A Study On The Binding Of MBD5 And MBD6 MBD Domain To Methylation DNA In Arabidopsis Thaliana

DNA methylation is a common epigenetic modification,which contains three main types of modifications:N4-methylcytosine(4mC),N6-methyladenine(6mA)and 5-methylcytosine(5mC).Among them,DNA 5-mC methylation is ubiquitous,and can be interpretated by MBD family proteins,which is first discovered in the human MeCP2 protein.In mammals,11 MBD domain proteins have been reported,including MeCP2,MBD1-6,SETDB1,SETDB2,BAZ2A and BAZ2B,which are involved in transcriptional repression,DNA mismatch repair and rRNA genes silencing.13 MBD members(AtMBD)have been identified in Arabidopsis thaliana,including AtMBD1-13.Among them,AtMBD1,2,4,and 8 do not display binding to 5-methylcytosine DNA,while AtMBD3,9,10,11,12,and 13 have not been reported yet.In contrast,AtMBD5,6 and 7 were reported to bind to DNA.Exceptionally,AtMBD7 contains three MBD structural domains,while AtMBD5 and AtMBD6 each have only one MBD structural domain each,and there is an extremely high sequence conservation between AtMBD5 and AtMBD6.The binding affinity of AtMBD5 and AtMBD6 to methylated DNA is controversial.It has been reported that both AtMBD5 and AtMBD6 are able to bind mCpG,on the other hand,AtMBD5 and AtMBD6 was found to bind to mCpHpG or mCpHpH(H=A,C,T)rather than mCpG DNA.Functionally,both AtMBD5 and AtMBD6 can be localized in the nucleolus,and may be associated with the transcriptional silencing of ribosomal RNAs.While AtMBD6 is able to bind to DRM2,interfering with NOR condensation.Meanwhile,AtMBD6 also participates in RNA-mediated gene silencing by binding to its partner proteins such as AtRPS2C,AtNTF2 and AtAGO4.These functions were not found for AtMBD5.In this thesis,I firstly expressed and purified AtMBD5 and AtMBD6 proteins,and then analyzed the binding affinity of AtMBD5 and AtMBD6 to different DNA using isothermal titration calorimetry(ITC)experiments combined with mass spectrometry.Furthermore,I crystallized the complex of AtMBD5 and AtMBD6 with different DNA,and finally determined the crystal structures of AtMBD5 in apo-state and AtMBD6 in complex with DNA.Our results showed that AtMBD5 does not bind either unmethylated DNA or mCpG,mCpHpG and mCpHpH(H=A,C,T),while AtMBD6 prefers to mCpG DNA.Structural analysis revealed that the MBD domains of AtMBD5 and AtMBD6 contain the conserved arginine fingers that have been reported to binding to methylated DNA.While one lysine K51 in the loop between β1 and β2 in AtMBD5 MBD domain might block the DNA binding due to the steric clash,which could be one important reason why AtMBD5 MBD domain do not display DNA binding ability.Moreover,it is also possible that the sequence of AtMBD5 binding to 5-methylcytosine is specific and the experiments in this thesis did not screen for DNA sequences capable of binding to AtMBD5.Taken together,the results in this thesis demonstrate the structural basis of MBD domain protein binding to DNA in Arabidopsis,and also provide important clues on the further studies of other MBD protein family in Arabidopsis thaliana.