Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains

Abstract

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 A 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.

Figure 1.

Preferential binding of Pc and HP1 chromodomains to different methyl-lysines on the histone H3 tail. (A) Sequence alignment of the chromodomains of HP1 and Pc. Three conserved aromatic residues forming an aromatic cage for methyl-lysine recognition in HP1 are highlighted. Secondary structure elements of the HP1 chromodomain fold are illustrated on top. (B) Sequence of the N terminus (residues 1-36) of histone H3. The “neighborhoods” of the Lys 9 and Lys 27 methylation sites are very similar; identical sequence stretches surrounding both sites are boxed. (C) Binding of the chromodomains of Pc (residues 1-98) and HP1 (residues 17-76) to different methylated and unmodified H3 peptides in fluorescence polarization assays. K_D (μM) values are listed in the bottom. Note that neither chromodomain interacts with the unmodified H3 tail.

Figure 2.

Colocalization of Pc with H3 Lys 27 trimethylation, but not H3 Lys 9 trimethylation on polytene chromosomes. (A) Immunostaining of salivary gland polytene chromosomes with anti-H3-Me₃K27-specific antibodies and anti-Pc antibodies. Many bands on the arms of the polytene chromosomes are labeled by both antibodies (as shown by yellow bands in the merged image), indicating colocalization of the Pc protein with sites of H3 Lys 27 trimethylation. It is not clear whether the weaker signal of H3 Lys 27 trimethylation occasionally observed around the chromocentric regions represents a true accumulation of this modification or might be caused by slight cross-reactivity of the antibodies with the H3 Lys 9 methyl mark. DNA was stained with DAPI, and the arrow points to the chromocenter. (B) Double labeling with anti-H3-Me₃K9-specific antibodies and anti-Pc-specific antibodies. The anti-H3-Me₃K9-specific antibodies stain mainly the chromocenter (denoted by an arrow in the DAPI staining), but not the many bands where the Pc protein is localized.

Figure 3.

Localization of HP1, Pc, and repressive H3 methyl-lysine marks to distinct regions of the nucleus in S2 cells. (A) Immunostaining of S2 cells with anti-H3-Me₂K9-specific (red) and anti-HP1-specific (green) antibodies shows colocalization to putatively heterochromatic regions inside the cell nucleus. Coimmunostaining of anti-HP1-specific antibodies with anti-H3-Me₃K27-specific (B) or anti-Pc-specific (C) antibodies, in contrast, shows strict exclusion from nuclear regions enriched in HP1. DNA inside the cell nucleus was stained with DAPI.

Figure 4.

Importance of the different chromodomains of Pc and HP1 for distinct subnuclear targeting in vivo. (A) Full-length HP1 fused to a V5-tag was transiently expressed in S2 cells. Immunostaining with antibodies specific for the V5-tag (green) shows colocalization with H3 dimethylated on Lys 9 (red). (B) The chromodomain of HP1 was replaced by the chromodomain of Pc (HP1^Pc-CD-V5). Coimmunostaining with anti-V5- and anti-H3-Me₂K9-specific antibodies shows exclusion of this chimeric fusion protein from the heterochromatic regions enriched in H3 Lys 9 dimethylation. (C) A transiently expressed Pc-V5 fusion protein localizes to regions that are low in anti-H3-Me₂K9 staining. (D) Replacement of the Pc chromodomain with that of HP1 (Pc^HP1-CD-V5) results in recruitment of this fusion protein to regions enriched in H3 Lys 9 dimethylation. Exemplary cells of a broader spectrum of phenotypes are shown.

Figure 5.

Structure of the Pc chromodomain (residues 15-77) in complex with the trimethylated-Lys 27 H3 tail (residues 15-32) at 1.8 Å resolution. (A) Stereo diagram of an |Fo - Fc| simulated annealing omit map contoured at 2.5σ in which the H3 tail peptide was omitted for map calculation. Side chains that make critical contacts are depicted; the chromodomain backbone is in brown, and the electron density of the H3 peptide is in magenta. Residues 15-19 and 29-32 of the peptide appear to be unstructured, suggesting that only Leu 20-Ser 28 of the histone H3 tail participate in binding to the chromodomain. Broken lines indicate intermolecular hydrogen bonds. (B) The chromodomains of both Pc (blue) and HP1 (green) contain three aromatic residues that form superimposable cages around the methyl-ammonium groups of Lys 27 (red) and Lys 9 (yellow), respectively. The van der Waals radii of the Pc aromatic rings and the methyl-Lys 27 methyl-ammonium atoms are shown. (C) Superposition of the backbone structures of the Pc (brown) and HP1 (blue) chromodomains bound to Me₃K27 and Me₃K9 H3 tail peptides, respectively.

Figure 6.

Structural basis for the recognition of methyl-Lys 27 by the Pc chromodomain. (A) Surface depictions of the Pc and HP1 chromodomain H3 tail-binding interfaces. (B) Closeup view of the surface of the Pc chromodomain (left) that interacts with residues 20-24 of the H3 tail, and the surface of the HP1 chromodomain (right) that interacts with residues 5-7 of the H3 tail.