Changes in Gene Expression Associated with FTO Overexpression in Mice

Single nucleotide polymorphisms in the first intron of the fat-mass-and-obesity-related gene FTO are associated with increased body weight and adiposity. Increased expression of FTO is likely underlying this obesity phenotype, as mice with two additional copies of Fto (FTO-4 mice) exhibit increased adiposity and are hyperphagic. FTO is a demethylase of single stranded DNA and RNA, and one of its targets is the m6A modification in RNA, which might play a role in the regulation of gene expression. In this study, we aimed to examine the changes in gene expression that occur in FTO-4 mice in order to gain more insight into the underlying mechanisms by which FTO influences body weight and adiposity. Our results indicate an upregulation of anabolic pathways and a downregulation of catabolic pathways in FTO-4 mice. Interestingly, although genes involved in methylation were differentially regulated in skeletal muscle of FTO-4 mice, no effect of FTO overexpression on m6A methylation of total mRNA was detected.     

m6A demethylation of cytidine deaminase APOBEC3B mRNA orchestrates arsenic-induced mutagenesis

The cytidine deaminase APOBEC3B (A3B) is an endogenous inducer of somatic mutations and causes chromosomal instability by converting cytosine to uracil in single-stranded DNA. Therefore, identification of factors and mechanisms that mediate A3B expression will be helpful for developing therapeutic approaches to decrease DNA mutagenesis. Arsenic (As) is one well-known mutagen and carcinogen, but the mechanisms by which it induces mutations have not been fully elucidated. Herein, we show that A3B is upregulated and required for As-induced DNA damage and mutagenesis. We found that As treatment causes a decrease of N6-methyladenosine (m6A) modification near the stop codon of A3B, consequently increasing the stability of A3B mRNA. We further reveal that the demethylase FTO is responsible for As-reduced m6A modification of A3B, leading to increased A3B expression and DNA mutation rates in a manner dependent on the m6A reader YTHDF2. Our in vivo data also confirm that A3B is a downstream target of FTO in As-exposed lung tissues. In addition, FTO protein is highly expressed and positively correlates with the protein levels of A3B in tumor samples from human non–small cell lung cancer patients. These findings indicate a previously unrecognized role of A3B in As-triggered somatic mutation and might open new avenues to reduce DNA mutagenesis by targeting the FTO/m6A axis.     

SIRT6 deficiency causes ovarian hypoplasia by affecting Plod1-related collagen formation

SIRT6 is a key member of the mammalian sirtuin family of conserved nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylases. Previous studies have shown that SIRT6 can regulate metabolism, DNA damage repair and aging. Ovarian aging process usually share similar mechanisms with general aging, which is characterized by decreases in both numbers of ovarian follicles and the quality of oocytes. It is reported that the expression level of SIRT6 was significantly decreased in the ovaries of aged mice, and the level of SIRT6 was positively correlated with ovarian reserve, indicating that SIRT6 may be potential markers of ovarian aging. However, its biological roles in follicular development are still unclear. Here, we explored the effect of SIRT6 on follicular development and found that ovarian development was interrupted in SIRT6 knockout (KO) mice, leading to disruptions of puberty and the estrus cycle, significant decreases in numbers of secondary and antral follicles, and decreased collagen in the ovarian stroma. Plod1, a lysyl hydroxylase that is vital for collagen crosslinking and deposition, was decreased at both the mRNA and protein levels in SIRT6-deficient ovaries and granulosa cells (GCs). Additionally, we found abnormal estrogen levels in both SIRT6 KO mice and SIRT6 KD GCs, accompanied by decreases in the levels of the estrogen biosynthesis genes Cyp11a1, Cyp19a1, Mgarp, and increases in the levels of TNF-α and NF-κB. These results confirmed the effect of SIRT6 on follicular development and revealed a possible molecular mechanism for SIRT6 involvement in follicular development via effects on estrogen biosynthesis and collagen formation.     

FTO-mediated autophagy promotes progression of clear cell renal cell carcinoma via regulating SIK2 mRNA stability

The progression of clear cell renal cell carcinoma (ccRCC) remains a major challenge in clinical practice, and elucidation of the molecular drivers of malignancy progression is critical for the development of effective therapeutic targets. Recent studies have demonstrated that N⁶-methyladenosine (m⁶A) is the most abundant modification of eukaryotic mRNA and plays a key role in tumorigenesis and progression. However, the biological roles and underlying mechanisms of m⁶A-mediated autophagy in cancers especially in ccRCC remain poorly elucidated. m⁶A dot blot assay, m⁶A RNA methylation assay kit and immunofluorescence analysis were used to profile m⁶A levels in tissue samples and their correlation with autophagic flux. Expression patterns and clinical significance of fat mass and obesity-associated protein (FTO) were determined through bioinformatics analysis, real-time PCR, western blotting, immunohistochemistry. RNA-seq, MeRIP-seq, MeRIP-qRT-PCR, RIP-qRT-PCR, transmission electron microscopy, immunofluorescence analysis and luciferase reporter assay were used to investigate the underlying mechanism of the FTO-autophagy axis. The role of FTO and autophagy in ccRCC progression was evaluated both in vitro and in vivo. Here we found that m⁶A modification was suppressed and closely related to autophagic flux in ccRCC. Elevated FTO was inhibited by rapamycin, whereas silencing FTO enhanced autophagic flux and impaired ccRCC growth and metastasis. SIK2 was identified as a functional target of m⁶A-mediated autophagy, thereby prompting FTO to play a conserved and important role in inhibiting autophagy and promoting tumorigenesis through an m⁶A-IGF2BP2 dependent mechanism. Moreover, the small molecule inhibitor FB23-2 targeting FTO inhibited tumor growth and prolonged survival in the patient-derived xenograft (PDX) model mice, suggesting that FTO is a potential effective therapeutic target for ccRCC. Our findings uncovered the crucial role of FTO/autophagy/SIK2 axis in modulating the progression of ccRCC, suggesting that FTO may serve as a valuable prognostic biomarker and promising therapeutic target in ccRCC.     

FTO overexpression inhibits apoptosis of hypoxia/reoxygenation-treated myocardial cells by regulating m6A modification of Mhrt

Heart failure (HF) is the end stage of many cardiovascular diseases and seriously threatens people’s health. This article aimed to explore the biological role of fat-mass and obesity-associated gene (FTO) in HF. We constructed HF mouse model by transverse aortic constriction or intraperitoneal injection of doxorubicin. Mouse myocardial cells were exposed to hypoxia/reoxygenation (H/R). FTO and Mhrt were downregulated in heart tissues of HF mice. HF mice exhibited an increase in the total levels of N⁶ methyladenosine (m⁶A) and the m⁶A levels of Mhrt. Moreover, FTO overexpression caused an upregulation of Mhrt and reduced m⁶A modification of Mhrt in the H/R-treated myocardial cells. FTO upregulation repressed apoptosis of H/R-treated myocardial cells. FTO knockdown had the opposite results. Mhrt overexpression reduced apoptosis of H/R-treated myocardial cells. Moreover, the influence conferred by FTO upregulation was abolished by Mhrt knockdown. In conclusion, our data demonstrate that FTO overexpression inhibits apoptosis of hypoxia/reoxygenation-treated myocardial cells by regulating m6A modification of Mhrt. Thus, FTO may be a target gene for HF treatment.

     

FSH mediates estradiol synthesis in hypoxic granulosa cells by activating glycolytic metabolism through the HIF-1α–AMPK–GLUT1 signaling pathway

Owing to the avascular environment within ovarian follicles, granulosa cells (GCs) are believed to live in a hypoxic niche. Follicle-stimulating hormone (FSH)-mediated steroidogenesis is crucial for normal growth and maturation of ovarian follicles, but it remains unclear how FSH stimulates estradiol (E2) synthesis under hypoxic conditions. Here, we aimed to explore whether FSH affects the ATP production required for estrogen synthesis from the perspective of glucose metabolism. It was observed that the levels of both E2 and HIF-1α were markedly increased in a dose-dependent manner in mouse ovarian GCs after the injection of FSH in vivo, indicating that hypoxia/HIF-1α may be relevant to FSH-induced E2 synthesis. By treating hypoxic GCs with FSH in vitro, we further revealed that the activation of the AMP-activated protein kinase (AMPK)–GLUT1 pathway, which in turn stimulates ATP generation, may be essential for FSH-mediated E2 production during hypoxia. In contrast, inhibition of AMPK or GLUT1 with siRNAs/antagonist both repressed glycolysis, ATP production, and E2 synthesis despite FSH treatment. Moreover, blocking HIF-1α activity using siRNAs/PX-478 suppressed AMPK activation, GLUT1 expression, and E2 levels in FSH-treated GCs. Finally, the in vitro findings were verified in vivo, which showed markedly increased AMPK activity, GLUT1 expression, glycolytic flux, ATP levels, and E2 concentrations in ovarian GCs following FSH injection. Taken together, these findings uncovered a novel mechanism for FSH-regulating E2 synthesis in hypoxic GCs by activating glycolytic metabolism through the HIF-1α–AMPK–GLUT1 pathway.     

N6-methyladenosine demethylase FTO impairs hepatic ischemia–reperfusion injury via inhibiting Drp1-mediated mitochondrial fragmentation

Despite N6-methyladenosine (m6A) is functionally important in various biological processes, its role and the underlying regulatory mechanism in the liver remain largely unexplored. In the present study, we showed that fat mass and obesity-associated protein (FTO, an m6A demethylase) was involved in mitochondrial function during hepatic ischemia–reperfusion injury (HIRI). We found that the expression of m6A demethylase FTO was decreased during HIRI. In contrast, the level of m6A methylated RNA was enhanced. Adeno-associated virus-mediated liver-specific overexpression of FTO (AAV8-TBG-FTO) ameliorated the HIRI, repressed the elevated level of m6A methylated RNA, and alleviated liver oxidative stress and mitochondrial fragmentation in vivo and in vitro. Moreover, dynamin-related protein 1 (Drp1) was a downstream target of FTO in the progression of HIRI. FTO contributed to the hepatic protective effect via demethylating the mRNA of Drp1 and impairing the Drp1-mediated mitochondrial fragmentation. Collectively, our findings demonstrated the functional importance of FTO-dependent hepatic m6A methylation during HIRI and provided valuable insights into the therapeutic mechanisms of FTO.Subject terms: Mechanisms of disease, RNA modification     

Mir-484 contributes to diminished ovarian reserve by regulating granulosa cell function via YAP1-mediated mitochondrial function and apoptosis

Women with diminished ovarian reserve (DOR) have reduced fertility, but the underlying regulation of ovarian function remains unknown. Although differential microRNA (miRNA) expression has been described in several ovarian disorders, little is known about the role of miRNAs in the pathogenesis of DOR. In this study, we investigated the expression levels of miR-484 in granulosa cells (GCs) derived from human follicular fluid, and explored their correlation with female ovarian reserve function as well as clinical outcomes of assisted reproduction technology (ART). Additionally, we investigated the effects of miR-484 on the biological functions of GC cell lines in vitro. We found that miR-484 was highly expressed in GCs from DOR patients and was correlated with decreasing AMH levels and AFC, as well as increasing FSH levels, but not with LH, progesterone, or estradiol. Additionally, miR-484 was negatively related to the number of retrieved oocytes and the ratio of high-quality embryos. Moreover, we found that miR-484 repressed the proliferation of GCs and induced apoptosis, which can in part be attributed to mitochondrial dysfunction. Conversely, silencing miR-484 had the opposite effect. Multiple approaches, including bioinformatic analysis, RNA-seq, qPCR, immunofluorescence, western blotting and luciferase reporter assays, identified YAP1 as a direct target of miR-484 in GCs. Additionally, reintroduction of YAP1 rescued the effects of miR-484 in GCs. The present study indicates that miR-484 can directly target the mRNA of YAP1, induce mitochondrial dysfunction, and consequently reduce the viability and promote the apoptosis of granulosa cells, which contributes to the pathogenesis of DOR.     

MiR-92a inhibits porcine ovarian granulosa cell apoptosis by targeting Smad7 gene

Smad7 has a key role in apoptosis of mammalian ovarian granulosa cells (GCs), as it antagonizes and fine-tunes transforming growth factor β (TGFβ) signaling. This study demonstrates that miR-92a regulates GC apoptosis in pig ovaries by targeting Smad7 directly. The expression level of miR-92a was down-regulated in atretic porcine follicles, whereas miR-92a expression led to inhibition of GC apoptosis. The Smad7 gene was identified as a direct target of miR-92a using a dual-luciferase reporter assay. Transfection of GCs with miR-92a mimics decreased Smad7 mRNA and protein levels, whereas expression of an miR-92a inhibitor in GCs had the opposite effect. In addition, knockdown of Smad7 prevented GC apoptosis in cells that expressed the miR-92a inhibitor.     

Fat mass and obesity-associated protein regulates lipogenesis via m6A modification in fatty acid synthase mRNA

As the first identified N⁶-methyladenosine (m⁶A) demethylase, fat mass and obesity-associated (FTO) protein is associated with fatty acid synthase (FASN) and lipid accumulation. However, little is known about the regulatory role of FTO in the expression of FASN and de novo lipogenesis through m⁶A modification. In this study, we used FTO small interfering RNA to explore the effects of FTO knockdown on hepatic lipogenesis and its underlying epigenetic mechanism in HepG2 cells. We found that knockdown of FTO increased m⁶A levels in total RNA and enhanced the expression of YTH domain family member 2 which serves as the m⁶A-binding protein. The de novo lipogenic enzymes and intracellular lipid content were significantly decreased under FTO knockdown. Mechanistically, knockdown of FTO dramatically enhanced m⁶A levels in FASN messenger RNA (mRNA), leading to the reduced expression of FASN mRNA through m⁶A-mediated mRNA decay. The protein expressions of FASN along with acetyl CoA carboxylase and ATP-citrate lyase were further decreased, which inhibited de novo lipogenesis, thereby resulting in the deficiency of lipid accumulation in HepG2 cells and the induction of cellular apoptosis. The results reveal that FTO regulates hepatic lipogenesis via FTO-dependent m⁶A demethylation in FASN mRNA and indicate the critical role of FTO-mediated lipid metabolism in the survival of HepG2 cells. This study provides novel insights into a unique RNA epigenetic mechanism by which FTO mediates hepatic lipid accumulation through m⁶A modification and indicates that FTO could be a potential target for obesity-related diseases and cancer.     

Type IV Collagen Controls the Axogenesis of Cerebellar Granule Cells by Regulating Basement Membrane Integrity in Zebrafish

Granule cells (GCs) are the major glutamatergic neurons in the cerebellum, and GC axon formation is an initial step in establishing functional cerebellar circuits. In the zebrafish cerebellum, GCs can be classified into rostromedial and caudolateral groups, according to the locations of their somata in the corresponding cerebellar lobes. The axons of the GCs in the caudolateral lobes terminate on crest cells in the dorsal hindbrain, as well as forming en passant synapses with Purkinje cells in the cerebellum. In the zebrafish mutant shiomaneki, the caudolateral GCs extend aberrant axons. Positional cloning revealed that the shiomaneki (sio) gene locus encodes Col4a6, a subunit of type IV collagen, which, in a complex with Col4a5, is a basement membrane (BM) component. Both col4a5 and col4a6 mutants displayed similar abnormalities in the axogenesis of GCs and retinal ganglion cells (RGCs). Although type IV collagen is reported to control axon targeting by regulating the concentration gradient of an axonal guidance molecule Slit, Slit overexpression did not affect the GC axons. The structure of the BM surrounding the tectum and dorsal hindbrain was disorganized in the col4a5 and col4a6 mutants. Moreover, the abnormal axogenesis of the caudolateral GCs and the RGCs was coupled with aberrant BM structures in the type IV collagen mutants. The regrowth of GC axons after experimental ablation revealed that the original and newly formed axons displayed similar branching and extension abnormalities in the col4a6 mutants. These results collectively suggest that type IV collagen controls GC axon formation by regulating the integrity of the BM, which provides axons with the correct path to their targets.     

Fat mass and obesity-related (FTO) shuttles between the nucleus and cytoplasm

SNPs (single nucleotide polymorphisms) on a chromosome 16 locus encompassing FTO, as well as IRX3, 5, 6, FTM and FTL are robustly associated with human obesity. FTO catalyses the Fe(II)- and 2OG-dependent demethylation of RNA and is an AA (amino acid) sensor that couples AA levels to mTORC1 (mammalian target of rapamycin complex 1) signalling, thereby playing a key role in regulating growth and translation. However, the cellular compartment in which FTO primarily resides to perform its biochemical role is unclear. Here, we undertake live cell imaging of GFP (green fluorescent protein)-FTO, and demonstrate that FTO resides in both the nucleus and cytoplasm. We show using ‘FLIP’ (fluorescence loss in photobleaching) that a mobile FTO fraction shuttles between both compartments. We performed a proteomic study and identified XPO2 (Exportin 2), one of a family of proteins that mediates the shuttling of proteins between the nucleus and the cytoplasm, as a binding partner of FTO. Finally, using deletion studies, we show that the N-terminus of FTO is required for its ability to shuttle between the nucleus and cytoplasm. In conclusion, FTO is present in both the nucleus and cytoplasm, with a mobile fraction that shuttles between both cellular compartments, possibly by interaction with XPO2.     

Suppression of SEMA6C promotes preantral follicles atresia with decreased cell junctions in mice ovaries

Mammalian oocytes go through a long and complex developmental process, while acquiring the competencies that are required for fertilization and embryogenesis. Recent studies revealed that the communication between oocytes and granulosa cells (GCs) is a critical process for female follicle development. In the current study, we aimed to study whether and how semaphorin 6C (Sema6c) regulated the cell junctions between oocytes and GCs in mice preantral follicles. The attenuation of SEMA6C expression by siRNA decreased the cell–cell junctions and accelerated follicle atresia in vitro. PI3K-AKT pathway was activated when SEMA6C expression was downregulated. And the LY294002, a PI3K inhibitor, could reverse the effect of low SEMA6C expression on cell junctions in preantral follicles. Our findings revealed that Sema6c was involved in follicle development, and the suppression of SEMA6C led to cell junction defection by activating the PI3K/AKT pathway, which might also provide valuable information for understanding premature ovarian failure and ovarian aging.     

The GDF11-FTO-PPARγ axis controls the shift of osteoporotic MSC fate to adipocyte and inhibits bone formation during osteoporosis

During osteoporosis, the shift of bone mesenchymal stem cell (BMSC) lineage commitment to adipocyte leads to the imbalance between bone mass and fat, which increases the risk of fracture. The mechanism underlying this process is not fully understood. Fat mass and obesity-associated protein (FTO) is an RNA demethylase that demethylates various methylated nucleic acids and participates in various physiological and pathological processes. Here we identified FTO as a regulator for BMSC fate determination during osteoporosis. FTO was up-regulated in bone marrow during aging or osteoporosis in human and mice in a GDF11(growth differentiation factor 11)-C/EBPα-dependent mechanism. The expression of FTO was also up-regulated during adipocyte differentiation of BMSCs whereas its expression was down-regulated during osteoblast differentiation. Gain-of-function and loss-of-function experiments showed that FTO favored the BMSCs to differentiate to adipocytes rather than osteoblasts. Further mechanism study demonstrated that FTO bound and demethylated the mRNA of the Peroxisome proliferator-activated receptor gamma (Pparg), leading to the increase in the expression of Pparg mRNA. Reversely, Pparg knockdown blocked the function of GDF11-FTO during osteoblast differentiation of BMSCs. Furthermore, conditionally genetic knockout of Fto in osteoblasts inhibited the development of osteopenia in mice. Collectively, our findings demonstrated that GDF11-FTO-Pparg axis promoted the shift of osteoporotic BMSC fate to adipocyte and inhibited bone formation during osteoporosis.