www.nature.com/scientificreports OPEN Regulation of Adipocyte Diferentiation by METTL4, a 6 mA Methylase Zhenxi Zhang1, Yingzi Hou1, Yao Wang1, Tao Gao1, Ziyue Ma1, Ying Yang2, Pei Zhang1, Fan Yi1, Jun Zhan1,3, Hongquan Zhang1,3 & Quan Du1 ✉ As one of the most abundant DNA methylation form in prokaryotes, N6-methyladenine nucleotide (6 mA) was however only recently identifed in eukaryotic genomes. To explore the implications of N6- adenine methylation in adipogenesis, genomic N6-adenine methylation was examined across adipocyte diferentiation stages of 3T3-L1 cells. When the N6-adenine methylation profles were analyzed and compared with the levels of gene expression, a positive correlation between the density of promoter 6 mA and gene expression level was uncovered. By means of in vitro methylation and gene knockdown assay, METTL4, a homologue of Drosophila methylase CG14906 and C. elegans methylase DAMT-1, was demonstrated to be a mammalian N6-adenine methylase that functions in adipogenesis. Knockdown of Mettl4 led to altered adipocyte diferentiation, shown by defective gene regulation and impaired lipid production. We also found that the efects of N6-adenine methylation on lipid production involved the regulation of INSR signaling pathway, which promotes glucose up-taking and lipid production in the terminal diferentiation stage. As a major epigenetic modifcation, genomic DNA methylation is of critical importance to many cellular pro- cesses such as expressional regulation, imprinting, X chromosome inactivation, and tumorigenesis1,2. Until very recently, 5-methylcytosine (5mC) has been known to be the only DNA methylation form in eukaryotic genomes3. Tis is however diferent from that of prokaryotes, in which 6 mA is another major DNA methylation form and involved in expressional regulation, genome replication as well as restriction-modifcation systems4,5. Te presence of 6 mA was recently revealed in diverse eukaryotic genomes, including Chlamydomonas reinhardtii, Caenorhabiditis elegans, Drosophila melanogaster, zebrafsh, pig, Arabidopsis thaliana, mouse and humans6–13. ALKBH1 was identifed in 2016 as a 6 mA demethylase involved in the regulation of long interspersed nuclear element1 (LINE-1)13. More recently, 6 mA was found to be involved in tumorigenesis, and its efects were medi- ated by ALKBH1 and N6AMT1. N6AMT1 was reported in 2018 to be the frst mammalian 6 mA methylase14. Despite those progresses, many essential aspects of the metabolism and biological consequences of 6 mA remain to be known. Adipocyte diferentiation of 3T3-L1 preadipocytes has been used as a classic in vitro model system of adi- pogenesis. Induced by an MDI cocktail consisting of insulin, dexamethasone and 3-isobutyl-1-methyxanthine, the preadipocytes exit from cell proliferation and enter into terminal diferentiation stage, to generate matured adipocytes. During this process, the cells undergo stages of growth-arrest’, hormone induction, mitotic clonal expansion, and terminal diferentiation. Widespread epigenetic modifcations have been recently revealed during the diferentiation, shown by dynamic chromosome modifcations and complex gene regulation15. Genomic 5mC methylations have been found to be involved in this process, regulating the expression of many adipogenic factors such as Fabp4, Glut4 and Lep16–18. However, whether N6-adenine methylation is also involved in the MDI-induced maturation of adipocytes is still unknown. In the present study, we examined the genomic distribution of 6 mA by means of immunoprecipitation/sequencing assay (6mA-IP-seq), and explored its roles in adipocyte diferenti- ation of 3T3-L1 cells and the underlying mechanisms. 1State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China. 2Department of Stomatology, Beijing Friendship Hospital, Capital Medical University, 95 Yong’an Road, Western District, Beijing, 100050, China. 3Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Health Science Center, Beijing, 100191, China. ✉e-mail: quan. [email protected] SCIENTIFIC REPORTS | (2020) 10:8285 | https://doi.org/10.1038/s41598-020-64873-w 1 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 1. Profling of N6-adenine methylation in adipocyte diferentiation of 3T3-L1 cells. (a) Staining of cellular lipid of preadipocytes on Day(0), diferentiating adipocytes on Day(+4), and mature adipocytes on Day(+8). Lipid staining was performed with Bodipy 493/503 (Green) and Oil red O (Red), nuclei were co- stained with Hoechst 33342 (Blue). (b) Expression profles of Cebpa and Pparg were determined by qRT-PCR, in relative to the expression of β-actin. (c) Workfow to quantify 6 mA abundance of genome DNA sample. (d) Dot blot assay of N6-adenine methylation. NC, a DNA oligo without N6-adenine methylation. 6mA-oligo, a DNA oligo containing one N6-adenine methylation. Genome DNA, genome DNAs extracted from 3T3-L1 preadipocytes. (e,f) Abundances of 6 mA and 5mC methylation were quantifed using HPLC-MS/MS analysis. Molar ratios of 6 mA to dA were calculated, and molar ratios of 5mC to dC were calculated. Data are presented as mean ± s.d, n > 3 independent assay. *P < 0.05; **P < 0.01; ***P < 0.001. Results Dynamic changes of genomic N6-adenine methylation during adipocyte diferentiation. To explore the involvement of N6-adenine methylation in adipogenesis, the extents of genome-wide modifcation were determined during the diferentiation of 3T3-L1 cells. Upon induction with MDI, proliferating 3T3-L1 cells undergo the process of diferentiation, which recapitulates the major events occurring in vivo. To facilitate the description of the process, the day to perform MDI induction is called day(0). Accordingly, the cells grew to confuence on day(−2) and entered into the “growth-arrest” stage from day(−2). Afer MDI induction on day(0), the cells proceeded to a mitotic clonal expansion stage and entered into a terminal diferentiation stage from day(+2) on. To monitor the progress of diferentiation, Bodipy and Oil red O staining were performed (Fig. 1a). In addi- tion to checking the production of cellular lipids, regulation of the major adipogenic factors were also examined by gene expression assay (Fig. 1b). By means of dot blot assay, the presence of genomic N6-adenine methylation was confrmed in proliferating 3T3-L1 cells (Fig. 1c,d). To rule out potential contamination of bacteria or myco- plasma, assays were performed with universal 16 S primer sets (Supplementary Fig. S1a)10, and a commercial mycoplasma detection kit (Supplementary Fig. S1b). To quantitatively determine the presence of 6 mA, genomic DNAs were extracted from the cells collected on day(−2) before MDI treatment and day(0), day(+2) and day(+4) afer MDI treatment, representing proliferating preadipocytes, growth-arresting cells, diferentiating adipocytes, and matured adipocytes. DNA samples were then sequentially degraded with RNase, DNase I and nuclease P1. Te resulting nucleoside products were sepa- rated and quantifed using a LC-MS/MS system19, with dA, dC, 5mC and 6 mA as the standards. Te abundance of genomic 6 mA was presented as the molar ratio to dA. In proliferating preadipocytes, a relatively low level (0.01%) of 6 mA was found. With the progress of diferentiation, 6 mA levels gradually increased and reached a peak of 0.06% at the terminal stage (Fig. 1e). Comparing with 6 mA, 5mC showed a relatively stable profle during the process (Fig. 1f). SCIENTIFIC REPORTS | (2020) 10:8285 | https://doi.org/10.1038/s41598-020-64873-w 2 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 2. 6mA-IP-seq and RNA-seq of mature adipocytes. (a) Genome-wide distribution of N6-adenine methylation. (b) Distribution profles of 6 mA in transposon elements. (c) Chromosome-wide distribution of 6 mA. For each chromosome, a chromosome length ratio was calculated by dividing its length in nucleotide against the total length of the genome, a 6 mA ratio was calculated by dividing the number of its 6 mA sites against the number of the total 6 mA sites. Ten, an 6 mA abundance index was calculated by subtracting the 6 mA ratio by its chromosome length ratio. (d) Sequence motifs of 6 mA sites. (e) Comparison of the median and interquartile range of gene expression between genes without 6 mA sites (Non-6mA), genes with 6 mA sites (6 mA) and genes with 6 mA sites around TSS region (TSS), in 3T3-L1 cell of Day(+4). (f) 6 mA profle around TSS region, for genes with high (FPKM > 50) and low (FPKM < 50) expression level. Reads densities were normalized by RPKM algorithm. Genomic N6-adenine methylation detected by immunoprecipitation and sequenc - ing. Genomic N6-adenine methylation was further investigated by immunoprecipitation and sequencing (6mA-IP-seq). 6mA-IP-seq was performed with DNA samples of preadipocytes and matured adipocytes. Te genomic DNAs were sonicated into fragments of 100–500 bp, ligated to adaptors with unique sequence index, denatured to single-stranded DNA, and immuno-precipitated to obtain fragments carrying N6-adenine meth- ylation. 6mA-containing fragments were PCR amplifed, and sequenced on an Illumina HiSeq. 2000 platform. From a total of 118 million sequencing reads, 114 million clean reads were obtained. Tis led to a clean read ratio of 0.9689, much higher than the threshold ratio of 0.75 that indicated a high-quality sequencing data. Sequencing reads were then mapped to mouse reference genome (NCBI37/mm10), using TopHat