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Science (New York, N.Y.) May 2022-methyladenosine (mA) is the most abundant internal modification on mammalian messenger RNA. It is installed by a writer complex and can be reversed by erasers such as...
-methyladenosine (mA) is the most abundant internal modification on mammalian messenger RNA. It is installed by a writer complex and can be reversed by erasers such as the fat mass and obesity-associated protein FTO. Despite extensive research, the primary physiological substrates of FTO in mammalian tissues and development remain elusive. Here, we show that FTO mediates mA demethylation of long-interspersed element-1 (LINE1) RNA in mouse embryonic stem cells (mESCs), regulating LINE1 RNA abundance and the local chromatin state, which in turn modulates the transcription of LINE1-containing genes. FTO-mediated LINE1 RNA mA demethylation also plays regulatory roles in shaping chromatin state and gene expression during mouse oocyte and embryonic development. Our results suggest broad effects of LINE1 RNA mA demethylation by FTO in mammals.
Topics: Adenosine; Alpha-Ketoglutarate-Dependent Dioxygenase FTO; Animals; Chromatin; Demethylation; Gene Expression Regulation, Developmental; Long Interspersed Nucleotide Elements; Mice; Mouse Embryonic Stem Cells; Oocytes; RNA, Messenger
PubMed: 35511947
DOI: 10.1126/science.abe9582 -
Science (New York, N.Y.) Dec 2022Neurons harbor high levels of single-strand DNA breaks (SSBs) that are targeted to neuronal enhancers, but the source of this endogenous damage remains unclear. Using...
Neurons harbor high levels of single-strand DNA breaks (SSBs) that are targeted to neuronal enhancers, but the source of this endogenous damage remains unclear. Using two systems of postmitotic lineage specification-induced pluripotent stem cell-derived neurons and transdifferentiated macrophages-we show that thymidine DNA glycosylase (TDG)-driven excision of methylcytosines oxidized with ten-eleven translocation enzymes (TET) is a source of SSBs. Although macrophage differentiation favors short-patch base excision repair to fill in single-nucleotide gaps, neurons also frequently use the long-patch subpathway. Disrupting this gap-filling process using anti-neoplastic cytosine analogs triggers a DNA damage response and neuronal cell death, which is dependent on TDG. Thus, TET-mediated active DNA demethylation promotes endogenous DNA damage, a process that normally safeguards cell identity but can also provoke neurotoxicity after anticancer treatments.
Topics: Cell Differentiation; DNA Demethylation; Induced Pluripotent Stem Cells; Neurons; DNA Breaks, Single-Stranded; Enhancer Elements, Genetic; Thymine DNA Glycosylase; DNA Repair; 5-Methylcytosine; Humans; Cell Transdifferentiation
PubMed: 36454826
DOI: 10.1126/science.add9838 -
Circulation Jan 2019Despite its functional importance in various fundamental bioprocesses, studies of N-methyladenosine (m6A) in the heart are lacking. Here, we show that the FTO (fat mass...
BACKGROUND
Despite its functional importance in various fundamental bioprocesses, studies of N-methyladenosine (m6A) in the heart are lacking. Here, we show that the FTO (fat mass and obesity-associated protein), an m6A demethylase, plays a critical role in cardiac contractile function during homeostasis, remodeling, and regeneration.
METHODS
We used clinical human samples, preclinical pig and mouse models, and primary cardiomyocyte cell cultures to study the functional role of m6A and FTO in the heart and in cardiomyocytes. We modulated expression of FTO by using adeno-associated virus serotype 9 (in vivo), adenovirus (both in vivo and in vitro), and small interfering RNAs (in vitro) to study its function in regulating cardiomyocyte m6A, calcium dynamics and contractility, and cardiac function postischemia. We performed methylated (m6A) RNA immunoprecipitation sequencing to map transcriptome-wide m6A, and methylated (m6A) RNA immunoprecipitation quantitative polymerase chain reaction assays to map and validate m6A in individual transcripts, in healthy and failing hearts, and in myocytes.
RESULTS
We discovered that FTO has decreased expression in failing mammalian hearts and hypoxic cardiomyocytes, thereby increasing m6A in RNA and decreasing cardiomyocyte contractile function. Improving expression of FTO in failing mouse hearts attenuated the ischemia-induced increase in m6A and decrease in cardiac contractile function. This is performed by the demethylation activity of FTO, which selectively demethylates cardiac contractile transcripts, thus preventing their degradation and improving their protein expression under ischemia. In addition, we demonstrate that FTO overexpression in mouse models of myocardial infarction decreased fibrosis and enhanced angiogenesis.
CONCLUSIONS
Collectively, our study demonstrates the functional importance of the FTO-dependent cardiac m6A methylome in cardiac contraction during heart failure and provides a novel mechanistic insight into the therapeutic mechanisms of FTO.
Topics: Adenosine; Adult; Aged; Alpha-Ketoglutarate-Dependent Dioxygenase FTO; Animals; Calcium Signaling; Case-Control Studies; Cell Line; Cell Proliferation; Demethylation; Disease Models, Animal; Female; Heart Failure; Humans; Male; Mice; Mice, Inbred C57BL; Middle Aged; Myocardial Infarction; Myocytes, Cardiac; RNA Processing, Post-Transcriptional; RNA Stability; RNA, Messenger; Rats, Sprague-Dawley; Regeneration; Sus scrofa; Ventricular Function, Left; Ventricular Remodeling
PubMed: 29997116
DOI: 10.1161/CIRCULATIONAHA.118.033794 -
Cancer Cell Jun 2023Cyclic GMP-AMP synthase (cGAS) is the major sensor for cytosolic DNA and activates type I interferon signaling and plays an essential role in antitumor immunity....
Cyclic GMP-AMP synthase (cGAS) is the major sensor for cytosolic DNA and activates type I interferon signaling and plays an essential role in antitumor immunity. However, it remains unclear whether the cGAS-mediated antitumor activity is affected by nutrient status. Here, our study reports that methionine deprivation enhances cGAS activity by blocking its methylation, which is catalyzed by methyltransferase SUV39H1. We further show that methylation enhances the chromatin sequestration of cGAS in a UHRF1-dependent manner. Blocking cGAS methylation enhances cGAS-mediated antitumor immunity and suppresses colorectal tumorigenesis. Clinically, cGAS methylation in human cancers correlates with poor prognosis. Thus, our results indicate that nutrient stress promotes cGAS activation via reversible methylation, and suggest a potential therapeutic strategy for targeting cGAS methylation in cancer treatment.
Topics: Humans; Chromatin; Methionine; Nucleotidyltransferases; DNA; Immunity, Innate; Demethylation; CCAAT-Enhancer-Binding Proteins; Ubiquitin-Protein Ligases
PubMed: 37267951
DOI: 10.1016/j.ccell.2023.05.005 -
Cell Metabolism May 2021Poor maternal diet increases the risk of obesity and type 2 diabetes in offspring, adding to the ever-increasing prevalence of these diseases. In contrast, we find that...
Poor maternal diet increases the risk of obesity and type 2 diabetes in offspring, adding to the ever-increasing prevalence of these diseases. In contrast, we find that maternal exercise improves the metabolic health of offspring, and here, we demonstrate that this occurs through a vitamin D receptor-mediated increase in placental superoxide dismutase 3 (SOD3) expression and secretion. SOD3 activates an AMPK/TET signaling axis in fetal offspring liver, resulting in DNA demethylation at the promoters of glucose metabolic genes, enhancing liver function, and improving glucose tolerance. In humans, SOD3 is upregulated in serum and placenta from physically active pregnant women. The discovery of maternal exercise-induced cross talk between placenta-derived SOD3 and offspring liver provides a central mechanism for improved offspring metabolic health. These findings may lead to novel therapeutic approaches to limit the transmission of metabolic disease to the next generation.
Topics: AMP-Activated Protein Kinases; Animals; Cells, Cultured; DNA Demethylation; Diet, High-Fat; Exercise; Female; Hepatocytes; Humans; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mixed Function Oxygenases; Placenta; Pregnancy; Proto-Oncogene Proteins; Receptors, Calcitriol; Signal Transduction; Superoxide Dismutase
PubMed: 33770509
DOI: 10.1016/j.cmet.2021.03.004 -
Molecular Cell Sep 2018FTO, the first RNA demethylase discovered, mediates the demethylation of internal N-methyladenosine (mA) and N, 2-O-dimethyladenosine (mA) at theĀ +1 position from the...
FTO, the first RNA demethylase discovered, mediates the demethylation of internal N-methyladenosine (mA) and N, 2-O-dimethyladenosine (mA) at theĀ +1 position from the 5' cap in mRNA. Here we demonstrate that the cellular distribution of FTO is distinct among different cell lines, affecting the access of FTO to different RNA substrates. We find that FTO binds multiple RNA species, including mRNA, snRNA, and tRNA, and can demethylate internal mA and cap mA in mRNA, internal mA in U6 RNA, internal and cap mA in snRNAs, and N-methyladenosine (mA) in tRNA. FTO-mediated demethylation has a greater effect on the transcript levels of mRNAs possessing internal mA than the ones with cap mA in the tested cells. We also show that FTO can directly repress translation by catalyzing mA tRNA demethylation. Collectively, FTO-mediated RNA demethylation occurs to mA and mA in mRNA and snRNA as well as mA in tRNA.
Topics: 3T3-L1 Cells; Adenosine; Alpha-Ketoglutarate-Dependent Dioxygenase FTO; Animals; Cell Nucleus; Cytoplasm; Demethylation; Gene Expression; HEK293 Cells; HeLa Cells; Humans; Methylation; Mice; RNA Processing, Post-Transcriptional; RNA, Messenger; RNA, Small Nuclear; RNA, Transfer
PubMed: 30197295
DOI: 10.1016/j.molcel.2018.08.011 -
Nature Aug 2023Cells undergo a major epigenome reconfiguration when reprogrammed to human induced pluripotent stem cells (hiPS cells). However, the epigenomes of hiPS cells and human...
Cells undergo a major epigenome reconfiguration when reprogrammed to human induced pluripotent stem cells (hiPS cells). However, the epigenomes of hiPS cells and human embryonic stem (hES) cells differ significantly, which affects hiPS cell function. These differences include epigenetic memory and aberrations that emerge during reprogramming, for which the mechanisms remain unknown. Here we characterized the persistence and emergence of these epigenetic differences by performing genome-wide DNA methylation profiling throughout primed and naive reprogramming of human somatic cells to hiPS cells. We found that reprogramming-induced epigenetic aberrations emerge midway through primed reprogramming, whereas DNA demethylation begins early in naive reprogramming. Using this knowledge, we developed a transient-naive-treatment (TNT) reprogramming strategy that emulates the embryonic epigenetic reset. We show that the epigenetic memory in hiPS cells is concentrated in cell of origin-dependent repressive chromatin marked by H3K9me3, lamin-B1 and aberrant CpH methylation. TNT reprogramming reconfigures these domains to a hES cell-like state and does not disrupt genomic imprinting. Using an isogenic system, we demonstrate that TNT reprogramming can correct the transposable element overexpression and differential gene expression seen in conventional hiPS cells, and that TNT-reprogrammed hiPS and hES cells show similar differentiation efficiencies. Moreover, TNT reprogramming enhances the differentiation of hiPS cells derived from multiple cell types. Thus, TNT reprogramming corrects epigenetic memory and aberrations, producing hiPS cells that are molecularly and functionally more similar to hES cells than conventional hiPS cells. We foresee TNT reprogramming becoming a new standard for biomedical and therapeutic applications and providing a novel system for studying epigenetic memory.
Topics: Humans; Cellular Reprogramming; Chromatin; DNA Demethylation; DNA Methylation; DNA Transposable Elements; Epigenesis, Genetic; Induced Pluripotent Stem Cells; Human Embryonic Stem Cells; Lamin Type B
PubMed: 37587336
DOI: 10.1038/s41586-023-06424-7 -
Experimental & Molecular Medicine Aug 2023Improving health and delaying aging is the focus of medical research. Previous studies have shown that mesenchymal stem cell (MSC) senescence is closely related to...
Improving health and delaying aging is the focus of medical research. Previous studies have shown that mesenchymal stem cell (MSC) senescence is closely related to organic aging and the development of aging-related diseases such as osteoarthritis (OA). m6A is a common RNA modification that plays an important role in regulating cell biological functions, and ALKBH5 is one of the key m6A demethylases. However, the role of m6A and ALKBH5 in MSC senescence is still unclear. Here, we found that the m6A level was enhanced and ALKBH5 expression was decreased in aging MSCs induced by multiple replications, HO stimulation or UV irradiation. Downregulation of ALKBH5 expression facilitated MSC senescence by enhancing the stability of CYP1B1 mRNA and inducing mitochondrial dysfunction. In addition, IGF2BP1 was identified as the m6A reader restraining the degradation of m6A-modified CYP1B1 mRNA. Furthermore, Alkbh5 knockout in MSCs aggravated spontaneous OA in mice, and overexpression of Alkbh5 improved the efficacy of MSCs in OA. Overall, this study revealed a novel mechanism of m6A in MSC senescence and identified promising targets to protect against aging and OA.
Topics: Animals; Mice; Demethylation; Hydrogen Peroxide; Mesenchymal Stem Cells; Osteoarthritis; RNA Stability; RNA, Messenger; AlkB Homolog 5, RNA Demethylase; Cytochrome P-450 CYP1B1
PubMed: 37524872
DOI: 10.1038/s12276-023-01059-0 -
The Plant Cell Dec 2017-methyladenosine (mA) is the most abundant, internal, posttranscriptional modification in mRNA among all higher eukaryotes. In mammals, this modification is reversible...
-methyladenosine (mA) is the most abundant, internal, posttranscriptional modification in mRNA among all higher eukaryotes. In mammals, this modification is reversible and plays broad roles in the regulation of mRNA metabolism and processing. Despite its importance, previous studies on the role and mechanism of mA methylation in have been limited. Here, we report that ALKBH10B is a demethylase that oxidatively reverses mA methylation in mRNA in vitro and in vivo. Depletion of ALKBH10B in the mutant delays flowering and represses vegetative growth. Complementation with wild-type , but not a catalytically inactive mutant (), rescues these effects in mutant plants, suggesting the observed phenotypes are controlled by the catalytic action of We show that -mediated mRNA demethylation affects the stability of target transcripts, thereby influencing floral transition. We identified 1190 mA hypermethylated transcripts in the mutant involved in plant development. The discovery and characterization of the archetypical RNA demethylase in Arabidopsis sheds light on the occurrence and functional role(s) of reversible mRNA methylation in plants and defines the role of mA RNA modification in Arabidopsis floral transition.
Topics: Adenosine; Arabidopsis; Arabidopsis Proteins; Demethylation; Flowers; Gene Expression Regulation, Plant; Genes, Plant; Methylation; Mutation; Oxidoreductases, N-Demethylating; Protein Stability; RNA, Messenger; RNA, Plant; RNA-Binding Proteins; Substrate Specificity; Up-Regulation
PubMed: 29180595
DOI: 10.1105/tpc.16.00912 -
Molecular Therapy : the Journal of the... Jul 2022Cancer cells respond to various stressful conditions through the dynamic regulation of RNA m6A modification. Doxorubicin is a widely used chemotherapeutic drug that...
Cancer cells respond to various stressful conditions through the dynamic regulation of RNA m6A modification. Doxorubicin is a widely used chemotherapeutic drug that induces DNA damage. It is interesting to know whether cancer cells regulate the DNA damage response and doxorubicin sensitivity through RNA m6A modification. Here, we found that doxorubicin treatment significantly induced RNA m6A methylation in breast cancer cells in both a dose- and a time-dependent manner. However, protein arginine methyltransferase 5 (PRMT5) inhibited RNA m6A modification under doxorubicin treatment by enhancing the nuclear translocation of the RNA demethylase AlkB homolog 5 (ALKBH5), which was previously believed to be exclusively localized in the nucleus. Then, ALKBH5 removed the m6A methylation of BRCA1 for mRNA stabilization and further enhanced DNA repair competency to decrease doxorubicin efficacy in breast cancer cells. Importantly, we identified the approved drug tadalafil as a novel PRMT5 inhibitor that could decrease RNA m6A methylation and increase doxorubicin sensitivity in breast cancer. The strategy of targeting PRMT5 with tadalafil is a promising approach to promote breast cancer sensitivity to doxorubicin through RNA methylation regulation.
Topics: Breast Neoplasms; Demethylation; Doxorubicin; Female; Humans; Protein-Arginine N-Methyltransferases; RNA; Tadalafil
PubMed: 35278676
DOI: 10.1016/j.ymthe.2022.03.003