Abstract
As one of the most abundant DNA methylation form in prokaryotes, N6-methyladenine nucleotide (6 mA) was however only recently identified in eukaryotic genomes. To explore the implications of N6-adenine methylation in adipogenesis, genomic N6-adenine methylation was examined across adipocyte differentiation stages of 3T3-L1 cells. When the N6-adenine methylation profiles 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 differentiation, shown by defective gene regulation and impaired lipid production. We also found that the effects 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 differentiation stage.
Highlights
As a major epigenetic modification, genomic DNA methylation is of critical importance to many cellular processes such as expressional regulation, imprinting, X chromosome inactivation, and tumorigenesis[1,2]
We examined the genomic distribution of 6 mA sites (6 mA) by means of immunoprecipitation/sequencing assay (6mA-IP-seq), and explored its roles in adipocyte differentiation of 3T3-L1 cells and the underlying mechanisms
After MDI induction on day(0), the cells proceeded to a mitotic clonal expansion stage and entered into a terminal differentiation stage from day(+2) on
Summary
As a major epigenetic modification, genomic DNA methylation is of critical importance to many cellular processes such as expressional regulation, imprinting, X chromosome inactivation, and tumorigenesis[1,2]. 5-methylcytosine (5mC) has been known to be the only DNA methylation form in eukaryotic genomes[3]. Induced by an MDI cocktail consisting of insulin, dexamethasone and 3-isobutyl-1-methyxanthine, the preadipocytes exit from cell proliferation and enter into terminal differentiation stage, to generate matured adipocytes. During this process, the cells undergo stages of growth-arrest’, hormone induction, mitotic clonal expansion, and terminal differentiation. We examined the genomic distribution of 6 mA by means of immunoprecipitation/sequencing assay (6mA-IP-seq), and explored its roles in adipocyte differentiation of 3T3-L1 cells and the underlying mechanisms
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