Family-wide Characterization Of Histone Binding Abilities Of PHD Domains Of AL Proteins In Arabidopsis Thaliana

Covalent modifications(such as methylation,acetylation,phosphorylation,and so on)of histone N-terminal tails play a critical role in regulating chromatin structure and controlling gene expression.These modifications are controlled by histone-modifying enzymes and read out by.histone-binding proteins.Numerous proteins have been identified as histone modification readers.For example,the Royal family proteins specially interact with methylated histones,the bromodomain proteins are readers of acetylated histones,and the 14-3-3 protein recognizes phosphorylated histones.The PHD domain is a zinc-binding domain with about 60 amino acid residues and uses its canonical CCCC-HCCC motif to coordinate two zinc ions in a cross-brace manner.PHD domain-containing proteins have been reported to bind to methylated and acetylated histones,which can recognize unmodified(H3K4me0)or trimethylated H3K4(H3K4me3)and acetylated H3K14 peptides(H3K14ac).AL(Alfinl-like)is a family of proteins homologous to the alfalfa Alfinl in plant and bears a PAL domain and a PHD domain at N-and C-terminus,respectively.There are 7 AL proteins in Arabidopsis thaliana,and the PHD domains of most AL proteins are reported to bind to histone H3K4me3.Here we report the family-wide characterization of histone binding abilities of PHD domains of AL proteins in Arabidopsis thaliana.In this study,we reported the gene cloning,protein expression and purification of the PHD domains of all AL family proteins in Arabidopsis.We then systematically characterized the histone binding abilities of these PHD domains by quantitative isothermal titration calorimetry(ITC)and fluorescence polarization(FP)binding assays.Our binding results indicate that all the PHD domains of the AL proteins bind to the histone H3K4me3 peptide,but they behave differently.Based on the binding affinity and selectivity,the PHD domains of AL proteins could be divided into 3 groups,preferential H3K4me3 binding group(AL1,AL2,AL4 and AL6),higher lysine methylation degree(me2/me3)of H3K4 binding group(AL3 and AL5),and weak H3K4me3 binding group(AL7).Our study presented here provides the foundation for further studies of the peptide state-specific recognition by PHD domains of AL proteins.