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Cells Mar 2022Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are characterized by genomic instability, which may arise from the global hypomethylation of the DNA....
Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are characterized by genomic instability, which may arise from the global hypomethylation of the DNA. The active DNA demethylation process may be linked with aberrant methylation and can be involved in leukemogenesis. The levels of 5-methylcytosine oxidation products were analyzed in minimally invasive material: the cellular DNA from peripheral blood cells and urine of patients with AML and MDS along with the control group, using isotope-dilution two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. The receiver operating characteristic curve analysis was used for the assessment of the ability to discriminate patients' groups from the control group, and AML from MDS. The most diagnostically useful for discriminating AML patients from the control group was the urinary excretion of 5-hydroxymethylcytosine (AUC = 0.918, sensitivity: 85%, and specificity: 97%), and 5-(hydroxymethyl)-2'-deoxyuridine (0.873, 74%, and 92%), while for MDS patients 5-(hydroxymethyl)-2'-deoxycytidine in DNA (0.905, 82%, and 98%) and urinary 5-hydroxymethylcytosine (0.746, 66%, and 92%). Multi-factor models of classification trees allowed the correct classification of patients with AML and MDS in 95.7% and 94.7% of cases. The highest prognostic value of the analyzed parameters in predicting the transformation of MDS into AML was observed for 5-carboxy-2'-deoxycytidine (0.823, 80%, and 97%) and 5-(hydroxymethyl)-2'-deoxyuridine (0.872, 100%, and 75%) in DNA. The presented research proves that the intermediates of the active DNA demethylation pathway determined in the completely non-invasive (urine) or minimally invasive (blood) material can be useful in supporting the diagnostic process of patients with MDS and AML. The possibility of an early identification of a group of MDS patients with an increased risk of transformation into AML is of particular importance.
Topics: DNA; DNA Demethylation; Deoxycytidine; Deoxyuridine; Humans; Leukemia, Myeloid, Acute; Myelodysplastic Syndromes; Prognosis
PubMed: 35269510
DOI: 10.3390/cells11050888 -
Hormones and Behavior May 2023Some of the best-studied neural sex differences depend on differential cell death in males and females, but other sex differences persist even if cell death is...
Some of the best-studied neural sex differences depend on differential cell death in males and females, but other sex differences persist even if cell death is prevented. These include sex differences in neurochemical phenotype (i.e., stable patterns of gene expression). Work in our laboratory over the last several years has tested the hypothesis that sex differences in DNA methylation early in life underlie sexual differentiation of neuronal phenotype. We have shown that 1) expression of enzymes that place or remove DNA methylation marks is greatest during the first week of life in the mouse brain and overlaps with the perinatal critical period of sexual differentiation; 2) a transient inhibition of DNA methylation during neonatal life abolishes several sex differences in cell phenotype in the mouse hypothalamus; 3) both DNA methylation and de-methylation contribute to the development of neural sex differences; and 4) the effects of DNA methylation and de-methylation are brain region- and cell type-specific.
Topics: Animals; Mice; Female; Male; DNA Methylation; Sex Differentiation; Phenotype; Neurons; Demethylation
PubMed: 37001316
DOI: 10.1016/j.yhbeh.2023.105349 -
BioMed Research International 2020Immunotherapy has been demonstrated as a promising strategy in controlling head and neck squamous cell carcinoma (HNSC). The AID/APOBEC family is well characterized as...
Immunotherapy has been demonstrated as a promising strategy in controlling head and neck squamous cell carcinoma (HNSC). The AID/APOBEC family is well characterized as DNA mutator and considered to play critical roles in immune responses in HNSC. However, the expression pattern and deamination-dependent demethylation roles of AID/APOBECs in HNSC are unclear. In this study, the RNA-seq and DNA methylation profiles of HNSC from TCGA database and cell-based experiments were applied to analyze the relationships between AID/APOBEC expression levels, patients' clinical outcomes, methylation alterations, and immune responses. Here, we found that APOBEC3H was abnormally upregulated in HNSC patients. HPV+ patients tended to have higher APOBEC3H levels than HPV- patients. Remarkably, patients with high APOBEC3H levels showed a favorable overall survival. Furthermore, tumors with high APOBEC3H levels exhibited a genome-wide DNA hypomethylation pattern. APOBEC3H was identified to demethylate and upregulate CXCL10 and improve CD8+ T cell tumor infiltration in the tumor microenvironment. Collectively, APOBEC3H plays critical roles in CD8+ T cell immune infiltration and activation in HNSC, which may be a potential biomarker for oncoimmunotherapy in HNSC.
Topics: Aminohydrolases; Biomarkers, Tumor; Databases, Nucleic Acid; Female; Head and Neck Neoplasms; Humans; Immunotherapy; Male; Methylation; Neoplasm Proteins; Squamous Cell Carcinoma of Head and Neck
PubMed: 32775421
DOI: 10.1155/2020/4612375 -
Pharmacology Research & Perspectives Feb 2021Mass drug administration of ivermectin has been proposed as a possible malaria elimination tool. Ivermectin exhibits a mosquito-lethal effect well beyond its biological... (Clinical Trial)
Clinical Trial
Mass drug administration of ivermectin has been proposed as a possible malaria elimination tool. Ivermectin exhibits a mosquito-lethal effect well beyond its biological half-life, suggesting the presence of active slowly eliminated metabolites. Human liver microsomes, primary human hepatocytes, and whole blood from healthy volunteers given oral ivermectin were used to identify ivermectin metabolites by ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry. The molecular structures of metabolites were determined by mass spectrometry and verified by nuclear magnetic resonance. Pure cytochrome P450 enzyme isoforms were used to elucidate the metabolic pathways. Thirteen different metabolites (M1-M13) were identified after incubation of ivermectin with human liver microsomes. Three (M1, M3, and M6) were the major metabolites found in microsomes, hepatocytes, and blood from volunteers after oral ivermectin administration. The chemical structure, defined by LC-MS/MS and NMR, indicated that M1 is 3″-O-demethyl ivermectin, M3 is 4-hydroxymethyl ivermectin, and M6 is 3″-O-demethyl, 4-hydroxymethyl ivermectin. Metabolic pathway evaluations with characterized cytochrome P450 enzymes showed that M1, M3, and M6 were produced primarily by CYP3A4, and that M1 was also produced to a small extent by CYP3A5. Demethylated (M1) and hydroxylated (M3) ivermectin were the main human in vivo metabolites. Further studies are needed to characterize the pharmacokinetic properties and mosquito-lethal activity of these metabolites.
Topics: Administration, Oral; Antiparasitic Agents; Cells, Cultured; Cytochrome P-450 Enzyme System; Demethylation; Hepatocytes; Humans; Hydroxylation; Ivermectin; Metabolic Networks and Pathways; Microsomes, Liver
PubMed: 33497030
DOI: 10.1002/prp2.712 -
Archives of Toxicology Sep 2023The risk of methylmercury (MeHg) toxicity following ingestion of contaminated foodstuffs (e.g., fish) is directly related to the kinetics of MeHg elimination among...
The risk of methylmercury (MeHg) toxicity following ingestion of contaminated foodstuffs (e.g., fish) is directly related to the kinetics of MeHg elimination among individuals. Yet, the factors driving the wide range of inter-individual variability in MeHg elimination within a population are poorly understood. Here, we investigated the relationship between MeHg elimination, gut microbiome demethylation activity, and gut microbiome composition using a coordinated human clinical trial and gnotobiotic mouse modeling approach together with metagenomic sequence analysis. We first observed MeHg elimination half-lives (t) ranging from 28 to 90 days across 27 volunteers. Subsequently, we found that ingestion of a prebiotic induced changes in the gut microbiome and mixed effects (increased, decrease, and no effect) on elimination in these same individuals. Nonetheless, elimination rates were found to correlate with MeHg demethylation activity in cultured stool samples. In mice, attempts to remove the microbiome via generation of germ-free (GF) animals or through antibiotic (Abx) treatment both diminished MeHg demethylation to a similar extent. While both conditions substantially slowed elimination, Abx treatment resulted in significantly slower elimination than the GF condition, indicating an additional role for host-derived factors in supporting elimination. Human fecal microbiomes transplanted to GF mice restored elimination rates to that seen in control mice. Metagenomic sequence analysis of human fecal DNA did not identify genes encoding proteins typically involved in demethylation (e.g., merB, organomercury lyase). However, the abundance of several anaerobic taxa, notably Alistipes onderdonkii, were positively correlated with MeHg elimination. Surprisingly, mono-colonization of GF free mice with A. onderdonkii did not restore MeHg elimination to control levels. Collectively, our findings indicate the human gut microbiome uses a non-conventional pathway of demethylation to increase MeHg elimination that relies on yet to be resolved functions encoded by the gut microbes and the hostClinical Trial NCT04060212, prospectively registered 10/1/2019.
Topics: Humans; Animals; Mice; Methylmercury Compounds; Gastrointestinal Microbiome; Microbiota; Kinetics; Demethylation
PubMed: 37392210
DOI: 10.1007/s00204-023-03548-7 -
ACS Chemical Biology Feb 2021-methyladenosine (mA) has emerged as the most abundant mRNA modification that regulates gene expression in many physiological processes. mA modification in RNA controls...
-methyladenosine (mA) has emerged as the most abundant mRNA modification that regulates gene expression in many physiological processes. mA modification in RNA controls cellular proliferation and pluripotency and has been implicated in the progression of multiple disease states, including cancer. RNA mA methylation is controlled by a multiprotein "writer" complex including the enzymatic factor methyltransferase-like protein 3 (METTL3) that regulates methylation and two "eraser" proteins, RNA demethylase ALKBH5 (ALKBH5) and fat mass- and obesity-associated protein (FTO), that demethylate mA in transcripts. FTO can also demethylate ,2'--dimethyladenosine (mA), which is found adjacent to the mG cap structure in mRNA. FTO has recently gained interest as a potential cancer target, and small molecule FTO inhibitors such as meclofenamic acid have been shown to prevent tumor progression in both acute myeloid leukemia and glioblastoma models. However, current FTO inhibitors are unsuitable for clinical applications due to either poor target selectivity or poor pharmacokinetics. In this work, we describe the structure-based design, synthesis, and biochemical evaluation of a new class of FTO inhibitors. Rational design of 20 small molecules with low micromolar IC's and specificity toward FTO over ALKBH5 identified two competitive inhibitors FTO-02 and FTO-04. Importantly, FTO-04 prevented neurosphere formation in patient-derived glioblastoma stem cells (GSCs) without inhibiting the growth of healthy neural stem cell-derived neurospheres. Finally, FTO-04 increased mA and mA levels in GSCs consistent with FTO inhibition. These results support FTO-04 as a potential new lead for treatment of glioblastoma.
Topics: Adenosine; Alpha-Ketoglutarate-Dependent Dioxygenase FTO; Antineoplastic Agents; Binding Sites; Demethylation; Drug Design; Enzyme Inhibitors; Humans; Molecular Docking Simulation; Neoplastic Stem Cells; Protein Binding; Pyrimidines; RNA
PubMed: 33412003
DOI: 10.1021/acschembio.0c00841 -
Cell Reports Dec 2021Regulatory T (T) cells play crucial roles in suppressing deleterious immune response. Here, we investigate how T cells are mechanistically induced in vitro (iT) and...
Regulatory T (T) cells play crucial roles in suppressing deleterious immune response. Here, we investigate how T cells are mechanistically induced in vitro (iT) and stabilized via transcriptional regulation of T lineage-specifying factor Foxp3. We find that acetylation of histone tails at the Foxp3 promoter is required for inducing Foxp3 transcription. Upon induction, histone acetylation signals via bromodomain-containing proteins, particularly targets of inhibitor JQ1, and sustains Foxp3 transcription via a global or trans effect. Subsequently, Tet-mediated DNA demethylation of Foxp3 cis-regulatory elements, mainly enhancer CNS2, increases chromatin accessibility and protein binding, stabilizing Foxp3 transcription and obviating the need for the histone acetylation signal. These processes transform stochastic iT induction into a stable cell fate, with the former sensitive and the latter resistant to genetic and environmental perturbations. Thus, sequential histone acetylation and DNA demethylation in Foxp3 induction and maintenance reflect stepwise mechanical switches governing iT cell lineage specification.
Topics: Acetylation; Animals; Cell Differentiation; DNA Demethylation; DNA Methylation; DNA-Binding Proteins; Female; Forkhead Transcription Factors; Gene Expression Regulation; Histones; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Promoter Regions, Genetic; Proto-Oncogene Proteins; Regulatory Sequences, Nucleic Acid; T-Lymphocytes, Regulatory
PubMed: 34910919
DOI: 10.1016/j.celrep.2021.110124 -
Cell Reports Mar 2020Histone methyl groups can be removed by demethylases. Although LSD1 and JmjC domain-containing proteins have been identified as histone demethylases, enzymes for many...
Histone methyl groups can be removed by demethylases. Although LSD1 and JmjC domain-containing proteins have been identified as histone demethylases, enzymes for many histone methylation states or sites are still unknown. Here, we perform a screening of a cDNA library containing 2,500 nuclear proteins and identify hHR23A as a histone H4K20 demethylase. Overexpression of hHR23A reduces the levels of H4K20me1/2/3 in cells. In vitro, hHR23A specifically demethylates H4K20me1/2/3 and generates formaldehyde. The enzymatic activity requires Fe(II) and α-ketoglutarate as cofactors and the UBA domains of hHR23A. hHR23B, a protein homologous to hHR23A, also demethylates H4K20me1/2/3 in vitro and in vivo. We further demonstrate that hHR23A/B activate the transcription of coding genes by demethylating H4K20me1 and the transcription of repetitive elements by demethylating H4K20me3. Nuclear magnetic resonance (NMR) analyses demonstrate that an HxxxE motif in the UBA1 domain is crucial for iron binding and demethylase activity. Thus, we identify two hHR23 proteins as histone demethylases.
Topics: Cell Cycle; DNA Repair Enzymes; DNA-Binding Proteins; Demethylation; Formaldehyde; Genetic Loci; Genome, Human; HEK293 Cells; HeLa Cells; Histones; Humans; Iron; Lysine; Peptides; Protein Domains; RNA, Messenger; Repetitive Sequences, Nucleic Acid; Substrate Specificity; Transcription, Genetic
PubMed: 32209475
DOI: 10.1016/j.celrep.2020.03.001 -
Neuroscience Research May 2024Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are characterized by nuclear depletion and cytoplasmic aggregation of TAR DNA-binding protein-43... (Review)
Review
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are characterized by nuclear depletion and cytoplasmic aggregation of TAR DNA-binding protein-43 (TDP-43). TDP-43 plays a key role in regulating the splicing of numerous genes, including TARDBP. This review aims to delineate two aspects of ALS/FTD pathogenesis associated with TDP-43 function. First, we provide novel mechanistic insights into the splicing of UNC13A, a TDP-43 target gene. Single nucleotide polymorphisms (SNPs) in UNC13A are the most common risk factors for ALS/FTD. We found that TDP-43 represses "cryptic exon" inclusion during UNC13A RNA splicing. A risk-associated SNP in this exon results in increased RNA levels of UNC13A retaining the cryptic exon. Second, we described the perturbation of the TDP-43 autoregulatory mechanism caused by age-related DNA demethylation. Aging is a major risk factor for sporadic ALS/FTD. Typically, TDP-43 levels are regulated via alternative splicing of TARDBP mRNA. We hypothesized that TARDBP methylation is altered by aging, thereby disrupting TDP-43 autoregulation. We found that demethylation reduces the efficiency of alternative splicing and increases TARDBP mRNA levels. Moreover, we demonstrated that, with aging, this region is demethylated in the human motor cortex and is associated with the early onset of ALS.
PubMed: 38723906
DOI: 10.1016/j.neures.2024.05.001 -
The New Phytologist Feb 2022The ripening of fleshy fruits is a unique developmental process that Arabidopsis and rice lack. This process is driven by hormones and transcription factors. However,...
The ripening of fleshy fruits is a unique developmental process that Arabidopsis and rice lack. This process is driven by hormones and transcription factors. However, the critical and early regulators of fruit ripening are still poorly understood. Here, we revealed that SlJMJ7, an H3K4 demethylase, is a critical negative regulator of fruit ripening in tomato. Combined genome-wide transcription, binding sites, histone H3K4me3 and DNA methylation analyses demonstrated that SlJMJ7 regulates a key group of ripening-related genes, including ethylene biosynthesis (ACS2, ACS4 and ACO6), transcriptional regulation (RIN and NOR) and DNA demethylation (DML2) genes, by H3K4me3 demethylation. Moreover, loss of SlJMJ7 function leads to increased H3K4me3 levels, which directly activates ripening-related genes, and to global DML2-mediated DNA hypomethylation in fruit, which indirectly prompts expression of ripening-related genes. Together, these effects lead to accelerated fruit ripening in sljmj7 mutant. Our findings demonstrate that SlJMJ7 acts as a master negative regulator of fruit ripening not only through direct removal of H3K4me3 from multiple key ripening-related factors, but also through crosstalk between histone and DNA demethylation. These findings reveal a novel crosstalk between histone methylation and DNA methylation to regulate gene expression in plant developmental processes.
Topics: DNA; DNA Demethylation; DNA Methylation; Ethylenes; Fruit; Gene Expression Regulation, Plant; Histones; Solanum lycopersicum; Plant Proteins
PubMed: 34729792
DOI: 10.1111/nph.17838