CAUCE

Paro and Hogness found that the Drosophila Polycomb protein shared a homologous domain with HP1a. Since both proteins are involved in chromatin regulation they named it the chromo domain (chromatin organization modifier) (Paro and Hogness, 1991). About 20 proteins in Drosophila contains chromo domain(s) (Li et al., 2002) and the molecular functions of the different chromo domains are diverse (See table 2.5). The HMTase SU(VAR)3-9 and the ATP-dependent chromatin remodeling factor Mi-2 described above, both contain a chromo domain (Table 2.3) (Brehm et al., 2000; Tschiersch et al., 1994). Best studied is the chromo domain of HP1. It was shown to interact specifically with a peptide resembling the N-terminus of H3 that was di- or trimethylated at K9 (Bannister et al., 2001; Jacobs and Khorasanizadeh, 2002; Jacobs et al., 2001; Lachner et al., 2001; Nielsen et al., 2002b). This discovery supported the histone code theory which postulated that different histone modifications are recognized by chromatin proteins (Jenuwein and Allis, 2001; Turner, 1993). Supporting this idea the chromo domains of Chd1 and Polycomb were shown to bind

the H3 tail methylated at lysine 4 and 27 respectively (Cao et al., 2002; Min et al., 2003; Pray-Grant et al., 2005).

Table 2.5 Chromo domain proteins studied in Drosophila and their molecular function Name Abbreviation Number of CD Molecular function of the CD Heterochromatin protein 1 HP1a, HP1b, HP1c 21 H3K9me binding

Polycomb Pc 1 H3K27me binding

Chromo-ATPase/helicase-DNA-binding CHD1 2 H3K4me binding

Mi-2 Mi-2 2

Nucleosomal DNA binding Males-absent on the first MOF 1 roX RNA binding Male-specific-lethal 3 MSL3 2 roX RNA binding Suppressor-of-position-effect variegation 3-9 SU(VAR)3-9 1 ?

Kismet KIS-L/-S 2 ?

Modified from (Brehm et al., 2004). CD, Chromo domain and 1) HP1 also contains a chromo shadow domain (see below).

The crystal structure of the HP1 chromo domain together with the methylated H3 tail revealed that the recognition of the methylated lysine involved a conserved aromatic pocket (Figure 2.10, residues are highlighted in purple) (Jacobs and Khorasanizadeh, 2002; Nielsen et al., 2002b). Four amino acids within the Drosophila HP1a chromo domain; Glu23, Val26, Asn60 and Asp62 interact with the H3 peptide to form a β-sheet (Figure 2.10; A) β-strands B0 and B4, B) residues highlighted in orange). The interaction with the H3K9 methylated tail is highly conserved among different HP1 like proteins and when a conserved Val26 was mutated to a methionine the binding to H3K9 methyl was abolished (Bannister et al., 2001; Lachner et al., 2001; Platero et al., 1995). The same mutation in a SU(VAR)2-502 allele showed diminished HP1a localization to centric regions, but retained association to euchromatic and telomeric sites (Fanti et al., 1998) resulting in a loss of function allele (Platero et al., 1995).

Figure 2.10 The chromo domain of Drosophila HP1a. Models of the chromo domain structure in complex with a histone H3 tail peptide (blue). Figure taken from (Brehm et al., 2004). The dimethylated K9 is shown in blue wireframe. (A) Secondary structures are shown; β-sheet (red arrows) and α-helix (green ribbon). (B) Residues that are structurally and functionally important are indicated with their carbon atoms shown as colored spheres. Labeled in yellow are conserved residues that form the hydrophobic core.

The affinity towards the H3K9 methylated peptide is rather weak (Table 2.6). Although it is 100-fold stronger then the affinity towards the H3K4 methylated peptide, (Table 2.4; compare rows 4 and 5 with 6). HP1a still binds H3-tail peptide containing both K4 and K9 methylation with a 2.5 fold weaker affinity than a peptide with only H3K9Me (Table 2.4; compare row 4 and 7). Isothermal titration calorimetry (ITC) measurements revealed that binding to H3K9 methylated peptide occurs in absence of significant change of the conformation of HP1a (Jacobs et al., 2001). The affinity for H3K9Me2 was improved when only the chromo domain was used compared to an N-terminal stretch including the chromo domain (Table 2.4; compare row 1 with 8 and 9), suggesting that the intact HP1 has a weaker affinity.

Table 2.6 In vitro HP1 binding studies

Isoforms Peptide Method kD Reference

1. HP1a CD (aa 17- 76) H3 (aa 1-15) K9Me2 ITC1 6.9 + 0.2 μM (Jacobs and Khorasanizadeh, 2002) 2. HP1a CD (aa 17-76) H3 (aa 1-15) K9Me3 ITC1 2.5 + 0.1 μM (Jacobs and Khorasanizadeh, 2002) 3. HP1a CD (aa 1-84) H3 (aa 1-15)

K9Me2 FA 2 120 + 12 μM (Jacobs et al., 2001) 4.

HP1a CD (aa 1-84)

H3 (aa 1-15)

K9Me2 FA 2 120 + 12 μM (Jacobs et al., 2001) 5.

HP1a intact

H3 (aa 1-15)

K9Me2 FA 2 133 + 11 μM (Jacobs et al., 2001) 6.

HP1a CD (aa 1-84)

H3 (aa 1-15)

K4Me2 FA 2 1.9 + 0.5 mM (Jacobs et al., 2001)

7.

HP1a CD (aa 1-84)

H3 (aa 1-15)

K4/K9Me2 FA2 268 + 25 μM (Jacobs et al., 2001) 8.

HP1a CD (aa 1-84)

H3 (aa 1-15)

K9Me2 ITC2 105 + 24 μM (Jacobs et al., 2001) 9.

HP1a CD (aa 1-84)

H3 (aa 1-15)

K9Me2 ITC1 59 + 8 μM (Jacobs et al., 2001)

Drosophila HP1a binding to premodified H3-tail peptides has been studied using different methods. Results were obtained at 15°C (1) and 25°C (2). kD, dissociation constant; ITC, isothermal titration calorimetry and FA, fluorescence anisotropy

Recently, the chromo domains of mammalian HP1α/β/γ have been shown to interact specifically with the linker histone isoform 1.4 when it is methylated at K26 (Daujat et al., 2005). The surrounding amino acids of K26 (KKARKSA) are similar to the amino acids surrounding K9 (QTARKST). H1.4 was shown to be methylated by Polycomb Repressive Complex 2 (PRC2) containing the HMTase Ezh2 and a specific EED isoform (Kuzmichev et al., 2004). This suggests that HP1 can be tethered to chromatin that lacks H3K9 methylation.

It has been reported that some proteins interact with the chromo domain of HP1 (See Table 2.7). Mouse HP1β was shown to interact with the nuclear envelope suggesting a role for HP1 in nuclear architecture (Kourmouli et al., 2000). The interaction with lamina-associated polypeptide 2β (LAP2β) and lamin B receptor (LBR) was mapped to the chromo domain. However, HP1β interaction with LBR was shown to be bridged by H3/H4 tetramers (Polioudaki et al., 2001). In addition, the chromo domain of mouse HP1α was shown to interact with the histone fold domain of bacterially expressed H3 (Nielsen et al., 2001a), suggesting that H3 may contribute to the interaction with the nuclear envelope.

The chromo domain of Drosophila HP1a interacts with origin recognition complexes (ORCs) (Pak et al., 1997). Another HP1/ORC associated protein (HOAP) (Shareef et al., 2001) has been described to interact with the hinge and chromo shadow domain (Badugu et al., 2003), suggesting that HP1 has a dual association with the ORC multi- protein complex. The ORC2 subunit is maternally deposited and enriched in centric heterochromatin of early embryos. In addition, HOAP and ORC2 mutants suppress heterochromatin-induced silencing and display defects in HP1 localization in centric heterochromatin (Huang et al., 1998a; Pak et al., 1997). These data suggest a role for ORC and HOAP in heterochromatin silencing.