The chromodomain of the HP1 family of proteins recognizes histone tails with specifically methylated lysines. Here, we present structural, energetic, and mutational analyses of the complex between the Drosophila HP1 chromodomain and the histone H3 tail with a methyllysine at residue 9, a modification associated with epigenetic silencing. The histone tail inserts as a beta strand, completing the beta-sandwich architecture of the chromodomain. The methylammonium group is caged by three aromatic side chains, whereas adjacent residues form discerning contacts with one face of the chromodomain. Comparison of dimethyl- and trimethyllysine-containing complexes suggests a role for cation-pi and van der Waals interactions, with trimethylation slightly improving the binding affinity.

On the histone H3 tail, Lys 9 and Lys 27 are both methylation sites associated with epigenetic repression, and reside within a highly related sequence motif ARKS. Here we show that the chromodomain proteins Polycomb (Pc) and HP1 (heterochromatin protein 1) are highly discriminatory for binding to these sites in vivo and in vitro. In Drosophila S2 cells, and on polytene chromosomes, methyl-Lys 27 and Pc are both excluded from areas that are enriched in methyl-Lys 9 and HP1. Swapping of the chromodomain regions of Pc and HP1 is sufficient for switching the nuclear localization patterns of these factors, indicating a role for their chromodomains in both target site binding and discrimination. To better understand the molecular basis for the selection of methyl-lysine binding sites, we solved the 1.8 Å structure of the Pc chromodomain in complex with a H3 peptide bearing trimethyl-Lys 27, and compared it with our previously determined structure of the HP1 chromodomain in complex with a H3 peptide bearing trimethyl-Lys 9. The Pc chromodomain distinguishes its methylation target on the H3 tail via an extended recognition groove that binds five additional residues preceding the ARKS motif. Chromatin structure contains the molecular imprint underlying cell memory and epigenetic inheritance, and emerging evidence suggests that covalent modifications of histones play a major role as carriers of epigenetic information (Felsenfeld and Groudine 2003). Histone modifications can be highly reversible, such as histone acetylation, or more stable, such as histone (lysine) methylation (Zhang and Reinberg 2001;Lachner and Jenuwein 2002). Thus, a wide range of chromatin-based regulatory options is available. These include dynamic marks permitting rapid changes in gene expression in response to physiological and environmental stimuli as well as more permanent indexing systems required for the passage of heritable patterns of epigenetic information from one cell generation to the next (Fischle et al. 2003). The identification of enzyme systems responsible for the steady-state balance of posttranslational histone modifications, together with the discovery of binding modules that "read" covalent marks on histones, have been key for our present understanding of gene regulation in the context of the chromatin polymer.Bromodomains have been the first modules implicated in the read-out of histone marks. They show affinity for acetylated lysines in histone and nonhistone proteins (for review, see Zeng and Zhou 2002), and local recruitment of bromodomain factors to certain regions of chromatin might function in mediating acetyl-histone-encoded antisilencing (Ladurner et al. 2003). In contrast, a second conserved module found in a variety of chromosomal proteins, the chromodomain, has been implicated in binding to methylated lysines on the histone tails (Bannister et al. 2001;Jacobs et al. 2001;Lachner et al. 2001). Indeed, recently a biochemical pathway of gene repression by heterochromatin assembly, involving methylation of Lys 9 of H3 by SE...

Recent studies show that heterochromatin‐associated protein‐1 (HP1) recognizes a ‘histone code’ involving methylated Lys9 (methyl‐K9) in histone H3. Using in situ immunofluorescence, we demonstrate that methyl‐K9 H3 and HP1 co‐localize to the heterochromatic regions of Drosophila polytene chromosomes. NMR spectra show that methyl‐K9 binding of HP1 occurs via its chromo (chromosome organization modifier) domain. This interaction requires methyl‐K9 to reside within the proper context of H3 sequence. NMR studies indicate that the methylated H3 tail binds in a groove of HP1 consisting of conserved residues. Using fluorescence anisotropy and isothermal titration calorimetry, we determined that this interaction occurs with a KD of ∼100 μM, with the binding enthalpically driven. A V26M mutation in HP1, which disrupts its gene silencing function, severely destabilizes the H3‐binding interface, and abolishes methyl‐K9 H3 tail binding. Finally, we note that sequence diversity in chromo domains may lead to diverse functions in eukaryotic gene regulation. For example, the chromo domain of the yeast histone acetyltransferase Esa1 does not interact with methyl‐ K9 H3, but instead shows preference for unmodified H3 tail.

Telomerase, a ribonucleoprotein enzyme, adds telomeric DNA repeats to the ends of linear chromosomes. Here we report the first high-resolution structure of any portion of the telomerase reverse transcriptase, the telomerase essential N-terminal (TEN) domain from Tetrahymena thermophila. The structure, which seems to represent a novel protein fold, shows phylogenetically conserved amino acid residues in a groove on its surface. These residues are crucial for telomerase catalytic activity, and several of them are required for sequence-specific binding of a single-stranded telomeric DNA primer. The positively charged C terminus, which becomes ordered upon interaction with other macromolecules, is involved in binding RNA in a non-sequence-specific manner. The TEN domain's ability to bind both RNA and telomeric DNA, coupled with the notably strong effects on activity upon mutagenesis of single surface residues, suggest how this domain contributes to telomerase catalysis.