M6a Mrna Methylation Controls Functional Maturation in Neonatal Murine B-Cells

1708 Diabetes Volume 69, August 2020

m6A mRNA Methylation Controls Functional Maturation in Neonatal Murine b-Cells

Yanqiu Wang,1 Jiajun Sun,1 Zhen Lin,2 Weizhen Zhang,3 Shu Wang,1 Weiqing Wang,1 Qidi Wang,1,4 and Guang Ning1

Diabetes 2020;69:1708–1722 | https://doi.org/10.2337/db19-0906

The N6-methyladenosine (m6A) RNA modification is es- Defects in either b-cell number and/or function can lead to sential during embryonic development of various organs. loss of functional b-cell mass and, eventually, to diabetes However, its role in embryonic and early postnatal islet (2). Establishment of adequate functional b-cell mass is development remains unknown. Mice in which RNA essential for glycemic control in adulthood, which is mainly methyltransferase-like 3/14 (Mettl3/14) were deleted in accomplished during neonatal period before weaning by Ngn31 endocrine progenitors (Mettl3/14nKO) developed b-cell differentiation, self-replication, and functional hyperglycemia and hypoinsulinemia at 2 weeks after maturation (3,4). Neonatal b-cells are immature and are birth. We found that Mettl3/14 specifically regulated both functional maturation and mass expansion of neonatal unable to secrete insulin appropriately in response to b-cells before weaning. Transcriptome and m6A meth- a glucose challenge (5). During the early postnatal period ylome analyses provided m6A-dependent mechanisms in (P0-P14), neonatal b-cells undergo dramatic changes to regulating cell identity, insulin secretion, and prolifera- acquire glucose-responsive insulin secretion ability and tion in neonatal b-cells. Importantly, we found that gain functional maturity (3,6,7). Previous studies have Mettl3/14 were dispensable for b-cell differentiation identified orchestrated mechanisms including molecular but directly regulated essential transcription factor MafA – ISLET STUDIES pathways (8), transcriptional signals (9 12), and epige- expression at least partially via modulating its mRNA netic regulators (5,13) in shaping the transcriptional net- fi stability. Failure to maintain this modi cation impacted works that reinforce the functional identity of mature fi b the ability to ful ll -cell functional maturity. In both b-cells. Epigenetic regulators, such as DNA methylation diabetic db/db mice and patients with type 2 diabetes and histone modifications, have profound effects on b-cell (T2D), decreased Mettl3/14 expression in b-cells was observed, suggesting its possible role in T2D. Our study development and functional maturation (5,13). We and unraveled the essential role of Mettl3/14 in neonatal others have reported that DNMT3A directs functional b-cell development and functional maturation, both of maturation in murine b-cells (5,8). Meanwhile, genome- which determined functional b-cell mass and glycemic wide analysis of histone marks during b-cell develop- control in adulthood. ment further underscored the importance of histone modifications in b-cell maturation (13). N6-methyladenosine (m6A) modification of mRNA is emerging as an important Mature b-cells have the unique ability to secrete insulin regulator of gene expression (14,15). However, its role in in response to extracellular glucose concentrations (1). neonatal b-cells is still unknown.

1Shanghai National Clinical Research Center for Endocrine and Metabolic Dis- Corresponding author: Guang Ning, [email protected], Qidi Wang, wqd11094@rjh eases, Key Laboratory for Endocrine and Metabolic Diseases of the National Health .com.cn, or Weiqing Wang, [email protected] Commission of the PR China, Shanghai Institute of Endocrine and Metabolic Received 10 September 2019 and accepted 8 May 2020 Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, This article contains supplementary material online at https://doi.org/10.2337/ Shanghai, China figshare.12272627. 2State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute Y.W. and J.S. contributed equally to this work. of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of © 2020 by the American Diabetes Association. Readers may use this article as Chinese Academy of Sciences, Shanghai, China long as the work is properly cited, the use is educational and not for profit, and the 3Department of Physiology and Pathophysiology, School of Basic Science, Peking work is not altered. More information is available at https://www.diabetesjournals University Health Science Center, Beijing, China .org/content/license. 4Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China diabetes.diabetesjournals.org Wang and Associates 1709

fl fl Being the most prevalent internal modification in Wei-Zhen Zhang. The Ngn3-cre;Mettl3 ox/ ox (Mettl3nKO) or fl fl mRNA, the m6A mRNA modification functionally modu- Ngn3-cre;Mettl14 ox/ ox (Mettl14nKO) mice were generated fl fl fl fl lates mRNA splicing, export, localization, translation, and by crossing Mettl3 ox/ ox or Mettl14 ox/ ox mice with Ngn3- stability and has crucial roles in various normal and Cre mice. Mettl3- and Mettl14- double knockout (KO) mice fl fl pathological processes (16,17). m6A methylation is dy- (Mettl3/14nKO) were generated by crossing Mettl3 ox/ ox fl fl fl 1 fl 1 namic and is accomplished by the orchestrated action of Mettl14 ox/ ox mice with Ngn3-cre;Mettl3 ox/ Mettl14 ox/ fl fl fl fl fl fl methyltransferase complex methyltransferase-like 3 (Mettl3), mice. Ngn3-Cre, Mettl3 ox/ ox, Mettl14 ox/ ox,orMettl3 ox/ ox fl fl methyltransferase-like 14 (Mettl14), and Wilms tumor Mettl14 ox/ ox mice were used as their littermate controls. 1-associated protein (WTAP), while it is removed by All mice were housed in the animal facility on a 12-h/12-h demethylases obesity-associated protein (FTO) and AlkB light/dark cycle. Normal chow and water were available ad homolog 5 (ALKBH5) (18). In particular, reversible m6A libitum. Blood glucose concentrations were measured by mRNA methylation plays critical roles in modulating dy- glucometers, and plasma insulin concentrations were de- namic transcriptome switching in response to diverse termined by using an ELISA kit (Mouse Ultrasensitive signals during embryonic development of various organs Insulin ELISA kit; Alpco). Intraperitoneal glucose tolerance and organisms (17). As the key writers of m6A, Mettl3 and/ tests were performed on 8-week-old mice after overnight or Mettl14 have been identified to regulate Drosophila fasting as previously described (8). Insulin tolerance tests neuronal function and sex determination (19), mouse stem were performed on 4-week-old Mettl3/14nKO mice and WT cell pluripotency (20), naive T cell differentiation (21), mice after 6 h of fasting. Male mice were used in all the cortical neurogenesis (22), spermatogenesis (23,24), and experiments in the current study, unless otherwise stated. hematopoietic stem/progenitor cell specification (25). All animal experiments were approved by the Animal Care Mettl3 and or Mettl14 were also reported to participate Committee of Shanghai Jiao Tong University. in the pathology and progression of multiple human cancer diseases, i.e., myeloid leukemia (26), liver cancer (27), lung Human Subjects cancer (28), and breast cancer (29). Very recently, m6A Paraffin sections of pancreas far from the margin of the modification was shown to regulate mature b-cell insulin pancreatectomy were collected from our previous research secretion and survival (30,31). It is currently unknown (32). In brief, all patients with partial pancreatectomy whether and how m6A RNA modifications regulate em- performed in Ruijin Hospital between 2013 and 2017 bryonic and early postnatal islet development. were enrolled. Those who had been reported as having To investigate the role of Mettl3/14-mediated m6A a malignant tumor were excluded, and then five patients modifications in islet development, we generated mice with T2D and five age- and BMI-matched subjects who 1 in which Mettl3 and/or Mettl14 were deleted in Ngn3 did not have diabetes (nondiabetic [ND]) were finally endocrine progenitors. Loss of Mettl3/14 in endocrine included in this study. Detailed information and clinical progenitors (Mettl3/14nKO) caused hyperglycemia at characteristics for each patient were listed in Supplemen- 2 weeks of age, with reduced cell number and impaired tary Table 1. This study was approved by the Institutional functional maturation of neonatal b-cells. Transcriptome Review Board of the Ruijin Hospital affiliated to Shanghai and m6A methylome analyses of primary islets from P14 Jiao Tong University School of Medicine and was in wild-type (WT) and Mettl3/14nKO mice identified m6A- accordance with the principles of the Declaration of dependent mechanisms in regulating neonatal b-cell iden- Helsinki II. tity and insulin secretion. Loss of Mettl3/14 silenced MafA expression, possibly via modulating its mRNA stability Cell Culture and Lentivirus Infection and, thus prevented neonatal b-cell to fulfill functional The mouse insulinoma cells (MIN6 cell line) purchased maturation. Importantly, as early as P0, reduced MafA from CAMS Cell Culture Center (Beijing, China) were protein abundance and impaired glucose-stimulated in- grown in DMEM medium (Gibco) containing 25.0 mmol/L sulin secretion (GSIS) were already present in mutant glucose, 15% FBS, 100 IU/mL penicillin, 100 mg/mL islets, which were prior to b-cell loss and changes in other streptomycin, 10.2 mmol/L L-glutamine, and 2.5 mmol/L transcription factors, i.e., Pdx1 and Nkx6.1. The findings b-mercaptoethanol at 37°C in a humidified 5% CO2 atmo- that Mettl3 and Mettl14 expression were significantly sphere. For knocking down Mettl3 and Mettl14, shRNA decreased in pancreatic b-cells of db/db mice and patients lentiviruses that targeted Mettl3, Mettl14, or a control with type 2 diabetes (T2D) further suggested the possible lentivirus were constructed, packaged, purified, and titrated role of m6A in T2D in both rodents and humans. at GeneChem Co. Ltd. MIN6 cells were infected with purified lentivirus at 50 multiplicity of infection for 48 h. RESEARCH DESIGN AND METHODS After infection, cells were harvested for RNA extraction and Mice further analysis. fl fl Mettl3- and Mettl14-floxed mice (Mettl3 ox/ ox and fl fl Mettl14 ox/ ox) were generated and provided by Prof. Immunostaining Analysis Ming-Han Tong (University of Chinese Academy of Sci- Pancreas sections were dissected, fixed, and processed as ences) (24). Ngn3-cre mice were a kind gift from Prof. described before (33). For the immunostaining analysis, 1710 m6A mRNA Methylation and Neonatal b-Cells Diabetes Volume 69, August 2020 entire pancreatic tissues were continuously sectioned at and RNA sequencing (RNA-seq) were performed by Cloud- 5-mm thickness. Immunochemistry procedures were per- seq Biotech Inc. (Shanghai, China) according to published formed on continuous sections (selected every 150-mm procedures. Briefly, m6A RNA immunoprecipitation was apart, 10–12 sections per animal) to obtain representative performed with the GenSeq m6A-MeRIP Kit (GenSeq Inc., b-cell mass information of the whole pancreas. Immuno- Shanghai, China) following the manufacturer’s instructions. chemistry staining of insulin for b-cell mass analysis was Both the input sample without immunoprecipitation and performed using a diaminobenzidine peroxidase substrate the m6A IP samples were used for library generation with kit (Vector Laboratories, Burlingame, CA) counterstaining NEBNext Ultra II Directional RNA Library Prep Kit (New with eosin. Digital images of whole pancreas were captured England Biolabs, Inc.). The library quality was evaluated by MZ 100 microscope (Nikon Corp., Tokyo, Japan). Total with BioAnalyzer 2100 system (Agilent Technologies, Inc.). pancreatic and insulin-positive areas of each section were Library sequencing was performed on an illumina Hiseq measured using Meta-Morph version 7.1 (Molecular Devi- instrument with 150 base pair (bp) paired-end reads. ces, Sunnyvale, CA). Paired-end reads were harvested from the Illumina HiSEq 4000 sequencer, and they were quality controlled by Q30 Immunoblot Analysis after 39 adaptor trimming and removal of low-quality reads P0 mouse islets and MIN6 cells were lysed, quantified, and by cutadapt software (v1.9.3). First, clean reads of all libraries blotted as described before. Primary antibodies are listed were aligned to the reference genome (MM10) by Hisat2 as following: rabbit anti-MAFA (1:1,000; Bethyl); rabbit software (v2.0.4). Methylated sites on RNAs (peaks) were anti-METTL3 (1:1,000; Proteintech); rabbit anti-METTL14 identified by MACS software. Differentially methylated sites (1:1,000; Proteintech); rabbit anti-Pdx1 (1:1,000; CST); were identified by diffReps. These peaks identified by both mouse anti-Nkx6.1 (1:1,000; DSHB); and mouse anti- software overlapping with exons of mRNA were figured out GAPDH (1:10,000; Proteintech) was used as an internal and chosen by homemade scripts. For RNA-seq, total RNA control to normalize band intensity. was used for removing the rRNAs with NEBNext rRNA Depletion Kit (New England Biolabs, Inc.) following the Extraction of RNA and Quantitative Real-Time PCR manufacturer’s instructions. RNA libraries were con- Analysis structed, and libraries were controlled for quality and Total cell RNA was extracted using the TRIzol reagent quantified. Library sequencing was performed on an illu- (Invitrogen). Reverse transcription and quantitative real- mina Hiseq instrument with 150 bp paired end reads. Then, time PCR were performed as previously described (8). guided by the Ensembl gtf gene annotation file, cuffdiff PCRs were performed in duplicate. The expression levels software (part of cufflinks) was used to get the gene-level were normalized to individual b-actin. Primers used in this FPKM (fragments per kilobase of exon model per million study were listed in Supplementary Table 2. mapped reads) as the expression profiles of mRNA. Fold m6A Quantification change and P value were calculated based on FPKM. nKO nKO Islets were isolated from 8-week-old Mettl3 , Mettl14 , Statistics Analyses nKO Mettl3/14 ,andWTmice,andthen,totalRNAwas The exact sample size for each experiment was indicated extracted using a RNeasy Micro kit (Qiagen) following the in the figure legends. All statistics comparing two groups 6 manufacturer’sprotocol.ThechangeofglobalmA levels in used two-sided Student t tests. ANOVA was used for 6 mRNA was measured using EpiQuik m A RNA Methylation multiple groups. Statistical analyses were performed Quantification Kit (Colorimetric) (Epigentek) following the with GraphPad Prism 7. P , 0.05 was considered as manufacturer’s protocol; 200-ng poly-A-purified RNA was statistically significant. used for each sample analysis. Data and Resource Availability mRNA Stability Assay TheP0andP14RNA-seqandm6A MeRIP-seq data are The mRNA half-life measurements were performed accord- deposited at the Gene Expression Omnibus (GEO) data- ing to previous publications (34). In brief, MIN6 cells with base under the accession numbers: GSE149193, GSE132323, or without Mettl3/14 knockdown were treated with 5-mg/ and GSE132319. mL actinomycin D (Sigma) for 1 or 3 h at the end of culture RESULTS and then collected for RNA extraction and real-time PCR 6 1 analysis. Since actinomycin D treatment results in tran- Loss of m A in Ngn3 Endocrine Progenitors Leads to scription stalling, the change of mRNA concentration at Hypoinsulinemia and Severe Diabetes 6 a given time (dC/dt) is proportional to the constant of To examine whether Mettl3/14-mediated m A mRNA methylation participates in b-cell development, we first mRNA decay (Kdecay) and mRNA concentration (C). checked the expression patterns of Mettl3 and Mettl14 in m6A MeRIP Sequencing, RNA Sequencing, and Data mouse pancreas at E17.5, P0, P4, P8, P14, and P56 by Analysis immunostaining (Fig. 1A). We found relatively weak Islet total RNA was prepared as previously described. The Mettl3 and Mettl14 expressions in b-cells at E17.5 and m6A mRNA immunoprecipitation sequencing (MeRIP-seq) P0, whereas the immunofluorescence intensities of both diabetes.diabetesjournals.org Wang and Associates 1711 the two methyltransferases increased dramatically during from WT and Mettl3/14nKO. We found similar cord-like b-cell maturation window P4-P8, reached maximal levels islet structures in newborn Mettl3/14nKO and WT mice at P14, and maintained at high levels throughout adult- (Fig. 2A). Then, we calculated the number of four different hood (Fig. 1B and C). These data suggested that Mettl3- islet endocrine cells per section and found the absolute and Mettl14-mediated m6A mRNA methylation might number of b, a, d, and PP cells remained constant at birth participate in postnatal b-cell functional maturation. between the two groups (Fig. 2B). Interestingly, after We then generated mice in which Mettl3 (Ngn3-cre; 14 days, Mettl3/14nKO islets exhibited significantly reduced fl fl Mettl3 ox/ ox, named as Mettl3nKO), Mettl14 (Ngn3-cre; b-cell number per section (369.6 6 45.1 vs. 819.4 6 217.2 fl fl Mettl14 ox/ ox, named as Mettl14nKO) or both Mettl3/ in WT, P , 0.05) (Fig. 2C and D). On the contrary, no fl fl fl fl Mettl14 (Ngn3-cre;Mettl3 ox/ ox/Mettl14 ox/ ox, named as differences were detected in the number of a, d and PP 1 Mettl3/14nKO) were specifically ablated in Ngn3 endo- cells per section between the two groups (Fig. 2C and D). fl fl crine progenitors (Fig. 1D). The Ngn3-Cre, Mettl3 ox/ ox, The above data indicated a preferential role of Mettl3/14 fl fl fl fl fl fl Mettl14 ox/ ox, and Mettl3 ox/ ox/Mettl14 ox/ ox mice were in b-cell development during early postnatal period (P0- all healthy and phenotypically normal, and thus, they were P14). In parallel, b-cell mass was 30% lower in Mettl3/ jointly used as WT littermate controls in the following 14nKO mice than in WT, while a-cell mass remained un- experiments. Successful KO of Mettl3 and Mettl14 were changed (Fig. 2E and F). The reduction in b-cell mass was confirmed by immunostaining: both METTL3 and METTL14 attributed to changes in both proliferation and apoptosis: 1 1 proteins were selectively absent in endocrine cells of P14 40% reduction in the proportion of Ki67 insulin cells Mettl3/14nKO mice (Fig. 1E). Moreover, m6A/A% levels in (Fig. 2G and Supplementary Fig. 2A) and a twofold increase 1 1 RNA extracted from Mettl3nKO and Mettl14nKO islets were in the percentage of TUNEL insulin cells (Fig. 2H and significantlyreducedcomparedwithWTcontrols,and Supplementary Fig. 2B) were found in 2-week-old Mettl3/ this decrease was more pronounced in Mettl3/14nKO islets 14nKO. b-Cell size was unchanged in mutant islets (Fig. 2I (Fig. 1F). and Supplementary Fig. 2C). Taken together, Mettl3/ At 2 weeks after birth, Mettl3nKO and Mettl14nKO had Mettl14 specifically regulated b-cell number via controlling comparable random blood glucose levels as WT controls neonatal b-cell proliferation and survival and, thus, de- (Fig. 1G). However, Mettl3nKO and Mettl14nKO began to termined postnatal b-cell mass formation during P0-P14. show significant increases in random blood glucose levels 6 6 Loss of Cell Identity and Function in Neonatal Mettl3/ at 4 weeks (14.34 3.26 vs. 9.95 1.18 mmol/L in WT, nKO P , 0.01) and 3 weeks (13.22 6 1.28 vs. 9.77 6 14 b-Cells 1.70 mmol/L in WT, P , 0.01) of age, respectively; and Neonatal b-cells are immature and undergo a dramatic their blood glucose rose gradually with age (Fig. 1G). At change in the early postnatal period to become functional 8 weeks of age, both Mettl3nKO and Mettl14nKO showed mature and acquire glucose responsiveness (3). We then a dramatic increase in glycemia after intraperitoneal glu- checked expression levels of the critical transcription cose injection (Fig. 1H and I). Notably, a more severe factors required for b-cell identity and function in WT nKO phenotype was observed in double KO mice: at 2 weeks and Mettl3/14 at P14. We found that Pdx1, MafA, and of age, Mettl3/14nKO mice already showed a slight increase Nkx6.1 were absent in a vast majority of Mettl3/14- 6 6 deficient b-cells at P14 (Fig. 3A–C). We then calculated in random blood glucose levels (9.00 1.45 vs. 7.88 1 1 1 1 1.22 mmol/L in WT, P 5 0.07), and they developed severe the percentages of Pdx1 insulin , MafA insulin , and 1 1 nKO hyperglycemia as early as 3 weeks of age (random blood Nkx6.1 insulin b-cells in P14 Mettl3/14 and WT glucose levels .20 mmol/L) and had persistently severe pancreas and found that they decreased about 63.3%, hyperglycemia (.30 mmol/L) thereafter (Fig. 1G). More- 49.0%, and 78.8%, respectively, in P14 mutants (Fig. – over, plasma insulin levels in 2-week-old Mettl3/14nKO 3D F). In parallel, the expression of Urocortin 3 (Ucn3) (3), a molecular marker of mature b-cells, was present in mice was reduced by 70%, suggesting b-cell failure pre- 1 dominated in these diabetic mutants (Fig. 1J). None of the a vast majority of insulin b-cells in WT, but it was mutants showed differences in body weight, compared remarkably decreased in P14 mutant b-cells (Fig. 3G). with their age-matched controls (Fig. 1K). Our data We also detected reduced expression of GLUT2 (35) in nKO also showed that insulin sensitivity was not impaired P14 Mettl3/14 (Fig. 3H). Then, we isolated primary nKO in 4-week-old male Mettl3/14nKO mice compared with WT islets from P14 WT and Mettl3/14 to evaluate their nKO (Supplementary Fig. 1A). Female Mettl3/14nKO mice were function in vitro. P14 Mettl3/14 islets displayed im- also diabetic at 2 weeks of age and manifested hypergly- paired insulin release at both basal (2.8 mmol/L) and high cemia thereafter (Supplementary Fig. 1B). glucose (16.7 mmol/L) levels (Fig. 3I). We further studied b-cell ultrastructure in P14 Mettl3/14nKO and WT pancreas Mettl3/14 Preferentially Regulates Postnatal b-Cell using transmission electron microscopy (Fig. 3J). On the Mass Establishment basis of the recognized criterion (36), we quantified the We performed immunostaining against four endocrine secretory vesicle subsets including mature (with dense core hormones including insulin (b), glucagon (a), somato- granules, red arrow), immature (with light core granules, statin (d), and pancreatic polypeptide (PP) on P0 pancreas green arrow), and empty vesicles (with no granules, blue 1712 m6A mRNA Methylation and Neonatal b-Cells Diabetes Volume 69, August 2020

Figure 1—Loss of m6A in Ngn31 endocrine progenitors leads to hypoinsulinemia and severe diabetes. A: Representative pancreatic sections from E17.5-P14 WT mice were immunostained for Mettl3 or Mettl14 (red) with insulin (green). B and C: Relative immune fluorescence intensity of Mettl3 (B) and Mettl14 (C) inside islets were determined at indicated times (n 5 3). D: Model illustration of the generation of Mettl3nKO, Mettl14nKO, and Mettl3/14nKO mice. E: Representative pancreatic sections from P14 Mettl3/14nKO and WT mice were immunostained for Mettl3 or Mettl14 (red) with Ins (white) and Gcg (green). F: Relative m6A amounts relative to adenosine (A) in mRNA extracted from the Mettl3nKO, Mettl14nKO, Mettl3/14nKO, and WT islets were quantified (n 5 3). G: Random blood glucose levels of Mettl3nKO, Mettl14nKO, Mettl3/ 14nKO, and WT mice were monitored weekly (n 5 4–9). H and I: Intraperitoneal glucose tolerance tests were performed on 8-week-old WT, Mettl3nKO (H), and Mettl14nKO (I) mice after overnight fasting (n 5 5). J: Plasma insulin levels of P14 Mettl3/14nKO and WT mice were determined (n 5 3–4). K: Body weights of Mettl3nKO, Mettl14nKO, Mettl3/14nKO, and WT mice were monitored weekly (n 5 4–9). Data are presented as mean 6 SEM of independent experiment indicated as above. *P , 0.05, **P , 0.01, ***P , 0.001 by Student t test. Nuclei were counterstained with DAPI (blue). Scale bars, 20 mm. arrow) (Fig. 3J). The proportion of mature vesicles was Mettl3/14 deficiency led to the loss of b-cell identity, significantly decreased and the ratios of immature and impaired glucose-induced insulin release, and defections empty vesicles were significantly increased in Mettl3/14- in secretory vesicle maturation in neonatal pancreatic 1 deficient b-cells (Fig. 3K). These results indicated that b-cells. Moreover, we detected substantial Aldh1a3 cells diabetes.diabetesjournals.org Wang and Associates 1713

Figure 2—Loss of m6A preferentially regulates postnatal b-cell mass establishment. A: Representative pancreatic sections immunostained for Gcg (red), Sst (red), PP (pancreatic polypeptide [Ppy], red) with Ins (green) in Mettl3/14nKO and WT pancreas at P0. B: The absolute cell number of a, b, d, and PP cells per pancreatic section of P0 Mettl3/14nKO and WT mice were determined (n 5 3). C: Representative pancreatic sections immunostained for Gcg (red), Sst (red), PP (Ppy, red) with Ins (green) in Mettl3/14nKO and WT pancreas at P14. D: The absolute cell number of a, b, d, and PP cells per pancreatic section at P14 Mettl3/14nKO and WT were determined (n 5 3). E–I: b-Cell mass (E), a-cell mass (F), the percentage of Ki671/insulin1 b-cells (G), the percentage of TUNEL1/insulin1 b-cells (H), and b-cell size (I) in P14 WT and Mettl3/14nKO mice were determined (n 5 3). Data are presented as mean 6 SEM of independent experiment indicated as above, *P , 0.05 by Student t test. Nuclei were counterstained with DAPI (blue). Scale bars, 20 mm. 1714 m6A mRNA Methylation and Neonatal b-Cells Diabetes Volume 69, August 2020

Figure 3—Loss of b-cell identity and function in P14 Mettl3/14nko mice. A–C: Representative pancreatic sections from P14 WT and Mettl3/ 14nKO were coimmunostained for Ins (green) and b-cell identity genes: Pdx1(red) (A); MafA (red) (B) and Nkx6.1 (red) (C). D–F: The percentages of Pdx11, MafA1, and Nkx6.11 b-cells in P14 WT and Mettl3/14nKO were determined (n 5 3). G: Representative pancreatic sections from P14 WT and Mettl3/14nKO were coimmunostained for Ins (green) and b-cell maturation marker Ucn3 (red). H: Representative pancreatic sections from P14 WT and Mettl3/14nKO mice were coimmunostained for Ins (green) and b-cell GLUT2 (red). I: Isolated P14 islets were incubated at 2.8 mmol/L or 16.7 mmol/L glucose for 1 h. Secreted insulin levels were measured and were normalized to total insulin in the islets (n 5 3). J and K: Representative transmission electron microscopy of islets from P14 WT and Mettl3/14nKO mice. Red and green arrows point to typical vesicles containing mature and immature granules. Blue arrows point to typical empty vesicles (J). Analysis of the percentage of mature, immature, and empty vesicles from P14 WT and Mettl3/14nKO mice (K)(n 5 3). L: Representative immunofluorescence stainings against Aldh1a3 (red) and insulin (green) in 8-week-old WT and Mettl3/14nKO pancreas sections are shown. M: Pancreatic sections diabetes.diabetesjournals.org Wang and Associates 1715 in 8-week-old Mettl3/14nKO islets (Fig. 3L). Meanwhile, we increased expression of proliferation inhibitors (Sfrp5, also performed immunostaining against chromogranin A Tlr2, Frzb, Lims2, and Klf11) (Supplementary Fig. 4B), (CGA) and four endocrine cell types (insulin [ins], glucagon diminished expression of genes related to ion transporta- [Gcg], somatostatin [Sst], and PP) in 8-week-old Mettl3/ tion and insulin secretion (Ero1lb, Nnat, Pcsk9, Trpm5, 14nKO and WT pancreas (Fig. 3M). We observed a dramatic Fkbp1b, Arrb1, Syt3, Flna, Sytl4, and Kcng3) (Supplemen- increase in the percentage of dedifferentiated cells (islet tary Fig. 4C). Taken together, both in vivo and in vitro, we hormone-negative and CGA-positive cells: 30.8 6 9.0% vs. showed that loss of Mettl3/14 impaired genes important 3.1 6 1.5%, Mettl3/14nKO vs. WT, P , 0.001, yellow for b-cell identity, insulin secretion, and proliferation. arrows in Fig. 3M) in 8-week-old Mettl3/14nKO pancreas sections. These results indicated that b-cells underwent Identification of m6A Targets in Neonatal Mettl3/14nKO dedifferentiation in 8-week-old Mettl3/14nKO mice. Islets 6 nKO To obtain insights into the transcriptome-wide m A Transcriptome Profile of P14 Mettl3/14 Islets distribution in neonatal b-cells, we performed MeRIP- To investigate the underlying mechanisms, we performed seq analysis using P14 mouse islets. Compared with the nKO RNA-seq analysis on P14 WT and Mettl3/14 islets. We unbound fractions, 8,987 methylation sites (correspond- fi detected signi cant differences in the expression levels of ing to 4,873 transcripts; fold change .2) were signifi- . , 544 genes (fold change 1.5, P 0.05), among which cantly enriched in m6A antibody-bound fractions and, 294 were upregulated and 250 were downregulated (Fig. thus, were identified as high-confidence m6Atargets.m6A 4A). We further analyzed alterations of known pathways peaks in islet transcripts were significantly enriched in using the ingenuity pathway analysis and listed the top GGACU motif (Fig. 4D). m6A was distributed throughout downregulated and upregulated signaling pathways in P14 islet mRNA transcripts and were abundant predomi- nKO Mettl3/14 islets (Fig. 4B). Pathways related to insulin nantly in the coding sequences (Fig. 4E). Gene Ontology secretion, ion transportation, and response to glucose were (GO) analysis showed m6A-containing mRNAs were among the top downregulated cellular biological processes, enriched in biological processes related to RNA Polymer- while the Wnt-signaling pathway, negative regulation of ase II Transcription, cell projection organization, RNA intracellular signal transduction, and negative regulation Metabolism, protein ubiquitination, mRNA metabolic fi of cell proliferation pathway were signi cantly upregulated process, and cell morphogenesis involved in differentia- in mutant islets (Fig. 4B and C). Interestingly, several tion (Fig. 4F). crucial b-cell transcription factors Pdx1, Nkx6.1, and To identify the direct m6A-modified targets, we corre- MafA were downregulated at P14, among which MafA lated MeRIP-seq results with RNA-seq data identified from fi was the most signi cantly repressed gene after loss of P14 islets. The correlation analysis eventually identified Mettl3/14 (Fig. 4C). Moreover, a cluster of genes involved 170 differentially expressed genes with m6A modifications in ion transportation and insulin secretion were reduced in (Fig. 4G). Several m6A-modified genes which were impor- mutant islets (i.e., Fkbp1b, Ero1lb, Sytl4, Maob, Nnat,and tant for b-cell function, such as MafA (11), Maob (37), Trpm5) (Fig. 4C). Loss of Mettl3/14 also affected expres- Fkbp1b (38), and Kcng3 (39) were identified as direct m6A sions of genes important for b-cell proliferation, including targets, and their expressions were significantly down- downregulation of several positive proliferation regulators regulated in neonatal Mettl3/14nKO islets (Fig. 4H and (i.e., Nkx6.1, Rtkn2, Nasp, and Plk3) and upregulation of Supplementary Fig. 5A–C). On the contrary, Sfrp5 (40), some proliferation inhibitors (i.e., Sfrp5, lims2, and Klf11) a well-established proliferation inhibitor in b-cells, was (Fig. 4C). The dramatic reduction in Pdx1, Nkx6.1, MafA, upregulated in mutants (Supplementary Fig. 5D). and Trpm5 mRNA levels were still observed in 8-week-old Mettl3/14nKO islets (Supplementary Fig. 3A). The above Mettl3/14-Mediated m6A Modification Regulates MafA results reinforced that methyltransferase-like Mettl3/14 Expression via Modulating mRNA Stability indeed regulated the expression of those critical b-cell We listed the top five downregulated and upregulated genes. m6A-modified genes that were preferentially changed in To identify the direct effects of Mettl3/14, we treated neonatal Mettl3/14nKO mice (Fig. 4H), among which MafA MIN6 cells with Mettl3 and Mettl14 shRNA lentiviruses. was identified as the most significantly reduced gene (Fig. Successful knockdown of Mettl3 and Mettl14 signifi- 4H). Integrative Genomics Viewer tracks showed MafA cantly reduced mRNA expression of b-cell transcription transcripts had abundantly enriched m6A peaks spread its factors Pdx1, Nkx6.1, and MafA (Supplementary Fig. 4A), mRNA, which indeed had the highest methylation score

of 8-week-old WT and Mettl3/14nKO immunostained with insulin (green), endocrine cocktail (Gcg/Sst/PP, red), and CGA (white) are shown. Yellow arrows indicated the dedifferentiated cells (endocrine hormone–negative/CGA-positive). N: The ratio of dedifferentiated (Dediff) cells was determined (n 5 3). Data are presented as mean 6 SEM of independent experiment indicated as above. **P , 0.01, ***P , 0.001 by Student t test. Nuclei were counterstained with DAPI (blue). Scale bars, 20 mm or 500 nm. 1716 m6A mRNA Methylation and Neonatal b-Cells Diabetes Volume 69, August 2020

Figure 4—Transcriptome and m6A methylome profile of P14 Mettl3/14nKO islets. A: Scatter plots of relative expression levels of 544 differentially expressed genes identified from RNA-seq of P14 WT and Mettl3/14nKO islets. Upregulated genes are marked in red, and downregulated genes are marked in blue (fold change .1.5, P , 0.05). B: GO analysis of differentially expressed genes as identified by RNA-seq of P14 WT and Mettl3/14nKO associated with b-cell function. TOR, target of rapamycin. C: The heatmap shows relative expression levels of genes critical for b-cell identity, insulin secretion, ion transportation, and proliferation inhibition between P14 WT and Mettl3/14nKO. D: Sequence motif identified within m6A peaks in P14 islets by HOMER database. E: Pie chart depicting the fraction of m6A peaks in five transcript segments in P14 WT islets. TSS, transcriptional start site. F: GO analysis of enriched signaling pathways of m6A target genes in P14 islets. G: Venn diagram showed the overlap between genes with m6A modifications and genes that were differentially expressed in RNA-seq of P14 Mettl3/14nkO. H: Top five downregulated and upregulated genes in the intersection of m6A-modified genes and differentially expressed diabetes.diabetesjournals.org Wang and Associates 1717 among all enriched transcripts (Fig. 4H and I). On the of P0 Mettl3/14nKO islets, whereas no reductions in protein contrary, the other two preferentially changed transcrip- levels were detected in Pdx1 and Nkx6.1 at this early time tion factors, Pdx1 and Nkx6.1, showed no m6A modifica- point (Fig. 5E). We also found slightly diminished GLUT2 tions in murine b-cells as identified in our MeRIP-seq. expression in P0 Mettl3/14nKO b-cells (Fig. 5E). However, To determine the direct effect of Mettl3/14 on MafA Ucn3 was rarely expressed in new-born b-cells (Supple- protein expression, we then treated MIN6 cells with mentary Fig. 7A). Moreover, we performed Western blot ShMettl3/14 virus for 48 h and found reduced MafA on isolated islets from P0 WT and mutant mice. Again, we protein abundance after loss of Mettl3 and/or Mettl14 found reduced MafA protein abundance in P0 Mettl3/14- in MIN6 cells (Fig. 4J). These results supported that deficient islets (Fig. 6F and H), whereas the expression Mettl3/14 directly regulated MafA expression in b-cells. levels of Pdx1 and Nkx6.1 were comparable to that of WT It is known that m6A modifications on mRNA tran- islets (Fig. 6G and H). The above data indicated that m6A- scripts might affect mRNA stability and translation (17). induced MafA change was prior to that of Pdx1 and Nkx6.1 We then treated control or Mettl3/14-deficient MIN6 cells at P0. Indeed, we found impaired glucose responsive in- with transcription inhibitor actinomycin D and checked sulin secretion ability in P0 Mettl3/14nKO islets. The the role of m6AonMafA mRNA decay. We found that mutant islet displayed comparable basal insulin secretion knockdown of Mettl3/14 significantly decreased the at2.8mmol/Lglucose,buttheyhadatrendtoward mRNA stability of MafA (Fig. 4K), but had no effect on reduced GSIS response under high glucose (16.7 mmol/ Pdx1, Nkx6.1 mRNA stability (Supplementary Fig. 6A and L) stimulation (P 5 0.059) (Fig. 5I). B). These findings indicated that Mettl3/14 directly regulates MafA expression in neonatal b-cells, at least partly, Reduced Mettl3 and Mettl14 Expression in db/db Mice via modulating its mRNA stability. and Patients With T2D To evaluate a possible role of Mettl3/14-mediated m6A Transcriptome Profile and Functional Analysis on P0 RNA methylation in the pathology process of T2D, we Mettl3/14nKO Islets checked the expression of these two methyltransferases To exclude the possibility that Mettl3/Mettl14 inactiva- in islets of lean control and db/db mice at 8 weeks of age tion compromised b-cell differentiation that contributed (Fig. 6A). The random blood glucose level of db/db mice fi 6 to later b-cell functional immaturity, comprehensive gene was signi cantly higher than lean controls (23.2 3.5 6 , expression analyses on P0 islets were performed. At birth, vs. 8.2 0.6 mmol/L, db/db vs. WT, P 0.05). We found mutant mice showed comparable blood glucose levels Mettl3 and Mettl14 were abundantly expressed in both 6 6 5 endocrine and exocrine cells in pancreas of lean control (5.37 0.98 vs. 4.23 0.92 mmol/L in WT, P 0.17) 1 mice; however, by counting the percentage of Mettl3 or and islet composition (Fig. 2A and B) compared with WT. 1 RNA-seq on isolated P0 islets identified 945 differentially Mettl14 b-cells in diabetic db/db mice, both of their fi expressed genes (fold change .1.5, P , 0.05), among expressions in b-cells were signi cantly reduced by 31.3% which 191 were upregulated and 754 were downregulated and 27.1%, respectively, compared with lean control mice – (Fig. 5A). Pathway analysis identified that pathways re- (Fig. 6A C). lated to the response to external stimulus, developmental We also evaluated the expression of METTL3 or fi process, metabolic process, and regulation of secretion METTL14 in b-cells in pancreas sections of ve patients fi were downregulated, while pathways like response to with T2D and ve age- and BMI-matched ND patients who stress, defense response, and the apoptosis process were had performed partial pancreatectomy (Supplementary Ta- significantly upregulated (Fig. 5B). We further compared ble 1). According to American Diabetes Association guide- fi these genes with the MeRIP-seq database and identified lines, all the ve patients with T2D were under adequate 326 differentially expressed genes with m6A modifications glucose control with antidiabetic therapy (Supplementary (Fig. 5C and D). Among them, genes related to ion trans- Table 1). We found METTL3 and METTL14 were present in portation and insulin secretion (i.e., Fxyd5, Slc3a2, Vamp8, islet endocrine cells in the ND group, including both and Syt9) were significantly downregulated (Fig. 5D). a-cells and b-cells. In contrast, islets from patients with T2D Importantly, although similar expression levels of MafA, showed dramatic reductions in METTL3 and METTL14 Nkx6.1,andPdx1 mRNA were observed in RNA-seq, by expression (Fig. 6D). We further calculated the percentage 1 1 in situ immunostaining, we still detected significantly of METTL3 or METTL14 b-cells in the two groups: 1 1 reduced MafA protein abundance in insulin-positive cells in diabetic islets, the ratio of METTL3 /insulin cells

genes in RNA-seq are presented in the table, respectively. I: Integrative Genomics Viewer tracks showed RNA-seq reads distribution in MafA mRNA of P14 WT and Mettl3/14nKO (upper panel) and showed MeRIP-seq reads distribution in MafA mRNA (lower panel). J: MIN6 cells were transfected with Lv-ShMettl3, Lv-ShMettl14, Lv-ShMettl3/14, or control virus for 48 h; then, MafA protein abundance was assayed by immunoblot. K: The MafA mRNA remaining after actinomycin D (ActD) treatment with or without ShMettl3/14 in MIN6 cells is shown. (n 5 3) Data are presented as mean 6 SEM of independent experiments indicated as above. *P , 0.05. **P , 0.01 by one-way ANOVA. CDS, coding sequences. 1718 m6A mRNA Methylation and Neonatal b-Cells Diabetes Volume 69, August 2020

Figure 5—Mettl3/14 regulates MafA expression to drive b-cell functional maturation. A: Scatter plots of relative expression levels of 945 differentially expressed genes identified from RNA-seq of P0 WT and Mettl3/14nKO islets (fold change .1.5, P , 0.05). B: GO analysis of differentially expressed genes as identified by RNA-seq of P0 WT and Mettl3/14nKO. C: Venn diagram shows the overlap between genes with m6A modifications and genes that were differentially expressed in RNA-seq of P0 Mettl3/14nkO. D: Heatmap shows relative expression levels of these 326 overlapped genes between P0 WT and Mettl3/14nKO. E: Representative pancreatic sections from P0 WT and Mettl3/14nKO mice were coimmunostained for Ins (green) and b-cell identity genes: MafA/Pdx1/Nkx6.1 (red), Ins (green), and GLUT2 (red). F and G: P0 WT and Mettl3/14nKO islets were isolated, and then Mettl3, Mettl14, and MafA (F) and Pdx1 and Nkx6.1 (G) protein abundances were assayed by immunoblot. H: Band intensities of MafA, Pdx1, and Nkx6.1 proteins were normalized based on the corresponding GAPDH intensity (n 5 3). I: P0 WT and Mettl3/14nKO islets were isolated and incubated with 2.8/16.7 mmol/L glucose for 1 h. Secreted insulin was normalized to total insulin in the islets (n 5 4–5). Data are presented as mean 6 SEM, **P , 0.01 by Student t test. Nuclei were counterstained with DAPI (blue). Scale bars, 20 mm. diabetes.diabetesjournals.org Wang and Associates 1719

Figure 6—Reduced Mettl3 and Mettl14 expression in db/db mice and patients with T2D. A: Representative pancreatic sections from db/ db and lean control mice were coimmunostained for Ins (white), Gcg (green), Mettl3 (red), or Mettl14 (red). B and C: The percentages of Mettl3 or Mettl14-positive/insulin-positive cells in db/db and lean control mice were calculated (n 5 3). D: Representative images of pancreatic sections from patients with T2D and those who were ND stained for Ins (white), Gcg (green), Mettl3 (red), or Mettl14 (red) are shown. BMI values are given in kg/m2. E and F: The ratio of Mettl3- or Mettl14-positive/insulin-positive cells in patients with T2D and in patients who were ND were determined (n 5 5). Data are presented as mean 6 SEM. **P , 0.01 by Student t test. Nuclei were counterstained with DAPI (blue). Scale bars, 20 mm.

(50.1 6 16.2% vs. 95.6 6 0.6%, T2D group vs. ND group, types (i.e., hematopoietic stem/progenitor cells or sper- 1 1 P , 0.01) (Fig. 6E) and METTL14 /insulin cells (55.3 6 matogenesis) (23–25). Germ cell–specific deletion of the 9.9% vs. 94.2 6 3.0%, T2D group vs. ND group, P , 0.01) m6A RNA methyltransferase Mettl3 or Mettl14 disrupted (Fig. 6F) declined substantially. These observations indi- spermiogenesis through inhibiting diploid spermatogonia cated that loss of Mettl3/14 might be involved in patho- stem cells proliferation/differentiation (24). Neural stem genesis of T2D in both rodents and human. cells lacking Mettl14 displayed markedly decreased proliferation and premature differentiation (42). During DISCUSSION zebrafish embryogenesis, Mettl3-depletion led to contin- Recent studies have reported the role of posttranscrip- uous activation of the Notch signaling pathway, ultimately tional mRNA modifications on organ development, cellular resulting in the blockage of endothelial-to-hematopoietic function, and human diseases, thus highlighting its in- transition to specify the earliest hematopoietic stem/pro- volvement in both physiological and pathological status genitor cells (25). Very recently, Men et al. (31) reported (19,23–25,41). Dynamic m6A modification was known to that Mettl14 deficiency in b-cells resulted in defects affect development and fate determination of various cell of b-cell survival and insulin secretion (30). However, 1720 m6A mRNA Methylation and Neonatal b-Cells Diabetes Volume 69, August 2020 whether m6A modification was implicated in neonatal analysis of neonatal islets demonstrated abundant m6A islet development and the detailed regulation mecha- modifications in MafA mRNA, which were mostly located nisms remained unknown. in the exon region, whereas Pdx1 and Nkx6.1 mRNA had In the current study, by using transgenic mice in which rather weak or no m6A modifications. Mechanical studies 1 Mettl3/14 were specifically ablated in Ngn3 endocrine proved that knockdown of Mettl3/14 reduced MafA mRNA progenitors, we observed that the composition of the four stability and shortened its mRNA half-life, while had no endocrine cell types (a, b, d, and PP cells) remained un- effect on that of Pdx1 and Nkx6.1. These observations changed in WT and Mettl3/14nKO at birth. Strikingly, a dra- suggest m6A/MafA serves as a direct trigger of functional matic drop in b-cell number and mass during the weaning maturation process in neonatal b-cells. period (P0-P14) led to the conclusion that m6Aisessential To exclude the possibility that Mettl3/Mettl14 inacti- for neonatal b-cell mass establishment. Interestingly, this vation compromised b-cell differentiation that contrib- regulation seems to be specifictob-cells, since Mettl3/14 uted to later b-cell functional immaturity, comprehensive deficiency had no effect on a, d,andPPcellformationand gene expression analyses in P0 b-cells were performed. In 1 expansion. Since Ngn3 cells would also give rise to enter- our P0 RNA-seq, we did not detect significant differences oendocrine cells, the interaction between enteroendocrine in MafA mRNA, which might be caused by their relatively cells and endocrine cells should also be considered. low expression level at this early time point. However, we The postnatal weaning period is pivotal for immature confirmed MafA downregulation at the protein level in P0 b-cells to acquire their glucose-responsive insulin-producing mutant islets by both immunofluorescence staining and mature phenotype (3). Previous studies have provided Western blot. The downregulation in MafA expression was evidence on transcriptional factors (i.e., Nkx6.1, MafA, accompanied with reduced GLUT2 expression and im- and Pdx1) (9–11) and cellular signals (i.e., mechanistic paired GSIS in P0 mutant islets, when the islet composi- target of rapamycin [mTOR], connective tissue growth tion and blood glucose level were unaffected. Interestingly, factor [CTGF], and estrogen-related receptor g [ERRg]) m6A-induced MafA alteration at P0 preceded and was (8,43,44) in modulating b-cell functional maturation followed by the changes in Nkx6.1 and Pdx1. It is well and/or postnatal expansion. In recent years, epigenetic accepted that Pdx1 and Nkx6.1 are essential for b-cell modifiers were confirmed to be involved in the functional differentiation (47–50). On the contrary, MafA is not maturation of b-cells in the postnatal period to promote involved in endocrine specification during embryonic de- adequate metabolic reprogramming (5,8,13). For the first velopment but is critical for b-cell functional maturation time, we demonstrated that Mettl3 and Mettl14 were (51,52). After birth, MafA expression remains low, and it essential for functional maturation of neonatal murine increases gradually with age (53). The role of MafA on b-cells. First, the loss of Mettl3/14 compromised expres- regulating replication/survival and function of b-cells is sion of critical b-cell transcription factors Pdx1, MafA, and gradually increased after birth, but it is not conspicuous at Nkx6.1, which were reported to modulate b-cell maturation birth (54). Our findings provide evidence that Mettl3/14 and function. Second, Mettl3/14-deficient b-cells exhibited regulates MafA expression and, thus, controls b-cell func- reduced expression of mature b-cell marker Ucn3 and tional maturation after birth. GLUT2. Third, impaired glucose-induced insulin release Indeed, our Mettl3/14nKO mice were to some extent was detected in Mettl3/14nKO b-cells, with defects in genes phenotypically similar to MafAKO mice (11): both of them responsible for glucose sensing (loss of GLUT2) (35), in- showed normal islet morphology and comparable b-cell sulin biosynthesis (loss of Ero1lb) (45), and granule dock- numbers at birth but a progressive loss of b-cell identity ing (loss of Sytl4) (46). Fourth, the secretory vesicles in and function with age. The link between m6A/MafA mod- Mettl3/14nKO b-cells were mostly immature. Interestingly, ulation in neonatal b-cells indicates that this finely tuned the pattern of Mettl3 and Mettl14 expression during the regulation network is crucial for proper b-cell maturation physiological maturation window in murine b-cells fur- during a critical time window. Interestingly, like other di- ther supported our observation. The relatively low levels abetic mice models, we observed a dramatic increase in the of Mettl3/14 at E17.5 and P0 might explain that they are percentage of dedifferentiated b-cells in 8-week-old Mettl3/ dispensable for cell lineage formation during differentiation. 14nKO islets. Considering the high percentage of dedifferen- The strong induction of Mettl3/14 during early postnatal tiation (30%) and the relatively short hyperglycemic period period P0-P14 and high expression level throughout adult- (8 weeks old), the possible role of Mettl3/14 on b-cell hood reinforced the importance of Mettl3/14 in driving dedifferentiation, independent of hyperglycemia, needs to b-cell functional maturation and maintaining b-cell function. be further clarified. Moreover, it would be more ideal to Combined analysis of P14 RNA-seq and MeRIP-seq perform RNA-seq analyses on purified b-cells from earlier identified that MafA was the direct target of Mettl3/14 developmental times instead of P14 islets to fully clarify the for identity maintenance and functional maturation of identical regulatory mechanisms in Mettl3/14 KO b-cells. neonatal b-cells. In Mettl3/14nKO pancreas, remarkably Using diabetic mouse models, we and others have diminished MafA expression was found as early as P0, shown that loss of Mettl3/14 in b-cells is one of the before changes of other transcriptional factors (Pdx1 and critical mechanisms in b-cell failure during the pathology Nkx6.1) and islet morphology. Moreover, our MeRIP-seq of diabetes (30,31). The current study provided observational diabetes.diabetesjournals.org Wang and Associates 1721 evidence on remarkably reduced METTL3 and METTL14 7. Otonkoski T, Andersson S, Knip M, Simell O. Maturation of insulin response to expression in islets from patients with T2D compared glucose during human fetal and neonatal development. Studies with perifusion of with patients who were ND, revealing the significance of pancreatic isletlike cell clusters. Diabetes 1988;37:286–291 Mettl3/14-mediated m6A signaling in the occurrence and 8. Ni Q, Gu Y, Xie Y, et al. 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Gene expression regulation mediated 6 Mettl3/14-mediated m Amodification in driving functional through reversible m⁶A RNA methylation. Nat Rev Genet 2014;15:293–306 maturation in neonatal b-cells, which might be a potential 17. Roundtree IA, Evans ME, Pan T, He C. Dynamic RNA modifications in gene target for diabetes therapy. expression regulation. Cell 2017;169:1187–1200 18. Lee M, Kim B, Kim VN. Emerging roles of RNA modification: m(6)A and U-tail. Cell 2014;158:980–987 19. Lence T, Akhtar J, Bayer M, et al. m6A modulates neuronal functions and sex Acknowledgments. The authors thank Prof. Ming-Han Tong (University of fl fl determination in Drosophila. Nature 2016;540:242–247 Chinese Academy of Sciences, Shanghai, China) for providing the Mettl3 ox/ ox and fl fl 20. Geula S, Moshitch-Moshkovitz S, Dominissini D, et al. Stem cells. m6A mRNA Mettl14 ox/ ox mice and for his valuable discussion and comments for the study. methylation facilitates resolution of naïve pluripotency toward differentiation. The authors thank Prof. Wei-Zhen Zhang (Department of Physiology and Path- Science 2015;347:1002–1006 ophysiology, School of Basic Science, Peking University Health Science Center, 21. Li HB, Tong J, Zhu S, et al. m6A mRNA methylation controls T cell ho- Beijing, China) for providing the Ngn3-cre mice. meostasis by targeting the IL-7/STAT5/SOCS pathways. Nature 2017;548:338– This work was supported by National Natural Sciences Foundation of Funding. 342 China grants 81670700 and 81870527. 22. Yoon KJ, Ringeling FR, Vissers C, et al. Temporal control of mammalian Duality of Interest. No potential conflicts of interest relevant to this article cortical neurogenesis by m6A methylation. Cell 2017;171:877–889.e17 were reported. 23. Xu K, Yang Y, Feng GH, et al. 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