Mechanism Of MAT4 On Regulating Gene Silencing Through Epigenetic Modification In Arabidopsis

DNA and histone methylation co-regulate heterochromatin formation and gene silencing in animals and plants.Numerous methyltransferases for DNA and histone methylation use S-adenosylmethionine(SAM)as methyl donor during the transmethylation reactions.The maintenance of SAM homeostasis in cell plays significant roles in regulating the epigenetic modifications,however,the mechanism is poorly understand in Arabidopsis.In this study,we carried out a forward genetic screening and identified methionine adenosyltransferase 4(MAT4)which is involved in maintaining gene silencing.MAT4 catalyzes the synthesis of SAM,a universal methyl donor,in the one-carbon metabolism cycle.By performing whole-genome bisulfite sequencing,we found that deficiency of MAT4 decreased DNA methylation,especially CHG and CHH methylation.Meanwhile,using histone immunoblotting and immunofluorescence assays,we discovered that H3K9me2 levels in mat4 decreased significantly compared with those in L119.We also detected obvious decrease of H3K9me2 at some loci by ChIP-qPCR assay,indicating that MAT4 plays significant roles in maintaining methylation modification.In addition,by RNA sequencing,we also found that mat4 mutation reactivated the expression of certain silenced transposons,and those loci were shown to be accompanied with decreased DNA methylation level,indicating that MAT4 maintains gene silencing by maintaining DNA and histone methylation.Exogenous addition of SAM could partially rescue the epigenetic defects of mat4,suggesting a direct impact of SAM on epigenetic modification.We did not find obvious alterations of DNA methylation levels in other three mat mutants.There was nearly 35%decrease of SAM content in mat4 compared with that in L119 revealed by analyses of liquid chromatography-electrospray ionization tandem mass spectrometry(LC-MS/MS)assay,while in other mat mutants less decrease of SAM content was detected.MAT1,2,and 4 proteins exhibited nearly equal activity in an in vitro assay,while MAT3 showed the strongest activity.MAT1,2 and 3 driven by the native MAT4 promoter can complement the defect of the mat4 mutant.However,MAT4 diven by the native promoters of MAT1,2,or 3 was unable to complement the mat4 mutant,possibly due to the low actitivy of their promoters in seedlings.These results indicate that MAT4 plays a predominant role in SAM production and epigenetic modifications partially caused by its higher expression.Genetic analyses indicated that matl mat4 double mutant was dwarf and mat2 mat4 double mutant was embryonic lethal.MAT4 knock-out mutations generated by CRISPR/Cas9 technique were lethal,indicating that MAT4 is an essential gene of embryogenesis in Arabidopsis.Interactions between MAT proteins by in vitro Pull-Down and in vivo Co-IP assays and the results of gel filtration assays suggested that they may form polymers with large molecular weight in vivo.In summary,our study identified a new factor involved in maintaining gene silencing in Arabidopsis,and demonstrated that MAT4 plays a predominant role in maintaining the proper concentrations of SAM in plant cells which are important for DNA and histone methylation and gene expression.These finding provides evidence showing the cooperative actions between metabolism and epigenetic regulation.