at Lys27 (H3K27me3) epigenetic marker and suppresses the expression of genes, including tumor suppressors. The function of the complex can be partially antagonized by the SWI/SNF chromatin-remodeling complex. Previous studies have suggested that PRC2 is important for the proliferation of tumors with SWI/SNF loss-of-function mutations. In the present study, we have developed an EED-directed allosteric inhibitor of PRC2 termed BR0063, which exhibits anti-proliferative properties in a subset of solid tumor cell lines harboring mutations of the SWI/SNF subunits, SMARCA4 or ARID1A. Tumor cells sensitive to BR0063 exhibited several distinct phenotypes, including cell senescence, which was mediated by the up-regulation of CDKN2A/p16. Further experiments revealed that the expression of p16 was suppressed in the BR0063-resistant cells via DNA hypermethylation in the CpG island (CGI) promoter region, rather than via PRC2 occupancy. The expression of TET1, which is required for DNA demethylation, was found to be inversely correlated with p16 CGI methylation, and this may serve as a biomarker for the prediction of resistance to PRC2 inhibitors in SWI/SNF LOF tumors.

Recruitment of homodimeric proneural factors by conserved CAT-CAT E-boxes drives major epigenetic reconfiguration in cortical neurogenesis.

Proneural factors of the basic helix-loop-helix family coordinate neurogenesis and neurodifferentiation. Among them, NEUROG2 and NEUROD2 subsequently act to specify neurons of the glutamatergic lineage. Disruption of these factors, their target genes and binding DNA motifs has been linked to various neuropsychiatric disorders. Proneural factors bind to specific DNA motifs called E-boxes (hexanucleotides of the form CANNTG, composed of two CAN half sites on opposed strands). While corticogenesis heavily relies on E-box activity, the collaboration of proneural factors on different E-box types and their chromatin remodeling mechanisms remain largely unknown. Here, we conducted a comprehensive analysis using chromatin immunoprecipitation followed bysequencing (ChIP-seq) data for NEUROG2 and NEUROD2, along with time-matched single-cell RNA-seq, ATAC-seq and DNA methylation data from the developing mouse cortex. Our findings show that these factors are highly enriched in transiently active genomic regions during intermediate stages of neuronal differentiation. Although they primarily bind CAG-containing E-boxes, their binding in dynamic regions is notably enriched in CAT-CAT E-boxes (i.e. CATATG, denoted as 5'3' half sites for dimers), which undergo significant DNA demethylation and exhibit the highest levels of evolutionary constraint. Aided by HT-SELEX data reanalysis, structural modeling and DNA footprinting, we propose that these proneural factors exert maximal chromatin remodeling influence during intermediate stages of neurogenesis by binding as homodimers to CAT-CAT motifs. This study provides an in-depth integrative analysis of the dynamic regulation of E-boxes during neuronal development, enhancing our understanding of the mechanisms underlying the binding specificity of critical proneural factors.

5-Hydroxymethylcytosine: Far Beyond the Intermediate of DNA Demethylation.

Epigenetics plays a pivotal role in regulating gene expression and cellular differentiation. DNA methylation, involving the addition of methyl groups to specific cytosine bases, is a well-known epigenetic modification. The recent discovery of 5-hydroxymethylcytosine (5hmC) has provided new insights into cytosine modifications. 5hmC, derived from the oxidation of 5-methylcytosine (5mC), serves as both an intermediate in demethylation and a stable chemical modification in the genome. In this comprehensive review, we summarize the recent research advancements regarding the functions of 5hmC in development and disease. We discuss its implications in gene expression regulation, cellular differentiation, and its potential role as a diagnostic and prognostic marker in various diseases. Additionally, we highlight the challenges associated with accurately detecting and quantifying 5hmC and present the latest methodologies employed for its detection. Understanding the functional role of 5hmC in epigenetic regulation and further advancing our understanding of gene expression dynamics and cellular processes hold immense promise for the development of novel therapeutic strategies and precision medicine approaches.

Copper Oxide Nanoparticles Impair Mouse Preimplantation Embryonic Development through Disruption of Mitophagy-Mediated Metabolism.

Copper oxide nanoparticles (CuONPs) have been widely applied, posing potential risks to human health. Although the toxicity of CuONPs on the liver and spleen has been reported, their effects on reproductive health remain unexplored. In this study, we investigate the effects of CuONPs on embryonic development and their potential mechanisms. Our results demonstrate that CuONPs exposure impairs mouse preimplantation embryonic development, particularly affecting the morula-to-blastocyst transition. Additionally, CuONPs were found to reduce the pluripotency of the inner cell mass (ICM) and mouse embryonic stem cells (mESCs). Mechanistically, CuONPs block autophagic flux and impair mitophagy, leading to the accumulation of damaged mitochondria. This mitochondrial dysfunction leads to reduced tricarboxylic acid (TCA) cycle activity and decreased α-ketoglutarate (α-KG) production. Insufficient α-KG induces the failure of DNA demethylation, reducing corresponding chromatin accessibility and consequently inhibiting ICM-specific genes expressions. Similar reduced development and inhibitions of pluripotency gene expression were observed in CuONPs-treated human blastocysts. Moreover, in women undergoing assisted reproductive technology (ART), a negative correlation was found between urinary Cu ion concentrations and clinical outcomes. Collectively, our study elucidates the mitophagy-mediated metabolic mechanisms of CuONPs embryotoxicity, improving our understanding of the potential reproductive toxicity associated with it.

Distinct Regions within SAP25 Recruit O-Linked Glycosylation, DNA Demethylation, and Ubiquitin Ligase and Hydrolase Activities to the Sin3/HDAC Complex.

Sin3 is an evolutionarily conserved repressor protein complex mainly associated with histone deacetylase (HDAC) activity. Many proteins are part of Sin3/HDAC complexes, and the function of most of these members remains poorly understood. SAP25, a previously identified Sin3A associated protein of 25 kDa, has been proposed to participate in regulating gene expression programs involved in the immune response but the exact mechanism of this regulation is unclear. SAP25 is not expressed in HEK293 cells, which hence serve as a natural knockout system to decipher the molecular functions uniquely carried out by this Sin3/HDAC subunit. Using molecular, proteomic, protein engineering, and interaction network approaches, we show that SAP25 interacts with distinct enzymatic and regulatory protein complexes in addition to Sin3/HDAC. Additional proteins uniquely recovered from the Halo-SAP25 pull-downs included the SCF E3 ubiquitin ligase complex SKP1/FBXO3/CUL1 and the ubiquitin carboxyl-terminal hydrolase 11 (USP11). Furthermore, mutational analysis demonstrates that distinct regions of SAP25 participate in its interaction with USP11, OGT/TETs, and SCF(FBXO3). These results suggest that SAP25 may function as an adaptor protein to coordinate the assembly of different enzymatic complexes to control Sin3/HDAC-mediated gene expression. The data were deposited with the MASSIVE repository with the identifiers MSV000093576 and MSV000093553.

Epigenetic Modeling of Jumping Translocations of 1q Heterochromatin in Acute Myeloid Leukemia After 5'-Azacytidine Treatment.

Jumping translocations (JT) are rare cytogenetic abnormalities associated with progression in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Typically, a tri-tetra-somic 1q chromosome is translocated to two or more recipient chromosomes. In multiple myeloma JT were shown to originate after DNA demethylation and decondensation. Using epigenomics, we investigated sequential samples in an SRSF2-mutated MDS and AML cohort with normal karyotype at diagnosis and 1qJT at disease evolution after 5'-azacytidine (AZA). 1qJT breakpoints fell within repetitive DNA at both 1q12 and the translocation partners, namely acrocentrics n. 14, 15, 21, and 22, chromosome 16, and chromosome Y. The global methylome at diagnosis showed hypermethylation at 61% of the differentially methylated regions (DMRs), followed by hypomethylation at 80% of DMRs under AZA, mostly affecting pathways related to immune system, chromatin organization, chromosome condensation, telomere maintenance, rRNA, and DNA repair. At disease evolution, a shift toward hypermethylation, intronic enhancers enrichment and epigenetic involvement of the PI3K/AKT and MAPK signaling emerged. In particular, AKT1 phosphorylation behaved as a hallmark of the progression. Overall, we provided new insights on the characterization of 1qJT in SRSF2-mutated myeloid neoplasms and first showed that epigenetics is a powerful tool to investigate the molecular landscape of repetitive DNA rearrangements.