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Nature Communications Mar 2021Cancer stem cells (CSCs) are a small but critical cell population for cancer biology since they display inherent resistance to standard therapies and give rise to...
Cancer stem cells (CSCs) are a small but critical cell population for cancer biology since they display inherent resistance to standard therapies and give rise to metastases. Despite accruing evidence establishing a link between deregulation of epitranscriptome-related players and tumorigenic process, the role of messenger RNA (mRNA) modifications in the regulation of CSC properties remains poorly understood. Here, we show that the cytoplasmic pool of fat mass and obesity-associated protein (FTO) impedes CSC abilities in colorectal cancer through its N,2'-O-dimethyladenosine (mA) demethylase activity. While mA is strategically located next to the mG-mRNA cap, its biological function is not well understood and has not been addressed in cancer. Low FTO expression in patient-derived cell lines elevates mA level in mRNA which results in enhanced in vivo tumorigenicity and chemoresistance. Inhibition of the nuclear mA methyltransferase, PCIF1/CAPAM, fully reverses this phenotype, stressing the role of mA modification in stem-like properties acquisition. FTO-mediated regulation of mA marking constitutes a reversible pathway controlling CSC abilities. Altogether, our findings bring to light the first biological function of the mA modification and its potential adverse consequences for colorectal cancer management.
Topics: Adaptor Proteins, Signal Transducing; Adenosine; Alpha-Ketoglutarate-Dependent Dioxygenase FTO; Cell Line, Tumor; Cell Nucleus; Colorectal Neoplasms; Cytoplasm; Demethylation; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Methyltransferases; Nuclear Proteins; RNA, Messenger
PubMed: 33741917
DOI: 10.1038/s41467-021-21758-4 -
The Plant Cell Dec 2017
Topics: Adenosine; Arabidopsis; Demethylation; Methylation; RNA; RNA, Messenger
PubMed: 29203635
DOI: 10.1105/tpc.17.00929 -
Clinical and Translational Medicine Sep 2023Cysteine dioxygenase 1 (CDO1) is frequently methylated, and its expression is decreased in many human cancers including breast cancer (BC). However, the functional and...
BACKGROUND
Cysteine dioxygenase 1 (CDO1) is frequently methylated, and its expression is decreased in many human cancers including breast cancer (BC). However, the functional and mechanistic aspects of CDO1 inactivation in BC are poorly understood, and the diagnostic significance of serum CDO1 methylation remains unclear.
METHODS
We performed bioinformatics analysis of publicly available databases and employed MassARRAY EpiTYPER methylation sequencing technology to identify differentially methylated sites in the CDO1 promoter of BC tissues compared to normal adjacent tissues (NATs). Subsequently, we developed a MethyLight assay using specific primers and probes for these CpG sites to detect the percentage of methylated reference (PMR) of the CDO1 promoter. Furthermore, both LentiCRISPR/dCas9-Tet1CD-based CDO1-targeted demethylation system and CDO1 overexpression strategy were utilized to detect the function and underlying mechanism of CDO1 in BC. Finally, the early diagnostic value of CDO1 as a methylation biomarker in BC serum was evaluated.
RESULTS
CDO1 promoter was hypermethylated in BC tissues, which was related to poor prognosis (p < .05). The CRISPR/dCas9-based targeted demethylation system significantly reduced the PMR of CDO1 promotor and increased CDO1 expression in BC cells. Consequently, this leads to suppression of cell proliferation, migration and invasion. Additionally, we found that CDO1 exerted a tumour suppressor effect by inhibiting the cell cycle, promoting cell apoptosis and ferroptosis. Furthermore, we employed the MethyLight to detect CDO1 PMR in BC serum, and we discovered that serum CDO1 methylation was an effective non-invasive biomarker for early diagnosis of BC.
CONCLUSIONS
CDO1 is hypermethylated and acts as a tumour suppressor gene in BC. Epigenetic editing of abnormal CDO1 methylation could have a crucial role in the clinical treatment and prognosis of BC. Additionally, serum CDO1 methylation holds promise as a valuable biomarker for the early diagnosis and management of BC.
Topics: Humans; Clustered Regularly Interspaced Short Palindromic Repeats; Cysteine Dioxygenase; Apoptosis; Cell Cycle; Demethylation; Neoplasms
PubMed: 37740473
DOI: 10.1002/ctm2.1423 -
Clinical and Translational Medicine Feb 2023Radiation-induced hepatic stellate cell (HSC) activation promotes radiation-induced liver fibrosis (RILF), a complication for hepatocellular carcinoma (HCC)...
BACKGROUND
Radiation-induced hepatic stellate cell (HSC) activation promotes radiation-induced liver fibrosis (RILF), a complication for hepatocellular carcinoma (HCC) radiotherapy. The demethylase alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) decreases N6-methyladenylate methylation (m A) modification of RNA, while its role in regulating RILF pathogenesis and HCC radiosensitivity remains unknown.
METHODS
Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-sequencing (RNA-seq) were used to screen target genes regulated by ALKBH5. HSC with altered ALKBH5 expression was used to assess irradiation-induced HSC activation and the effect of HSC on recruitment and polarisation of monocytes. Key cytokines in medium from irradiated HSC-educated monocytes were identified by cytokine array detection. The effects of blocking ALKBH5 and key cytokines on RILF and HCC radiosensitivity were also evaluated.
RESULTS
Radiation-induced ALKBH5 expression in HSC mediated m A demethylation of toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) mRNA and activated its downstream NF-κB and JNK/Smad2 pathways to promote HSC activation. Additionally, ALKBH5 regulated CCL5 secretion by irradiated HSC to promote monocyte recruitment and M2 macrophage polarisation. Notably, polarised monocytes secreted CCL20 to up-regulate ALKBH5 expression in HSC, and reduce HCC radiosensitivity by activating ALKBH5/TIRAP axis in HCC cells. ALKBH5 knockdown-combined CCR6 (CCL20 receptor) inhibitor significantly alleviated RILF and improved HCC radiosensitivity in mice. HCC patients with high ALKBH5 and TIRAP expression were prone to radiation-induced liver injury and poor tumour response to radiotherapy.
CONCLUSIONS
Collectively, irradiation up-regulates ALKBH5 in HSC to mediate monocyte recruitment and M2 polarisation and form positive feedback to promote RILF and reduce HCC radiosensitivity. The dual roles of ALKBH5 as a microenvironmental regulator and radiosensitisation target provide new ideas for RILF prevention and radiosensitisation of HCC.
Topics: Animals; Mice; Carcinoma, Hepatocellular; Demethylation; Liver Cirrhosis; Liver Neoplasms; Membrane Glycoproteins; Receptors, Interleukin-1; RNA; RNA, Messenger
PubMed: 36792369
DOI: 10.1002/ctm2.1198 -
Communications Biology Dec 2023N-methyladenosine (mA) plays a crucial role in the development and functional homeostasis of the central nervous system. The fat mass and obesity-associated (FTO) gene,...
N-methyladenosine (mA) plays a crucial role in the development and functional homeostasis of the central nervous system. The fat mass and obesity-associated (FTO) gene, which is highly expressed in the hypothalamus, is closely related to female pubertal development. In this study, we found that mA methylation decreased in the hypothalamus gradually with puberty and decreased in female rats with precocious puberty. FTO expression was increased at the same time. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) showed that the mA methylation of PLCβ, a key enzyme of the Ca signalling pathway, was decreased significantly in the hypothalamus in precocious rats. Upregulating FTO increased PLCβ3 expression and activated the Ca signalling pathway, which promoted GnRH expression. Dual-luciferase reporter and MeRIP-qPCR assays confirmed that FTO regulated mA demethylation of PLCβ and promoted PLCβ expression. Upon overexpressing FTO in the hypothalamic arcuate nucleus (ARC) in female rats, we observed advanced puberty onset. Meanwhile, PLCβ and GnRH expression in the hypothalamus increased significantly, and the Ca signalling pathway was activated. Our study demonstrates that FTO enhances GnRH expression, which promotes puberty onset, by regulating mA demethylation of PLCβ3 and activating the Ca signalling pathway.
Topics: Animals; Female; Rats; Demethylation; Gonadotropin-Releasing Hormone; Hypothalamus; Methylation; Signal Transduction
PubMed: 38129517
DOI: 10.1038/s42003-023-05677-2 -
Epigenetics & Chromatin Oct 2023Vitamin C (vitC) enhances the activity of 2-oxoglutarate-dependent dioxygenases, including TET enzymes, which catalyse DNA demethylation, and Jumonji-domain histone...
BACKGROUND
Vitamin C (vitC) enhances the activity of 2-oxoglutarate-dependent dioxygenases, including TET enzymes, which catalyse DNA demethylation, and Jumonji-domain histone demethylases. The epigenetic remodelling promoted by vitC improves the efficiency of induced pluripotent stem cell derivation, and is required to attain a ground-state of pluripotency in embryonic stem cells (ESCs) that closely mimics the inner cell mass of the early blastocyst. However, genome-wide DNA and histone demethylation can lead to upregulation of transposable elements (TEs), and it is not known how vitC addition in culture media affects TE expression in pluripotent stem cells.
RESULTS
Here we show that vitC increases the expression of several TE families, including evolutionarily young LINE-1 (L1) elements, in mouse ESCs. We find that TET activity is dispensable for L1 upregulation, and that instead it occurs largely as a result of H3K9me3 loss mediated by KDM4A/C histone demethylases. Despite increased L1 levels, we did not detect increased somatic insertion rates in vitC-treated cells. Notably, treatment of human ESCs with vitC also increases L1 protein levels, albeit through a distinct, post-transcriptional mechanism.
CONCLUSION
VitC directly modulates the expression of mouse L1s and other TEs through epigenetic mechanisms, with potential for downstream effects related to the multiple emerging roles of L1s in cellular function.
Topics: Humans; Animals; Mice; Ascorbic Acid; Mouse Embryonic Stem Cells; Long Interspersed Nucleotide Elements; DNA Methylation; Histone Demethylases; DNA; Demethylation; Jumonji Domain-Containing Histone Demethylases
PubMed: 37845773
DOI: 10.1186/s13072-023-00514-6 -
Neuroendocrinology 2022Neurons expressing estrogen receptor (ER) ɑ in the arcuate (ARC) and ventromedial (VMH) nuclei of the hypothalamus sex-specifically control energy homeostasis, sexual...
INTRODUCTION
Neurons expressing estrogen receptor (ER) ɑ in the arcuate (ARC) and ventromedial (VMH) nuclei of the hypothalamus sex-specifically control energy homeostasis, sexual behavior, and bone density. Females have more ERɑ neurons in the VMH and ARC than males, and the sex difference in the VMH is eliminated by neonatal treatment with testosterone or a DNA methylation inhibitor.
OBJECTIVE
Here, we tested the roles of testosterone and DNA methylation/demethylation in development of ERɑ in the ARC.
METHODS
ERɑ was examined at birth and weaning in mice that received vehicle or testosterone subcutaneously, and vehicle or DNA methyltransferase inhibitor intracerebroventricularly, as neonates. To examine effects of DNA demethylation on the ERɑ cell number in the ARC, mice were treated neonatally with small interfering RNAs against ten-eleven translocase enzymes. The methylation status of the ERɑ gene (Esr1) was determined in the ARC and VMH using pyrosequencing of bisulfite-converted DNA.
RESULTS
A sex difference in ERɑ in the ARC, favoring females, developed between birth and weaning and was due to programming effects of testosterone. Neonatal inhibition of DNA methylation decreased ERɑ in the ARC of females, and an inhibition of
de methylation increased ERɑ in the ARC of males. The promoter region of Esr1 exhibited a small sex difference in percent of total methylation in the ARC (females > males) that was opposite to that in the VMH (males > females).CONCLUSION
DNA methylation and demethylation regulate ERɑ cell number in the ARC, and methylation correlates with activation of Esr1 in this region.
Topics: Animals; Arcuate Nucleus of Hypothalamus; DNA Methylation; Demethylation; Estrogen Receptor alpha; Female; Male; Mice; Sex Characteristics; Testosterone
PubMed: 34547753
DOI: 10.1159/000519671 -
JCI Insight Oct 2023Abnormal macrophage polarization is generally present in autoimmune diseases. Overwhelming M1 macrophage activation promotes the continuous progression of inflammation,...
Abnormal macrophage polarization is generally present in autoimmune diseases. Overwhelming M1 macrophage activation promotes the continuous progression of inflammation, which is one of the reasons for the development of autoimmune diseases. However, the underlying mechanism is still unclear. Here we explore the function of Regulatory factor X1 (RFX1) in macrophage polarization by constructing colitis and lupus-like mouse models. Both in vivo and in vitro experiments confirmed that RFX1 can promote M1 and inhibit M2 macrophage polarization. Furthermore, we found that RFX1 promoted DNA demethylation of macrophage polarization-related genes by increasing APOBEC3A/Apobec3 expression. We identified a potential RFX1 inhibitor, adenosine diphosphate (ADP), providing a potential strategy for treating autoimmune diseases.
Topics: Animals; Mice; Autoimmune Diseases; DNA Demethylation; Inflammation; Macrophage Activation; Macrophages; Regulatory Factor X1
PubMed: 37733446
DOI: 10.1172/jci.insight.165546 -
Clinical Epigenetics Aug 2023Promoter hypermethylation of tumour suppressor genes is frequently observed during the malignant transformation of colorectal cancer (CRC). However, whether this...
BACKGROUND
Promoter hypermethylation of tumour suppressor genes is frequently observed during the malignant transformation of colorectal cancer (CRC). However, whether this epigenetic mechanism is functional in cancer or is a mere consequence of the carcinogenic process remains to be elucidated.
RESULTS
In this work, we performed an integrative multi-omic approach to identify gene candidates with strong correlations between DNA methylation and gene expression in human CRC samples and a set of 8 colon cancer cell lines. As a proof of concept, we combined recent CRISPR-Cas9 epigenome editing tools (dCas9-TET1, dCas9-TET-IM) with a customized arrayed gRNA library to modulate the DNA methylation status of 56 promoters previously linked with strong epigenetic repression in CRC, and we monitored the potential functional consequences of this DNA methylation loss by means of a high-content cell proliferation screen. Overall, the epigenetic modulation of most of these DNA methylated regions had a mild impact on the reactivation of gene expression and on the viability of cancer cells. Interestingly, we found that epigenetic reactivation of RSPO2 in the tumour context was associated with a significant impairment in cell proliferation in p53 cancer cell lines, and further validation with human samples demonstrated that the epigenetic silencing of RSPO2 is a mid-late event in the adenoma to carcinoma sequence.
CONCLUSIONS
These results highlight the potential role of DNA methylation as a driver mechanism of CRC and paves the way for the identification of novel therapeutic windows based on the epigenetic reactivation of certain tumour suppressor genes.
Topics: Humans; DNA Methylation; DNA Demethylation; Epigenesis, Genetic; Carcinogenesis; Colonic Neoplasms; Mixed Function Oxygenases; Proto-Oncogene Proteins
PubMed: 37612734
DOI: 10.1186/s13148-023-01546-1 -
Toxicological Sciences : An Official... Nov 2022Methylmercury (MeHg) persists today as a priority public health concern. Mechanisms influencing MeHg metabolism, kinetics, and toxicity outcomes are therefore essential...
Methylmercury (MeHg) persists today as a priority public health concern. Mechanisms influencing MeHg metabolism, kinetics, and toxicity outcomes are therefore essential knowledge for informing exposure risks. Evidence points to different toxic potencies of MeHg and inorganic mercury (Hg2+), highlighting the role for biotransformation (demethylation) in regulating MeHg toxicokinetics/dynamics. Whereas microbial MeHg demethylation in the gut is seen to influence elimination kinetics, the potential for systemic demethylation in tissues and target organs to influence MeHg toxicity remains uncertain. To investigate the consequences of systemic MeHg demethylation across development, we engineered transgenic Drosophila to express the bacterial organomercurial lyase enzyme (merB) in a targeted and tissue-specific manner. With all combinations of merB-induced demethylation, ubiquitously (via an actin promoter) or in a tissue-specific manner (ie, gut, muscle, neurons), we observe a rescue of MeHg-induced eclosion failure at the pupal to adult transition. In MeHg-fed larvae with ubiquitous or targeted (gut and muscle) merB expression, we see a significant decrease in MeHg body burden at the pupal stage relative to control flies. We also observe a significant increase in the MeHg elimination rate with merB demethylation induced in adults (control, t1/2 = 7.2 days; merB flies, t1/2 = 3.1 days). With neuronal-specific merB expression, we observe a rescue of MeHg-induced eclosion failure without a decrease in Hg body burden, but a redistribution of Hg away from the brain. These results demonstrate the previously unidentified potential for intracellular MeHg demethylation to promote transport and elimination of Hg, and reduce developmental MeHg toxicity. Impact Statement: These findings demonstrate the potential for MeHg demethylation in situ to contribute significantly to the MeHg elimination and distribution kinetics of whole animals and thereby affords a means of protection against the toxic insult of MeHg. Therefore, this study reveals important insight into processes that can determine an individual's resistance or susceptibility to MeHg and provides rationale for therapies targeting a novel metabolism-based pathways to alleviate toxicity risk stemming from MeHg exposure.
Topics: Animals; Methylmercury Compounds; Kinetics; Drosophila; Mercury; Animals, Genetically Modified; Demethylation
PubMed: 36200918
DOI: 10.1093/toxsci/kfac105