Knockout Of Mettl3 Research Articles

Alternative splicing of METTL3 explains apparently METTL3-independent m6A modifications in mRNA.

N6-methyladenosine (m6A) is a highly prevalent mRNA modification that promotes degradation of transcripts encoding proteins that have roles in cell development, differentiation, and other pathways. METTL3 is the major methyltransferase that catalyzes the formation of m6A in mRNA. As 30% to 80% of m6A can remain in mRNA after METTL3 depletion by CRISPR/Cas9-based methods, other enzymes are thought to catalyze a sizable fraction of m6A. Here, we reexamined the source of m6A in the mRNA transcriptome. We characterized mouse embryonic stem cell lines that continue to have m6A in their mRNA after Mettl3 knockout. We show that these cells express alternatively spliced Mettl3 transcript isoforms that bypass the CRISPR/Cas9 mutations and produce functionally active methyltransferases. We similarly show that other reported METTL3 knockout cell lines express altered METTL3 proteins. We find that gene dependency datasets show that most cell lines fail to proliferate after METTL3 deletion, suggesting that reported METTL3 knockout cell lines express altered METTL3 proteins rather than have full knockout. Finally, we reassessed METTL3’s role in synthesizing m6A using an exon 4 deletion of Mettl3 and found that METTL3 is responsible for >95% of m6A in mRNA. Overall, these studies suggest that METTL3 is responsible for the vast majority of m6A in the transcriptome, and that remaining m6A in putative METTL3 knockout cell lines is due to the expression of altered but functional METTL3 isoforms.

Deletion of Mettl3 at the Pro-B Stage Marginally Affects B Cell Development and Profibrogenic Activity of B Cells in Liver Fibrosis.

N6-methyladenosine (m6A) modification plays a pivotal role in cell fate determination. Previous studies show that eliminating m6A using Mb1-Cre dramatically impairs B cell development. However, whether disturbing m6A modification at later stages affects B cell development and function remains elusive. Here, we deleted m6A methyltransferase Mettl3 from the pro-B stage on using Cd19-Cre (Mettl3 cKO) and found that the frequency of total B cells in peripheral blood, peritoneal cavity, and liver is comparable between Mettl3 cKO mice and wild-type (WT) littermates, while the percentage of whole splenic B cells slightly increases in Mettl3 cKO individuals. The proportion of pre-pro-B, pro-B, pre-B, immature, and mature B cells in the bone marrow were minimally affected. Loss of Mettl3 resulted in increased apoptosis but barely affected B cells' proliferation and IgG production upon LPS, CD40L, anti-IgM, or TNF-α stimulation. Different stimuli had different effects on B cell activation. In addition, B cell-specific Mettl3 knockout had no influence on the pro-fibrogenic activity of B cells in liver fibrosis, evidenced by comparable fibrosis in carbon tetrachloride- (CCl4-) treated Mettl3 cKO mice and WT controls. In summary, our study demonstrated that deletion of Mettl3 from the pro-B stage on has minimal effects on B cell development and function, as well as profibrogenic activity of B cells in liver fibrosis, revealing a stage-specific dependence on Mettl3-mediated m6A of B cell development.

METTL3 Inhibits Antitumor Immunity by Targeting m6A-BHLHE41-CXCL1/CXCR2 Axis to Promote Colorectal Cancer

N6-Methyladenosine (m6A) is the most prevalent RNA modification and recognized as an important epitranscriptomic mechanism in colorectal cancer (CRC). We aimed to exploit whether and how tumor-intrinsic m6A modification driven by methyltransferase like 3 (METTL3) can dictate the immune landscape of CRC. Mettl3 knockout mice, CD34+ humanized mice, and different syngeneic mice models were used. Immune cell composition and cytokine level were analyzed by flow cytometry and Cytokine 23-Plex immunoassay, respectively. M6A sequencing and RNA sequencing were performed to identify downstream targets and pathways of METTL3. Human CRC specimens (n= 176) were used to evaluate correlation between METTL3 expression and myeloid-derived suppressor cell (MDSC) infiltration. We demonstrated that silencing of METTL3 in CRC cells reduced MDSC accumulation to sustain activation and proliferation of CD4+ and CD8+ T cells, and eventually suppressed CRC in ApcMin/+Mettl3+/- mice, CD34+ humanized mice, and syngeneic mice models. Mechanistically, METTL3 activated the m6A-BHLHE41-CXCL1 axis by analysis of m6A sequencing, RNA sequencing, and cytokine arrays. METTL3 promoted BHLHE41 expression in an m6A-dependent manner, which subsequently induced CXCL1 transcription to enhance MDSC migration invitro. However, the effect was negligible on BHLHE41 depletion, CXCL1 protein or CXCR2 inhibitor SB265610 administration, inferring that METTL3 promotes MDSC migration via BHLHE41-CXCL1/CXCR2. Consistently, depletion of MDSCs by anti-Gr1 antibody or SB265610 blocked the tumor-promoting effect of METTL3 invivo. Importantly, targeting METTL3 by METTL3-single guide RNA or specific inhibitor potentiated the effect of anti-programmed cell death protein 1 (anti-PD1) treatment. Our study identifies METTL3 as a potential therapeutic target for CRC immunotherapy whose inhibition reverses immune suppression through the m6A-BHLHE41-CXCL1 axis. METTL3 inhibition plus anti-PD1 treatment shows promising antitumor efficacy against CRC.

METTL3-mediated m6A RNA methylation regulates dorsal lingual epithelium homeostasis

The dorsal lingual epithelium, which is composed of taste buds and keratinocytes differentiated from K14+ basal cells, discriminates taste compounds and maintains the epithelial barrier. N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotic cells. How METTL3-mediated m6A modification regulates K14+ basal cell fate during dorsal lingual epithelium formation and regeneration remains unclear. Here we show knockout of Mettl3 in K14+ cells reduced the taste buds and enhanced keratinocytes. Deletion of Mettl3 led to increased basal cell proliferation and decreased cell division in taste buds. Conditional Mettl3 knock-in mice showed little impact on taste buds or keratinization, but displayed increased proliferation of cells around taste buds in a protective manner during post-irradiation recovery. Mechanically, we revealed that the most frequent m6A modifications were enriched in Hippo and Wnt signaling, and specific peaks were observed near the stop codons of Lats1 and FZD7. Our study elucidates that METTL3 is essential for taste bud formation and could promote the quantity recovery of taste bud after radiation.

METTL3-mediated m6A modification of TIMP2 mRNA promotes podocyte injury in diabetic nephropathy

Epigenetic changes are present in many physiological and pathological processes. The N6-methyladenosine (m6A) modification is the most common modification in eukaryotic mRNA. However, the role of m6A modification in diabetic nephropathy (DN) remains elusive. Here, we found that m6A modification was significantly upregulated in the kidney of type 1 and type 2 diabetic mice, which was caused by elevated levels of METTL3. Moreover, METTL3 is increased in podocyte of renal biopsy from patients with DN, which is related to renal damage. METTL3 knockout significantly reduced the inflammation and apoptosis in high glucose (HG)-stimulated podocytes, while its overexpression significantly aggravated these responses invitro. Podocyte-conditional knockout METTL3 significantly alleviated podocyte injury and albuminuria in streptozotocin (STZ)-induced diabetic mice. Therapeutically, silencing METTL3 with adeno-associated virus serotype-9 (AAV9)-shMETTL3 invivo mitigated albuminuria and histopathological injury in STZ-induced diabetic mice and db/db mice. Mechanistically, METTL3 modulated Notch signaling via the m6A modification of TIMP2 in an insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2)-dependent manner and exerted pro-inflammatory and pro-apoptotic effects. In summary, this study suggested that METTL3-mediated m6A modification is an important mechanism of podocyte injury in DN. Targeting m6A through the writer enzyme METTL3 is a potential approach for the treatment of DN.

Mettl3 deficiency leads to the upregulation of Cav1.2 and increases arrhythmia susceptibility in mice.

Methyltransferase-like 3 (Mettl3) is a component of methyltransferase complex that mediates mA modification of RNAs, and participates in multiple biological processes. However, the role of Mettl3 in cardiac electrophysiology remains unknown. This study aims to explore the ventricular arrhythmia susceptibility of Mettl3 mice and the underlying mechanisms. Mice were anesthetized with 2% avertin (0.1 mL/ body weight) for echocardiography and programmed electrical pacing. Whole-cell patch clamp technique was used to examine the electrophysiological property of cardiomyocytes. The expression of Cav1.2 was determined by qRT-PCR and western blot analysis. The mA medication of mRNA was examined by MeRIP-Seq and MeRIP-qPCR. No differences are found in the morphology and function of the hearts between Mettl3 mice and wild-type (WT) controls. The QT and QTc intervals of Mettl3 mice are significantly longer. High-frequency electrical stimulation showed that heterozygous knockout of Mettl3 increases ventricular arrhythmia susceptibility. The whole-cell patch-clamp recordings showed that the APD is prolonged in Mettl3 ventricular myocytes and more EADs were observed. The density of is substantially increased in ventricular myocytes of Mettl3 mice. The pore-forming subunit of L-type calcium channel Cav1.2 is upregulated in Mettl3 mice, while the mRNA of its coding gene does not change. MeRIP-Seq and MeRIP-qPCR showed that the mA methylation of mRNA is decreased in cultured Mettl3-knockdown cardiomyocytes and Mettl3 hearts. Collectively, deficiency of Mettl3 increases ventricular arrhythmia susceptibility due to the upregulation of Cav1.2 by reducing mA modification onmRNA in mice. This study highlights the role of mA modification in the regulation of cardiac electrophysiology.

Neutrophil extracellular traps mediate m6A modification and regulates sepsis-associated acute lung injury by activating ferroptosis in alveolar epithelial cells.

Neutrophil extracellular traps (NETs) production is a major strategy employed by polymorphonuclear neutrophils (PMNs) to fight against microbes. NETs have been implicated in the pathogenesis of various lung injuries, although few studies have explored NETs in sepsis-associated acute lung injury (SI-ALI). Here, we demonstrate a major contribution of NETs to the pathology of sepsis-associated ALI by inducing ferroptosis of alveolar epithelial cells. Using both in vitro and in vivo studies, our findings show enhanced NETs accumulation in sepsis-associated ALI patients and mice, as well as the closely related upregulation of ferroptosis, the induction of which depends on METTL3-induced m6A modification of GPX4. Using a CLP-induced sepsis-associated ALI mouse model established with METTL3-/- versus WT mice, in addition to METTL3 knockout and overexpression in vitro, we elucidated and confirmed the critical role of ferroptosis in NETs-induced ALI. These findings support a role for NETs-induced METTL3 modification and the subsequent induction of ferroptosis in the pathogenesis of sepsis-associated ALI.

N6-methyladenosine (m6A) depletion regulates pluripotency exit by activating signaling pathways in embryonic stem cells

N6-methyladenosine (m6A) deposition on messenger RNA (mRNA) controls embryonic stem cell (ESC) fate by regulating the mRNA stabilities of pluripotency and lineage transcription factors (TFs) [P. J. Batista etal., Cell Stem Cell 15, 707-719 (2014); Y. Wang etal., Nat. Cell Biol. 16, 191-198 (2014); and S. Geula etal., Science 347, 1002-1006 (2015)]. If the mRNAs of these two TF groups become stabilized, it remains unclear how the pluripotency or lineage commitment decision is implemented. We performed noninvasive quantification of Nanog and Oct4 TF protein levels in reporter ESCs to define cell-state dynamics at single-cell resolution. Long-term single-cell tracking shows that immediate m6A depletion by Mettl3 knock-down in serum/leukemia inhibitory factor supports both pluripotency maintenance and its departure. This is mediated by differential and opposing signaling pathways. Increased FGF5 mRNA stability activates pErk, leading to Nanog down-regulation. FGF5-mediated coactivation of pAkt reenforces Nanog expression. In formative stem cells poised toward differentiation, m6A depletion activates both pErk and pAkt, increasing the propensity for mesendodermal lineage induction. Stable m6A depletion by Mettl3 knock-out also promotes pErk activation. Higher pErk counteracts the pluripotency exit delay exhibited by stably m6A-depleted cells upon differentiation. At single-cell resolution, we illustrate that decreasing m6A abundances activates pErk and pAkt-signaling, regulating pluripotency departure.

METTL3 induces PLX4032 resistance in melanoma by promoting m6A-dependent EGFR translation.

Acquired resistance often limits therapeutic efficacy of the BFAF (V600E) kinase inhibitor PLX4032 in patients with advanced melanoma. Epitranscriptomic modification of mRNAs by N6-methyladenosine (m6A) modification contributes to melanoma pathogenesis; however, its role in acquired PLX4032 resistance remains unexplored. Here, we showed that m6A methyltransferase METTL3 expression is upregulated in A375R cells, a PLX4032-resistant subline of A375 melanoma cells, compared with the parental cells. Moreover, METTL3 increased the m6A modification of epidermal growth factor receptor (EGFR) mRNA in A375R cells, which promoted its translation efficiency. In turn, increased EGFR expression facilitated rebound activation of the RAF/MEK/ERK pathway in A375R cells, inducing PLX4032 resistance. In contrast, knockout of METTL3 in A375R cells reduced EGFR expression and restored PLX4032 sensitivity. PLX4032 treatment following METTL3 knockout induced apoptosis and reduced colony formation in A375R cells and reduced A375R cell-derived tumor growth in BALB/c nude mice. These findings indicate that METTL3 promotes rebound activation of the RAF/MEK/ERK pathway through EGFR upregulation and highlight a critical role for METTL3-induced m6A modification in acquired PLX4032 resistance in melanoma, implicating METTL3 as a potential candidate for targeted chemotherapy.

Dynamic regulation of N6,2\u2032-O-dimethyladenosine (m6Am) in obesity

The prevalent m6Am mRNA cap modification was recently identified as a valid target for removal by the human obesity gene FTO along with the previously established m6A mRNA modification. However, the deposition and dynamics of m6Am in regulating obesity are unknown. Here, we investigate the liver m6A/m methylomes in mice fed on a high fat Western-diet and in ob/ob mice. We find that FTO levels are elevated in fat mice, and that genes which lost m6Am marking under obesity are overly downregulated, including the two fatty-acid-binding proteins FABP2, and FABP5. Furthermore, the cellular perturbation of FTO correspondingly affect protein levels of its targets. Notably, generally m6Am- but not m6A-methylated genes, are found to be highly enriched in metabolic processes. Finally, we deplete all m6A background via Mettl3 knockout, and unequivocally uncover the association of m6Am methylation with increased mRNA stability, translation efficiency, and higher protein expression. Together, these results strongly implicate a dynamic role for m6Am in obesity-related translation regulation.

The methyltransferase METTL3 negatively regulates nonalcoholic steatohepatitis (NASH) progression

Nonalcoholic steatohepatitis (NASH) is a key step in the progression of nonalcoholic fatty liver (NAFL) to cirrhosis. However, the molecular mechanisms of the NAFL-to-NASH transition are largely unknown. Here, we identify methyltransferase like 3 (METTL3) as a key negative regulator of NASH pathogenesis. Hepatocyte-specific deletion of Mettl3 drives NAFL-to-NASH progression by increasing CD36-mediated hepatic free fatty acid uptake and CCL2-induced inflammation, which is due to increased chromatin accessibility in the promoter region of Cd36 and Ccl2. Antibody blockade of CD36 and CCL2 ameliorates NASH progression in hepatic Mettl3 knockout mice. Hepatic overexpression of Mettl3 protects against NASH progression by inhibiting the expression of CD36 and CCL2. Mechanistically, METTL3 directly binds to the promoters of the Cd36 and Ccl2 genes and recruits HDAC1/2 to induce deacetylation of H3K9 and H3K27 in their promoters, thus suppressing Cd36 and Ccl2 transcription. Furthermore, METTL3 is translocated from the nucleus to the cytosol in NASH, which is associated with CDK9-mediated phosphorylation of METTL3. Our data reveal a mechanism by which METTL3 negatively regulates hepatic Cd36 and Ccl2 gene transcription via a histone modification pathway for protection against NASH progression.

METTL3 Dysregulates RNA Splicing by Translational Control of Splicing Factors via m 6A Modification in CLL

METTL3 Dysregulates RNA Splicing by Translational Control of Splicing Factors via m 6A Modification in CLL

METTL3-Mediated m6A RNA Modification Regulates Corneal Injury Repair

Limbal stem cells are essential for continuous corneal regeneration and injury repair. METTL3-catalyzed N6-methyladenosine (m6A) mRNA modifications are involved in many biological processes and play a specific role in stem cell regeneration, while the role of m6A modifications in corneal injury repair remains unknown. In this study, we generated a limbal stem cell-specific METTL3 knockout mouse model and studied the role of m6A in repairing corneal injury caused by alkali burn. The results showed that METTL3 knockout in the limbal stem cells promotes the in vivo cell proliferation and migration, leading to the fast repair of corneal injury. In addition, m6A modification profiling identified stem cell regulatory factors AHNAK and DDIT4 as m6A targets. Our study reveals the essential functions of m6A RNA modification in regulating injury repair and provides novel insights for clinical therapy of corneal diseases.

METTL3 Regulates Liver Homeostasis, Hepatocyte Ploidy, and Circadian Rhythm–Controlled Gene Expression in Mice

N6-methyladenosine (m6A), the most abundant internal modifier of mRNAs installed by the methyltransferase 13 (METTL3) at the (G/A)(m6A)C motif, plays a critical role in the regulation of gene expression. METTL3 is essential for embryonic development, and its dysregulation is linked to various diseases. However, the role of METTL3 in liver biology is largely unknown. In this study, METTL3 function was unraveled in mice depleted of Mettl3 in neonatal livers (Mettl3fl/fl; Alb-Cre). Liver-specific Mettl3 knockout (M3LKO) mice exhibited global decrease in m6A on polyadenylated RNAs and pathologic features associated with nonalcoholic fatty liver disease (eg, hepatocyte ballooning, ductular reaction, microsteatosis, pleomorphic nuclei, DNA damage, foci of altered hepatocytes, focal lobular and portal inflammation, and elevated serum alanine transaminase/alkaline phosphatase levels). Mettl3-depleted hepatocytes were highly proliferative, with decreased numbers of binucleate hepatocytes and increased nuclear polyploidy. M3LKO livers were characterized by reduced m6A and expression of several key metabolic transcripts regulated by circadian rhythm and decreased nuclear protein levels of the core clock transcription factors BMAL1 and CLOCK. A significant decrease in total Bmal1 and Clock mRNAs but an increase in their nuclear levels were observed in M3LKO livers, suggesting impaired nuclear export. Consistent with the phenotype, methylated (m6A) RNA immunoprecipitation coupled with sequencing and RNA sequencing revealed transcriptome-wide loss of m6A markers and alterations in abundance of mRNAs involved in metabolism in M3LKO. Collectively, METTL3 and m6A modifications are critical regulators of liver homeostasis and function.

Distinct roles of Fto and Mettl3 in controlling development of the cerebral cortex through transcriptional and translational regulations

Proper development of the mammalian cerebral cortex relies on precise gene expression regulation, which is controlled by genetic, epigenetic, and epitranscriptomic factors. Here we generate RNA demethylase Fto and methyltransferase Mettl3 cortical-specific conditional knockout mice, and detect severe brain defects caused by Mettl3 deletion but not Fto knockout. Transcriptomic profiles using RNA sequencing indicate that knockout of Mettl3 causes a more dramatic alteration on gene transcription than that of Fto. Interestingly, we conduct ribosome profiling sequencing, and find that knockout of Mettl3 leads to a more severe disruption of translational regulation of mRNAs than deletion of Fto and results in altered translation of crucial genes in cortical radial glial cells and intermediate progenitors. Moreover, Mettl3 deletion causes elevated translation of a significant number of mRNAs, in particular major components in m6A methylation. Our findings indicate distinct functions of Mettl3 and Fto in brain development, and uncover a profound role of Mettl3 in regulating translation of major mRNAs that control proper cortical development.

Deep and accurate detection of m6A RNA modifications using miCLIP2 and m6Aboost machine learning.

N6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic mRNAs and influences many aspects of RNA processing. miCLIP (m6A individual-nucleotide resolution UV crosslinking and immunoprecipitation) is an antibody-based approach to map m6A sites with single-nucleotide resolution. However, due to broad antibody reactivity, reliable identification of m6A sites from miCLIP data remains challenging. Here, we present miCLIP2 in combination with machine learning to significantly improve m6A detection. The optimized miCLIP2 results in high-complexity libraries from less input material. Importantly, we established a robust computational pipeline to tackle the inherent issue of false positives in antibody-based m6A detection. The analyses were calibrated with Mettl3 knockout cells to learn the characteristics of m6A deposition, including m6A sites outside of DRACH motifs. To make our results universally applicable, we trained a machine learning model, m6Aboost, based on the experimental and RNA sequence features. Importantly, m6Aboost allows prediction of genuine m6A sites in miCLIP2 data without filtering for DRACH motifs or the need for Mettl3 depletion. Using m6Aboost, we identify thousands of high-confidence m6A sites in different murine and human cell lines, which provide a rich resource for future analysis. Collectively, our combined experimental and computational methodology greatly improves m6A identification.

METTL3-Mediated m6A Methylation Regulates Muscle Stem Cells and Muscle Regeneration by Notch Signaling Pathway.

The Pax7+ muscle stem cells (MuSCs) are essential for skeletal muscle homeostasis and muscle regeneration upon injury, while the molecular mechanisms underlying muscle stem cell fate determination and muscle regeneration are still not fully understood. N6-methyladenosine (m6A) RNA modification is catalyzed by METTL3 and plays important functions in posttranscriptional gene expression regulation and various biological processes. Here, we generated muscle stem cell-specific METTL3 conditional knockout mouse model and revealed that METTL3 knockout in muscle stem cells significantly inhibits the proliferation of muscle stem cells and blocks the muscle regeneration after injury. Moreover, knockin of METTL3 in muscle stem cells promotes the muscle stem cell proliferation and muscle regeneration in vivo. Mechanistically, METTL3-m6A-YTHDF1 axis regulates the mRNA translation of Notch signaling pathway. Our data demonstrated the important in vivo physiological function of METTL3-mediated m6A modification in muscle stem cells and muscle regeneration, providing molecular basis for the therapy of stem cell-related muscle diseases.

Role of epigenetic m6 A RNA methylation in vascular development: mettl3 regulates vascular development through PHLPP2/mTOR-AKT signaling.

N6 -methyladenosine (m6A) methylation is the most prevalent RNA modification, and it emerges as an important regulatory mechanism of gene expression involved in many cellular and biological processes. However, the role of m6 A methylation in vascular development is not clear. The m6 A RNA methylation is regulated by dynamic interplay among methyltransferases, binding proteins, and demethylases. Mettl3 is a member of the mettl3-mettl14 methyltransferase complex, referred to as writers that catalyze m6A RNA methylation. Here, we used CRISPR-Cas9 genome editing to develop two lines of knockout (KO) zebrafish for mettl3. Heterozygous mettl3+/- KO embryos show defective vascular development, which is directly visible in fli-EGFP and flk-EGFP zebrafish. Alkaline phosphatase staining and whole mount in situ hybridization with cdh5, and flk markers demonstrated defective development of intersegmental vessels (ISVs), subintestinal vessels (SIVs), interconnecting vessels (ICVs) and dorsal longitudinal anastomotic vessels (DLAV) in both heterozygous mettl3+/- and homozygous mettl3-/- KO zebrafish embryos. Similar phenotypes were observed in zebrafish embryos with morpholino knockdown (KD) of mettl3; however, the vascular defects were rescued fully by overexpression of constitutively active AKT1. KD of METTL3 in human endothelial cells inhibited cell proliferation, migration, and capillary tube formation. Mechanistically, mettl3 KO and KD significantly reduced the levels of m6 A RNA methylation, and AKT phosphorylation (S473) by an increase in the expression of phosphatase enzyme PHLPP2 and reduction in the phosphorylation of mTOR (S2481), a member of the phosphatidylinositol 3-kinase-related kinase family of protein kinases. These data suggest that m6 A RNA methylation regulates vascular development via PHLPP2/mTOR-AKT signaling.

Deficiency of Mettl3 in Bladder Cancer Stem Cells Inhibits Bladder Cancer Progression and Angiogenesis

RNA N6-methyladenosine is a key step of posttranscriptional modulation that is involved in governing gene expression. The m6A modification catalyzed by Mettl3 has been widely recognized as a critical epigenetic regulation process for tumorigenic properties in various cancer cell lines, including bladder cancer. However, the in vivo function of Mettl3 in bladder cancer remains largely unknown. In our study, we found that ablation of Mettl3 in bladder urothelial attenuates the oncogenesis and tumor angiogenesis of bladder cancer using transgenic mouse model. In addition, conditional knockout of Mettl3 in K14+ bladder cancer stem cell population leads to inhibition of bladder cancer progression. Coupled with the global transcriptome sequencing and methylated RNA immunoprecipitation sequencing results, we showed that deletion of Mettl3 leads to the suppression of tyrosine kinase endothelial (TEK) and vascular endothelial growth factor A (VEGF-A) through reduced abundance of m6A peaks on a specific region. In addition, the depletion of Mettl3 results in the decrease in both messenger RNA (mRNA) and protein levels of TEK and VEGF-A in vitro. Taken together, Mettl3-mediated m6A modification is required for the activation of TEK–VEGF-A-mediated tumor progression and angiogenesis. Our findings may provide theoretical basis for bladder cancer treatment targeting Mettl3.

METTL3 regulates heterochromatin in mouse embryonic stem cells.

METTL3 (methyltransferase-like 3) mediates the N6-methyladenosine (m6A) methylation of mRNA, which affects the stability of mRNA and its translation into protein1. METTL3 also binds chromatin2-4, but the role of METTL3 and m6A methylation in chromatin is not fully understood. Here we show that METTL3 regulates mouse embryonic stem-cell heterochromatin, the integrity of which is critical for silencing retroviral elements and for mammalian development5. METTL3 predominantly localizes to the intracisternal A particle (IAP)-type family of endogenous retroviruses. Knockout of Mettl3 impairs the deposition of multiple heterochromatin marks onto METTL3-targeted IAPs, and upregulates IAP transcription, suggesting that METTL3 is important for the integrity of IAP heterochromatin. We provide further evidence that RNA transcripts derived from METTL3-bound IAPs are associated with chromatin and are m6A-methylated. These m6A-marked transcripts are bound by the m6A reader YTHDC1, which interacts with METTL3 and in turn promotes the association of METTL3 with chromatin. METTL3 also interacts physically with the histone 3 lysine 9 (H3K9) tri-methyltransferase SETDB1 and its cofactor TRIM28, and is important for their localization to IAPs. Our findings demonstrate that METTL3-catalysed m6A modification of RNA is important for the integrity of IAP heterochromatin in mouse embryonic stem cells, revealing a mechanism of heterochromatin regulation in mammals.