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Proceedings of the National Academy of... Mar 2022RNA modifications regulate a variety of cellular processes including DNA repair.The RNA methyltransferase TRDMT1 generates methyl-5-cytosine (m5C) on messen-ger RNA...
RNA modifications regulate a variety of cellular processes including DNA repair.The RNA methyltransferase TRDMT1 generates methyl-5-cytosine (m5C) on messen-ger RNA (mRNA) at DNA double-strand breaks (DSBs) in transcribed regions, pro-moting transcription-coupled homologous recombination (HR). Here, we identifiedthat Fragile X mental retardation protein (FMRP) promotes transcription-coupled HRvia its interaction with both the m5C writer TRDMT1 and the m5C eraser ten-eleventranslocation protein 1 (TET1). TRDMT1, FMRP, and TET1 function in a temporalorder at the transcriptionally active sites of DSBs. FMRP displays a higher affinity forDNA:RNA hybrids containing m5C-modified RNA than for hybrids without modifica-tion and facilitates demethylation of m5C by TET1 in vitro. Loss of either the chroma-tin- or RNA-binding domain of FMRP compromises demethylation of damage-inducedm5C in cells. Importantly, FMRP is required for R-loop resolving in cells. Due to unre-solved R-loop and m5C preventing completion of DSB repair, FMRP depletion or lowexpression leads to delayed repair of DSBs at transcriptionally active sites and sensitizescancer cells to radiation in a BRCA-independent manner. Together, ourfindings presentan m5C reader, FMRP, which acts as a coordinator between the m5C writer and eraserto promote mRNA-dependent repair and cell survival in cancer.
Topics: Cytosine; Demethylation; Fragile X Mental Retardation Protein; Fragile X Syndrome; Homologous Recombination; Humans; Mixed Function Oxygenases; Proto-Oncogene Proteins; RNA; RNA, Messenger
PubMed: 35290126
DOI: 10.1073/pnas.2116251119 -
Journal of Experimental & Clinical... Sep 2021Chemotherapy resistance remains a barrier to improving the prognosis of epithelial ovarian cancer (EOC). ALKBH5 has recently been shown to be one of the RNA...
BACKGROUND
Chemotherapy resistance remains a barrier to improving the prognosis of epithelial ovarian cancer (EOC). ALKBH5 has recently been shown to be one of the RNA N6-methyladenosine (m6A) demethyltransferases associated with various cancers, but its role in cancer therapeutic resistance remains unclear. This study aimed to investigate the role of AlkB homolog 5 (ALKBH5) in cisplatin-resistant EOC.
METHODS
Functional assays were performed both in vitro and in vivo. RNA sequencing (RNA-seq), m6A-modified RNA immunoprecipitation sequencing (MeRIP-seq), chromatin immunoprecipitation, RNA immunoprecipitation, and luciferase reporter and actinomycin-D assays were performed to investigate RNA/RNA interaction and m6A modification of the ALKBH5-HOXA10 loop.
RESULTS
ALKBH5 was upregulated in cisplatin-resistant EOC and promoted cancer cell cisplatin resistance both in vivo and in vitro. Notably, HOXA10 formed a loop with ALKBH5 and was found to be the upstream transcription factor of ALKBH5. HOXA10 overexpression also facilitated EOC cell chemoresistance both in vivo and in vitro. Collective results of MeRIP-seq and RNA-seq showed that JAK2 is the m6A-modified gene targeted by ALKBH5. The JAK2/STAT3 signaling pathway was activated by overexpression of the ALKBH5-HOXA10 loop, resulting in EOC chemoresistance. Cell sensitivity to cisplatin was rescued by ALKBH5 and HOXA10 knockdown or inhibition of the JAK2/STAT3 signaling pathway in EOC cells overexpressing ALKBH5-HOXA10.
CONCLUSIONS
The ALKBH5-HOXA10 loop jointly activates the JAK2/STAT3 signaling pathway by mediating JAK2 m6A demethylation, promoting EOC resistance to cisplatin. Thus, inhibition of the expression of the ALKBH5-HOXA10 loop may be a potential strategy to overcome cisplatin resistance in EOC.
Topics: Animals; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Demethylation; Drug Resistance, Neoplasm; Female; Homeobox A10 Proteins; Humans; Janus Kinase 2; Mice; Mice, Nude; Transfection
PubMed: 34496932
DOI: 10.1186/s13046-021-02088-1 -
Nature Communications Jun 2023N6-methyladenosine (mA) modification plays important roles in bioprocesses and diseases. AlkB homolog 5 (ALKBH5) is one of two mA demethylases. Here, we reveal that...
N6-methyladenosine (mA) modification plays important roles in bioprocesses and diseases. AlkB homolog 5 (ALKBH5) is one of two mA demethylases. Here, we reveal that ALKBH5 is acetylated at lysine 235 (K235) by lysine acetyltransferase 8 and deacetylated by histone deacetylase 7. K235 acetylation strengthens the mA demethylation activity of ALKBH5 by increasing its recognition of mA on mRNA. RNA-binding protein paraspeckle component 1 (PSCP1) is a regulatory subunit of ALKBH5 and preferentially interacts with K235-acetylated ALKBH5 to recruit and facilitate the recognition of mA mRNA by ALKBH5, thereby promoting mA erasure. Mitogenic signals promote ALKBH5 K235 acetylation. K235 acetylation of ALKBH5 is upregulated in cancers and promotes tumorigenesis. Thus, our findings reveal that the mA demethylation activity of ALKBH5 is orchestrated by its K235 acetylation and regulatory subunit PSPC1 and that K235 acetylation is necessary for the mA demethylase activity and oncogenic roles of ALKBH5.
Topics: Humans; Acetylation; RNA, Messenger; Carcinogenesis; Cell Transformation, Neoplastic; AlkB Homolog 5, RNA Demethylase; Demethylation; RNA-Binding Proteins
PubMed: 37369679
DOI: 10.1038/s41467-023-39414-4 -
American Journal of Human Genetics Feb 2020Germline pathogenic variants in chromatin-modifying enzymes are a common cause of pediatric developmental disorders. These enzymes catalyze reactions that regulate...
Germline pathogenic variants in chromatin-modifying enzymes are a common cause of pediatric developmental disorders. These enzymes catalyze reactions that regulate epigenetic inheritance via histone post-translational modifications and DNA methylation. Cytosine methylation (5-methylcytosine [5mC]) of DNA is the quintessential epigenetic mark, yet no human Mendelian disorder of DNA demethylation has yet been delineated. Here, we describe in detail a Mendelian disorder caused by the disruption of DNA demethylation. TET3 is a methylcytosine dioxygenase that initiates DNA demethylation during early zygote formation, embryogenesis, and neuronal differentiation and is intolerant to haploinsufficiency in mice and humans. We identify and characterize 11 cases of human TET3 deficiency in eight families with the common phenotypic features of intellectual disability and/or global developmental delay; hypotonia; autistic traits; movement disorders; growth abnormalities; and facial dysmorphism. Mono-allelic frameshift and nonsense variants in TET3 occur throughout the coding region. Mono-allelic and bi-allelic missense variants localize to conserved residues; all but one such variant occur within the catalytic domain, and most display hypomorphic function in an assay of catalytic activity. TET3 deficiency and other Mendelian disorders of the epigenetic machinery show substantial phenotypic overlap, including features of intellectual disability and abnormal growth, underscoring shared disease mechanisms.
Topics: Adult; Amino Acid Sequence; Autistic Disorder; Child; Child, Preschool; DNA Demethylation; Developmental Disabilities; Dioxygenases; Embryonic Development; Female; Gene Expression Regulation, Developmental; Growth Disorders; Humans; Infant; Male; Middle Aged; Movement Disorders; Pedigree; Protein Conformation; Sequence Homology; Young Adult
PubMed: 31928709
DOI: 10.1016/j.ajhg.2019.12.007 -
Theranostics 2023Lymph node (LN) metastasis is common in patients with epithelial ovarian cancer (EOC) and is associated with poor prognosis. Tumor-associated lymphangiogenesis is the...
Lymph node (LN) metastasis is common in patients with epithelial ovarian cancer (EOC) and is associated with poor prognosis. Tumor-associated lymphangiogenesis is the first stage of LN metastasis. Research on lymphangiogenesis and lymph node metastases can help develop new anti-LN-targeted therapies. Aberrant N6-methyladenosine (m6A) modifications have been reported to be linked to LN metastasis in several cancers, however, their role in EOC lymphangiogenesis and LN metastasis remains unclear. m6A levels in EOC tissues with or without LN metastases were evaluated by dot blot analysis. Real-time polymerase chain reaction (PCR) and immunofluorescence were used to examine the expression of m6A-related enzymes. Additionally, and functional studies were performed to discover the importance of the AlkB homolog 5 () gene in EOC lymphatic metastasis. To identify the downstream target genes regulated by ALKBH5, we performed RNA pulldown, RNA-binding protein immunoprecipitation-quantitative PCR, co-immunoprecipitation, m6A-modified RNA immunoprecipitation-quantitative PCR, and luciferase reporter assays. m6A modification was reduced in ovarian cancers with LN metastases. ALKBH5 overexpression increased tumor-associated lymphangiogenesis and LN metastasis both and . ALKBH5 overexpression also reversed the m6A modification in mRNA and suppressed the YTHDF2 protein-mediated m6A-dependent mRNA degradation, which resulted in increased expression of ITGB1 and phosphorylation of the focal adhesion kinase (FAK) and Src proto-oncogene proteins, thereby increasing LN metastasis. Furthermore, hypoxia induced the expression of hypoxia inducible factor 1 subunit alpha, which increased ALKBH5 expression and enhanced LN metastasis in EOC. The ALKBH5/m6A-ITGB1/FAK signalling axis is important in ovarian cancer lymphangiogenesis and LN metastasis. Antibodies that block ITGB1 and FAK kinase-inhibitors are promising anti-metastatic agents.
Topics: Female; Humans; AlkB Homolog 5, RNA Demethylase; Carcinoma, Ovarian Epithelial; Demethylation; Focal Adhesion Protein-Tyrosine Kinases; Lymphangiogenesis; Lymphatic Metastasis; Ovarian Neoplasms; RNA, Messenger
PubMed: 36632222
DOI: 10.7150/thno.77441 -
European Heart Journal May 2023Epicardium and epicardium-derived cells are critical players in myocardial fibrosis. Mesenchymal stem cell-derived extracellular vesicles (EVs) have been studied for...
AIMS
Epicardium and epicardium-derived cells are critical players in myocardial fibrosis. Mesenchymal stem cell-derived extracellular vesicles (EVs) have been studied for cardiac repair to improve cardiac remodelling, but the actual mechanisms remain elusive. The aim of this study is to investigate the mechanisms of EV therapy for improving cardiac remodelling and develop a promising treatment addressing myocardial fibrosis.
METHODS AND RESULTS
Extracellular vesicles were intrapericardially injected for mice myocardial infarction treatment. RNA-seq, in vitro gain- and loss-of-function experiments, and in vivo studies were performed to identify targets that can be used for myocardial fibrosis treatment. Afterward, a lipid nanoparticle-based long non-coding RNA (lncRNA) therapy was prepared for mouse and porcine models of myocardial infarction treatment. Intrapericardial injection of EVs improved adverse myocardial remodelling in mouse models of myocardial infarction. Mechanistically, Tcf21 was identified as a potential target to improve cardiac remodelling. Loss of Tcf21 function in epicardium-derived cells caused increased myofibroblast differentiation, whereas forced Tcf21 overexpression suppressed transforming growth factor-β signalling and myofibroblast differentiation. LncRNA-Tcf21 antisense RNA inducing demethylation (TARID) that enriched in EVs was identified to up-regulate Tcf21 expression. Formulated lncRNA-TARID-laden lipid nanoparticles up-regulated Tcf21 expression in epicardium-derived cells and improved cardiac function and histology in mouse and porcine models of myocardial infarction.
CONCLUSION
This study identified Tcf21 as a critical target for improving cardiac fibrosis. Up-regulating Tcf21 by using lncRNA-TARID-laden lipid nanoparticles could be a promising way to treat myocardial fibrosis. This study established novel mechanisms underlying EV therapy for improving adverse remodelling and proposed a lncRNA therapy for cardiac fibrosis.
Topics: Mice; Animals; Swine; RNA, Long Noncoding; RNA, Antisense; Ventricular Remodeling; Myocardial Infarction; Fibrosis; Demethylation
PubMed: 36916305
DOI: 10.1093/eurheartj/ehad114 -
Cell Death & Disease Oct 2022Cellular senescence is characterized by a tumor-suppressive program as well as a pro-inflammatory secretome. Neutrophils constitute significant compositions of...
Cellular senescence is characterized by a tumor-suppressive program as well as a pro-inflammatory secretome. Neutrophils constitute significant compositions of malignancies and play key roles in tumor development. However, the role of senescent neutrophils in cancer progression is presently unexplored. Here, we demonstrate that neutrophils display enhanced senescence in breast cancer patients receiving chemotherapy. The senescent neutrophils produce increased number of exosomes, which confer drug resistance to tumor cells in vitro and in vivo. Mechanistically, senescent neutrophils-derived exosomal piRNA-17560 enhances the expression of fat mass and obesity-associated protein (FTO) in breast cancer cells. The upregulation of FTO further strengthens ZEB1 transcripts stability and expression by decreasing N6-methyladenosine (m6A) RNA methylation, leading to chemoresistance and epithelial-mesenchymal transition (EMT) of tumor cells. Clinically, the level of exosomal piR-17560 correlates with poor chemotherapy response in patients with breast cancer. In addition, YTHDF2 is essential for the posttranscriptional regulation of ZEB1 by piRNA-17560/FTO signaling. Senescent neutrophils secret exosomal piR-17560 in a STAT3-dependent manner. Altogether, this study suggests that senescent neutrophils-derived exosomal piR-17560 confers chemoresistance to tumor cells and senescent neutrophils may serve as a potential therapeutic target in breast cancer.
Topics: Humans; Female; Epithelial-Mesenchymal Transition; Breast Neoplasms; RNA, Small Interfering; Drug Resistance, Neoplasm; Neutrophils; Demethylation; Alpha-Ketoglutarate-Dependent Dioxygenase FTO
PubMed: 36302751
DOI: 10.1038/s41419-022-05317-3 -
Experimental & Molecular Medicine Apr 2017
Topics: Animals; Chromatin; Cytosine; DNA Demethylation; DNA Methylation; Epigenesis, Genetic; Genetic Markers; Histones; Humans; Neoplasms
PubMed: 28450735
DOI: 10.1038/emm.2017.38 -
Nucleic Acids Research Jun 2021Faithful genome integrity maintenance plays an essential role in cell survival. Here, we identify the RNA demethylase ALKBH5 as a key regulator that protects cells from...
Faithful genome integrity maintenance plays an essential role in cell survival. Here, we identify the RNA demethylase ALKBH5 as a key regulator that protects cells from DNA damage and apoptosis during reactive oxygen species (ROS)-induced stress. We find that ROS significantly induces global mRNA N6-methyladenosine (m6A) levels by modulating ALKBH5 post-translational modifications (PTMs), leading to the rapid and efficient induction of thousands of genes involved in a variety of biological processes including DNA damage repair. Mechanistically, ROS promotes ALKBH5 SUMOylation through activating ERK/JNK signaling, leading to inhibition of ALKBH5 m6A demethylase activity by blocking substrate accessibility. Moreover, ERK/JNK/ALKBH5-PTMs/m6A axis is activated by ROS in hematopoietic stem/progenitor cells (HSPCs) in vivo in mice, suggesting a physiological role of this molecular pathway in the maintenance of genome stability in HSPCs. Together, our study uncovers a molecular mechanism involving ALKBH5 PTMs and increased mRNA m6A levels that protect genomic integrity of cells in response to ROS.
Topics: AlkB Homolog 5, RNA Demethylase; Animals; Apoptosis; Bone Marrow Cells; Cell Line, Tumor; DNA Damage; DNA Repair; Demethylation; Fanconi Anemia Complementation Group Proteins; Gene Expression Regulation; Hematopoietic Stem Cells; Humans; Hydrogen Peroxide; MAP Kinase Signaling System; Methylation; Mice; Phosphorylation; Protein Processing, Post-Translational; RNA, Small Interfering; RNA-Binding Proteins; RNA-Seq; Reactive Oxygen Species; Sumoylation; Tandem Mass Spectrometry; X-linked Nuclear Protein
PubMed: 34048572
DOI: 10.1093/nar/gkab415 -
International Journal of Molecular... Sep 2019Methylation of cytosine (5-meC) is a critical epigenetic modification in many eukaryotes, and genomic DNA methylation landscapes are dynamically regulated by opposed... (Review)
Review
Methylation of cytosine (5-meC) is a critical epigenetic modification in many eukaryotes, and genomic DNA methylation landscapes are dynamically regulated by opposed methylation and demethylation processes. Plants are unique in possessing a mechanism for active DNA demethylation involving DNA glycosylases that excise 5-meC and initiate its replacement with unmodified C through a base excision repair (BER) pathway. Plant BER-mediated DNA demethylation is a complex process involving numerous proteins, as well as additional regulatory factors that avoid accumulation of potentially harmful intermediates and coordinate demethylation and methylation to maintain balanced yet flexible DNA methylation patterns. Active DNA demethylation counteracts excessive methylation at transposable elements (TEs), mainly in euchromatic regions, and one of its major functions is to avoid methylation spreading to nearby genes. It is also involved in transcriptional activation of TEs and TE-derived sequences in companion cells of male and female gametophytes, which reinforces transposon silencing in gametes and also contributes to gene imprinting in the endosperm. Plant 5-meC DNA glycosylases are additionally involved in many other physiological processes, including seed development and germination, fruit ripening, and plant responses to a variety of biotic and abiotic environmental stimuli.
Topics: 5-Methylcytosine; DNA Demethylation; DNA Glycosylases; DNA Methylation; DNA, Plant; Endosperm; Gene Expression Regulation, Plant; Genomic Instability; Ovule; Plants; Pollen; Stress, Physiological
PubMed: 31546611
DOI: 10.3390/ijms20194683