| DNA methylation is a conserved epigenetic marker that is important in genome organization,gene expression,genomic imprinting,paramutation,and X chromosome inactivation.DNA methylation patterns are coordinately determined by methylation and demethylation process in plants and animals.In Arabidopsis thaliana,DNA methylation are established by the RNA-directed DNA methylation(RdDM)pathway,and are maintained via the action of several DNA methyltransferases,i.e.,MET1,CMT3,DRM2,and CMT2.The active removal of 5mC in Arabidopsis is carried out by a subfamily of bifunctional DNA glycosylases/lyases represented by REPRESSOR OF SILENCING 1(ROS1)and DEMETER(DME).ROS1 family proteins bind DNA nonspecifically and depend on other factors to help them find their targets.ROS4/INCREASED DNA METHYLATION 1(IDM1)is a plant homeodomain-finger-containing histone acetyltransferase that catalyzes the acetylation of histone H3 lysine 18(H3K18)and lysine 23(H3K23)to create a favorable chromatin environment for the recruitment of ROS1 at some loci.ROS4/IDM1,together with other factors such as ROS5/IDM2,IDM3,methyl-CPG-binding domain 7(MBD7),Harbinger transposon-derived protein 1(HDP1),and HDP2,forms a complex to regulate active DNA demethylation.To identify new anti-silencing factors in regulating DNA methylation,a forward genetic screen was perfonned on the ProRD29A:LUC/Pro35S:NPT? transgenic C24 in which DNA methylation levels in the NOS terminator of NPT? was regulated by ROS1 and ROS4.We identified a new anti-silencing factor,Acyl-CoA oxidases 4(ACX4),an enzyme in the first step of the fatty acid ?-oxidation pathway.Significant increase in CG,CHG,and CHH DNA methylation levels in the NOS terminator was observed in acx4.Whole-genome bisulfite sequencing studies revealed that acx4-hyper-DMRs are also regulated by rosl and ros4.The fact that the expression levels of genes near the acx4-hyper-DMRs were reduced in the acx4 and rosl mutants,and no additive effect on gene expression in acx4rosl relative to acx4 and rosl,suggested that ACX4 regulate transcriptional gene silencing in ROS1 demethylation pathway.In addition,total histone acetylation levels was reduced in acx4.In acx4 hyper-DMRs,the levels of H3K18Ac were reduced relative to the WT,suggesting a close connection between DNA methylation and histone acetylation at these loci.Furthermore,the hyper-DMRs caused by the mutations in multifunctional protein 2(MFP2)and 3-ketoacyl-CoA thiolase-2(KAT2/PED1/PKT3),two enzymes in the last two steps of the ?-oxidation pathway,are highly overlapped with acx4-hyper DMRs.And the Pro35S:NPT? report gene was repressed in mfp2 CRISPR/Cas9 mutants with increased DNA methylation levels in the NOS terminator.All of these results indicates that metabolites from fatty acid ?-oxidation in peroxisomes are closely linked to nuclear epigenetic modifications,which may affect diverse cellular processes in plants.Our study reveals that the ?-oxidation of fatty acids affects transcriptional gene silencing and DNA methylation,likely through maintaining global histone acetylation in vivo.Since acetyl-CoA can be produced in various metabolic pathways in plant cells and other organisms,the novel connection between fatty acid ?-oxidation and epigenetic regulation in plants provides a pioneer study in understanding the mechanism of the metabolic pathways in regulating histone acetylation and DNA methylation. |
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