Bacterially expressed HP1 dimerizes and binds H3 peptides methylated at lysine 9. (A) Untagged recombinant HP1 was purified over four successive columns. A Coomassie-stained SDS-12% polyacrylamide (PAA) gel of 5 μl of fractions 9 to 19 from the last column, a MonoQ, is depicted. fxn, fractions. (B) Purified recombinant HP1 was loaded onto a gel filtration column (Superdex 200), and the elution profile (A₂₈₀) of HP1 is shown. Molecular mass (MM) standards (bovine serum albumin [66 kDa] and carbonic anhydrase [29 kDa]) are labeled with arrows. (C) In vitro cross-linking of HP1 using DTSSP (DSS). Recombinant HP1 before (lane 3) or after (lanes 1 and 2) cross-linking was subjected to SDS-12% PAGE, transferred to a PVDF membrane, and detected with HP1 (C1A9) antibody. The DTSSP cross-linking can be partially reversed by reductive cleavage of the disulfide-containing cross-linking molecule (lane 2). The cross-linking revealed dimeric HP1. (D) Recombinant HP1 was assayed for binding to H3 peptides containing the first 19 amino acids of H3 immobilized onto Sulfolink Sepharose. The substrates were unmodified peptide (lane 3), peptide dimethylated at K4 (K4_Me2; lane 4), and peptide dimethylated at K9 (K9_Me2; lane 5). Bound HP1 was visualized by Coomassie staining.

Reconstitution of methylated chromatin using an S150 Drosophila assembly extract and HP1 binding. (A) Scheme of the assay. (B) Micrococcal digestion pattern of chromatin assembly reactions as described for panel C, without HP1 added. MNase digestions were stopped after 30, 60, and 300 s. Assembly of circular DNA was used as a control. MW indicates lanes containing the 123-bp ladder as size marker. Unmod., unmodified. (C) Chromatin was reconstituted on a 2-μg linearized biotinylated fragment containing 12 repeats of the 5S nucleosome positioning sequence bound to paramagnetic beads in the presence or absence of 2 μg of HP1 for 6 h at 26°C. Before assembly, 2 μg of unmodified (lane 4) or H3K9Me (lane 5) histone octamers was supplemented to the extract. The paramagnetic beads were washed, and proteins remaining on the beads were separated on an SDS-15% polyacrylamide gel. HP1 was visualized with an HP1 polyclonal antibody. The corresponding histones were stained by Coomassie blue. Endogenous (endog.) HP1 from the assembly extract and recombinantly added HP1 are labeled. InP, input. (D) The graph corresponds to quantification of bound HP1 from panel C. Recombinant and endogenous HP1 are included in the quantification. The y axis displays the percentage of input bound. The graph is representative of three or more different experiments.

Expression of HP1 mutant proteins and binding of these to H3K9Me chromatin during assembly. (A) Scheme of HP1 mutants generated. (B) Coomassie blue-stained 15% SDS-polyacrylamide gel of the purified HP1 proteins. MM, molecular mass. (C) Peptide pulldown of the recombinant HP1 WT and mutants using H3 peptide aa 1 to 21, unmodified (WT) versus trimethylated at K9 (H3K9_triMe). Bound HP1s were visualized by use of Coomassie blue. InP, input. (D) Drosophila assembly reaction with 2 μg H3K9Me octamer as described for Fig. 3A. In lanes 1 to 3, 2.5% HP1 input was used. Lanes 4 to 7 correspond to proteins bound after 6 h of incubation. HP1 was detected with HP1 polyclonal antibody, and the corresponding histones were stained with Coomassie blue. Bound exogenous and endogenous (endog.) HP1 present in the Drosophila assembly extract are labeled. (E) The graph corresponds to quantification of bound HP1 from panel D. Recombinant and endogenous HP1 are included in the quantification. The y axis displays percentage of input bound. This quantification is representative of at least three different experiments.

HP1 interacts with SU(VAR)3-9. (A) SU(VAR)3-9 constructs used for in vitro translation and GST constructs are shown at the top. The GST pulldown is shown at the bottom. SU(VAR)3-9 was detected by autoradiography. CHROMO SH., chromo shadow. (B) The upper panel shows GST constructs used for the pulldown experiment. The lower panel represents an HP1 Western blot of GST pulldown with recombinant HP1 WT (lanes 1 to 3), HP1 (V26M) (lanes 4 to 6), and HP1 (W200A) (lanes 7 to 9). HP1 was detected with HP1 polyclonal antibody.

Generation of H3K9-methylated chromatin. (A) To the left is a Coomassie blue-stained SDS-15% PAA gel of reconstituted unmodified (lanes 1 and 2) and H3K9-methylated (lanes 3 and 4) octamers. Displayed in the lower panel is a MALDI-TOF analysis of H3 peptide 9-17 from unmodified H3 (top) and H3 methylated at K9 (bottom). As an internal standard we show H3 peptide 41-49. Unmodified and mono-, di-, and trimethylated peptides with corresponding mass are labeled with arrows. The masses of unmodified and monomethylated peptide 9-17 are higher than those of di- and trimethylated peptide because the free N-terminal amines are propionylated. The quantification of the MALDI-TOF analysis is shown in the upper right panel. (B) Scheme of our chromatin reconstitution protocol. The DNA used for chromatin reconstitution is a linearized biotinylated fragment containing 12 repeats of the 5S nucleosome positioning sequence (69). A micrococcal digestion pattern of salt-reconstituted chromatin with unmodified or in vitro methylated histones is shown on the right. MW, molecular weight. (C) HP1 was assayed for binding to unmodified chromatin (lane 2), H3K9Me chromatin (lane 3), DNA immobilized on paramagnetic beads (lane 4), and beads alone (lane 5). Bound HP1 was separated by SDS-15% PAGE and visualized with an HP1 polyclonal antibody. The bottom panel shows the corresponding histones stained with Coomassie blue. Boiling of the streptavidin-coated beads resulted in the release of a strongly stained band with an apparent molecular weight similar to that of H4, which is therefore labeled H4/streptavidin (strep.). InP, input. (D) The same assay as described for panel C was performed with histone H1, also visualized by Coomassie blue. MM, molecular mass.

HP1 is bound to salt-assembled chromatin in the presence of Drosophila assembly extract. (A) Scheme of the assay. (B) Salt-assembled unmodified or H3K9Me chromatin attached to paramagnetic beads was incubated for 1 h at 26°C with HP1, plus and minus Drosophila (D.) assembly extract. The reactions were carried out in the presence of ATP or nonhydrolyzable ATP-γ-S analog (γS ATP). The assembly extract added was less than 5% of what is needed for the assembly reaction in Fig. 3. HP1 was detected with HP1 polyclonal antibody, and corresponding histones were detected with Coomassie blue. Nucl, nucleosome. (C) The graph corresponds to quantification of bound HP1 as shown in Fig. 3B. The y axis displays the percentage of input bound. The graph is representative of at least four individual experiments.

HP1 interacts with the ACF complex and ACF1. (A) Coomassie-stained SDS-8% PAA gel of FLAG affinity-purified recombinant ACF1 and ACF complex from Sf9 cells coinfected with FLAG-ACF1 in the presence or absence of untagged ISWI. MM, molecular mass. (B) HP1 pulldown with FLAG beads incubated with mock Sf9 extract or extract containing FLAG-ACF1 and untagged ISWI. After extensive washing, the protein remaining on the beads was separated by SDS-12% PAGE, imunoblotted, and detected with HP1 antibody. Asterisks indicate FLAG antibody light chains. Inp, input. (C) Western blot of HP1 pulldown using FLAG beads incubated with mock Sf9 extract or extract containing FLAG-ACF1. Asterisks indicate FLAG antibody light chains. (D) ACF1 constructs used for in vitro translation. (E) GST and GST-HP1 pulldown with in vitro translated ACF1 constructs. (F) Quantification of the binding affinities of the various ACF constructs. Error bars represent standard deviations from three independent pulldown experiments.

SU(VAR) 3-9 and ACF facilitate HP1 binding to H3K9Me chromatin. (A) Scheme of the assay. (B) Salt-assembled unmodified or H3K9Me chromatin bound to paramagnetic beads was incubated with HP1 in the presence or absence of ACF and ATP for 1 h at 26°C. After washing, the proteins remaining on the paramagnetic beads were separated by SDS-15% PAGE. ACF1 was detected with FLAG antibody, and HP1 was detected with HP1 polyclonal antibody (upper panels). The corresponding histones were detected with Coomassie blue (bottom panel). Lane 1 corresponds to 50% ACF input (InP) and 2.5% HP1 input. Nucl, nucleosome. (C) The graph displays bound HP1 as a percentage of input as shown in panel B. The y axis corresponds to percent input bound. The graph is representative of at least two individual experiments. (D) SU(VAR)3-9 couples HP1 to chromatin. HP1 was incubated with unmodified or H3K9Me chromatin in the presence of recombinant SU(VAR)3-9 WT (lanes 4 to 7) or Δ213 (lanes 8 and 9). The SU(VAR)3-9-specific HMTase inhibitor Chaetocin was added to a concentration of 1 μM (lanes 6 and 7). The remaining SU(VAR)3-9 and HP1 on paramagnetic beads was detected by Western analysis, and the histones were detected with Coomassie blue. Lane 1 corresponds to 100% SU(VAR)3-9 input and 2.5% HP1 input. (E) Quantification of HP1 bound as shown in panel D. The graph is representative of at least three individual experiments and displays percent input bound. (F) SU(VAR)3-9 was added in the presence of HP1 WT (lanes 1 to 3), HP1 (V26M) (lanes 4 to 6), and HP1 (W200A) (lanes 7 to 9) to unmodified and H3K9Me chromatin. Bound SU(VAR)3-9 and HP1 were detected by Western analysis, and histones were detected with Coomassie blue. (G) The graph corresponds to total HP1 binding as shown in panel F. The y axis displays percent input bound. The graph is representative of at least two individual experiments.