Structural Basis For The Recognition Of Methylated Histone H3 By Chromodomain Of Arabidopsis LHP1

In eukaryotic,gene expression is dynamically regulated by epigenetics,including histone modifications.Histone modification refers to post-translational covalent modification of the N-terminal tail of histones,such as methylation,acetylation,ubiquitination,etc.Methylation generally occurs on the lysine or arginine residues.And lysines can be mono-,di-or tri-methylated.These modifications are dynamically regulated by various histone modification enzymes and recognized by histone-modified recognition proteins.Members of the Royal famlily have been extensively reported as methylation-modified histone readers.LHP1(Like Heterochromatin Protein 1)in Arabidopsis,the only homologous to HP1 in Drosophila,contains both the chromodomain(CD)and chromo shadow domain(CSD).Chromodomain is a major member of the Royal family and was first found in Drosophila HP1 and Pc proteins.It is involved in the regulation of gene expression by binding to histone H3K9me3(HP1)and H3K27me3(Pc),respectively.The human genome encodes three HP1 homologous proteins(CBX1,3,5)and five Pc homologous proteins(CBX2,4,6,7,8),Biochemical experiments showed that the chromodomain of human HP1 protein selectively binds to H3K9me3 and has strong binding ability.Chromodomain of human Pc protein can weakly bind to H3K9/K27me3 with no obvious selectivity.Although the structural composition of AtLHP1 is similar to HP 1,it has been reported that the chromodomain of AtLHP1 can bind to H3K9me2/3 or H3K27me3,which is functionally similar to Pc homologue,but the specific molecular mechanism is still unclear.In order to further study the recognition specificity of AtLHP1 with histone H3 methylation site,we first cloned and expressed AtLHP1 chromodomain protein,and systematically conducted the ITC assays to detect the binding affinity of AtLHP1-CD with H3K9me0-me3 and H3K27me0-me3 peptides.Our results showed that AtLHP1 chromodomain binds to H3K9me2/3 and H3K27me2/3 with similar binding affinity to H3K9me3 and H3K27me2/3,but 3-4 times weaker to H3K9me2.In order to further investigate the recognition mechanism of AtLHP1 chromodomain with methylated histone H3,we attempted to screen the crystallization conditions of AtLHP1 chromodomain with different methylation states and length of H3K9/K27 peptides and successfully resolved the AtLHP1 chromodomain apo structure and AtLHPl chromodomain-H3K9me3 complex structure.By the alignment of sequences and structures with HP1 and Pc chromodomain,we found that unlike the polar fingers residue(D-E)that selectively binds to methylated H3K9 in HP1,the amino acid composition in AtLHP1 is F-A,which is similar to the hydrophobic clasp(V-L)of Pc protein.This hydrophobic clasp explains the possible mechanism for AtLHP1 binding to methylated H3K9 and H3K27.On the other hand,the AtLHP1 chromodomain bares a negative electrostatic potential surface,which may enhance its binding to positive histone peptide same as HP1.Our results support that although the AtLHP1 protein is similar in composition to the HP1 protein,it is functionally closer to the Pc protein and shed light on the further study of the biological function of AtLHP1.