Specific modifications to histones are essential epigenetic markers---heritable changes in gene expression that do not affect the DNA sequence. Methylation of lysine 9 in histone H3 is recognized by heterochromatin protein 1 (HP1), which directs the binding of other proteins to control chromatin structure and gene expression. Here we show that HP1 uses an induced-fit mechanism for recognition of this modification, as revealed by the structure of its chromodomain bound to a histone H3 peptide dimethylated at Nzeta of lysine 9. The binding pocket for the N-methyl groups is provided by three aromatic side chains, Tyr21, Trp42 and Phe45, which reside in two regions that become ordered on binding of the peptide. The side chain of Lys9 is almost fully extended and surrounded by residues that are conserved in many other chromodomains. The QTAR peptide sequence preceding Lys9 makes most of the additional interactions with the chromodomain, with HP1 residues Val23, Leu40, Trp42, Leu58 and Cys60 appearing to be a major determinant of specificity by binding the key buried Ala7. These findings predict which other chromodomains will bind methylated proteins and suggest a motif that they recognize.

HP1 family proteins are adaptor molecules, containing two related chromo domains that are required for chromatin packaging and gene silencing. Here we present the structure of the chromo shadow domain from mouse HP1b bound to a peptide containing a consensus PXVXL motif found in many HP1 binding partners. The shadow domain exhibits a novel mode of peptide recognition, where the peptide binds across the dimer interface, sandwiched in a b-sheet between strands from each monomer. The structure allows us to predict which other shadow domains bind similar PXVXL motif-containing peptides and provides a framework for predicting the sequence specificity of the others. We show that targeting of HP1b to heterochromatin requires shadow domain interactions with PXVXL-containing proteins in addition to chromo domain recognition of Lys-9-methylated histone H3. Interestingly, it also appears to require the simultaneous recognition of two Lys-9-methylated histone H3 molecules. This finding implies a further complexity to the histone code for regulation of chromatin structure and suggests how binding of HP1 family proteins may lead to its condensation.

The heterochromatin protein 1 (HP1) family of proteins is involved in gene silencing via the formation of heterochromatic structures. They are composed of two related domains: an N-terminal chromo domain and a C-terminal shadow chromo domain. Present results suggest that chromo domains may function as protein interaction motifs, bringing together different proteins in multi-protein complexes and locating them in heterochromatin. We have previously determined the structure of the chromo domain from the mouse HP1β protein, MOD1. We show here that, in contrast to the chromo domain, the shadow chromo domain is a homodimer. The intact HP1β protein is also dimeric, where the interaction is mediated by the shadow chromo domain, with the chromo domains moving independently of each other at the end of flexible linkers. Mapping studies, with fragments of the CAF1 and TIF1β proteins, show that an intact, dimeric, shadow chromo domain structure is required for complex formation. Keywords: chromatin structure/chromo domain/ heterochromatin protein 1/protein complex/protein structure

SummaryRbAp46 and RbAp48 (pRB-associated proteins p46 and p48, also known as RBBP7 and RBBP4, respectively) are highly homologous histone chaperones that play key roles in establishing and maintaining chromatin structure. We report here the crystal structure of human RbAp46 bound to histone H4. RbAp46 folds into a seven-bladed β propeller structure and binds histone H4 in a groove formed between an N-terminal α helix and an extended loop inserted into blade six. Surprisingly, histone H4 adopts a different conformation when interacting with RbAp46 than it does in either the nucleosome or in the complex with ASF1, another histone chaperone. Our structural and biochemical results suggest that when a histone H3/H4 dimer (or tetramer) binds to RbAp46 or RbAp48, helix 1 of histone H4 unfolds to interact with the histone chaperone. We discuss the implications of our findings for the assembly and function of RbAp46 and RbAp48 complexes.

Structure of the chromatin binding (chromo) domain from mouse modifier protein 1 protein, an activator, might also be localized to chromatin Linda J.Ball, Natalia V.Murzina, via its chromo domain (Koonin et al., 1995).