A Systematic Study On The Binding Specificity Of MBD Domains To Methylated DNA In Arabidopsis Thaliana

The variability and plasticity of some important ecological traits of plants are essential for their response to environmental change.For example,plant can adapt to the abiotic and biological changes by means of selective absorption,in combination with passivation,metabolic transformation,and altered metabolic pathways,in which plants actually try to regulate the expression of related genes.Previous studies have reported that DNA methylation,as a conservative epigenetic marker,plays an important role in the regulation of expression.So far,three kinds of DNA methylation,including symmetric methylated CG,methylated CHG and CHH(H represents A,T,C)have been identified in plant.Among them,the symmetric methylated CG dinucleotides mainly play roles through its recognition by MBD domain proteins.Based on the amino acid sequence alignment,a total of 13 MBD domain proteins,AtMBDl-AtMBD13,are identified in Arabidopsis thaliana.AtMBD5,AtMBD6 and AtMBD7 proteins have been reported to recruit the chromatin remodeling complex,histone deacetylases and histone methyltransferases to mediate the transcription repression by their recognition of methylated DNA.While AtMBD4 and AtMBD11 are able to non-specifically bind the symmetric methylated CG dinucleotides,and also methylated CHH and CHG DNA.However,AtMBD1,AtMBD2 and AtMBD8 do not have the ability to recognize DNA,and the binding affinity of MBD domains of AtMBD3,AtMBD9,AtMBD10,AtMBD12 and AtMBD13 are yet reported so far.At present,MBD proteins,such as hMeCP2,have been extensively studied in animals.The MBD domain of hMeCP2 can specifically recognize the symmetric methylated CG.Compared with the MBD domain of hMeCP2,we found that hMeCP2,AtMBD1,AtMBD2,AtMBD3,AtMBD4,AtMBD5,AtMBD6,AtMBD7 and AtMBD12 share the conserved amino acid residues related methylated DNA recognition,which is not found in AtMBD8,AtMBD9,AtMBD10,AtMBD11 and AtMBD13.In this case,we plan to analyze if the MBD domains of AtMBDl-AtMBD13 have ability to recognize DNA in Arabidopsis?What is the binding specificity to different methylated DNA?What are the structural mechanisms of MBD domain recognition with methylated DNA in Arabidopsis thaliana?In this paper,we systematically studied the binding affinity of MBD domain of AtMBD1-AtMBD13 protein.First,we constructed 58 MBD domain expression plasmids by gene recombination,and also obtained 32 soluble proteins,including the MBD domains of AtMBD2,AtMBD4,AtMBD5,AtMBD6,AtMBD7,AtMBD8,AtMBD9,AtMBD10,AtMBD11 and AtMBD13.Furthermore,we studied the specificity of these MBD domain proteins in binding with DNA via EMSA and ITC.The results show that MBD6 has the ability to recognize the symmetric and Hemi-methylated CG DNA,while MBD4,MBD5 and MBD7 only can bind against the symmetric methylated CG DNA.However,we did not detect any binding affinity for MBD domain of AtMBD2,AtMBD8,AtMBD9,AtMBD10,AtMBD11,and AtMBD13.In order to understand the structural mechanism of MBD domain binding with methylated DNA,we selected AtMBD6 and AtMBD11 protein to analyze their protein structures using nuclear magnetic resonance experiments(NMR).Although we did not get the structure so far,our 1H-15N HSQC maps show that the MBD domains of AtMBD6 and AtMBD11 have better stability and spectral dispersion,implying that they can be used to do the NMR study.We are preparing the experiment for structural study.In this paper,we first time systematically studied the binding specificity of 10 MBD domains with methylated DNA using two different methods,and expected to illuminate the structural mechanisms in which the MBD proteins recognizing different methylated DNA.The results provide some clues for the understanding of how MBD domain recognizing methylated DNA to regulate the plants response to environmental stress.