(A) Experimental strategy for the preparation of immobilised and modified nucleosomes for pulldown studies. (B) The native chemical ligation strategy for generating post-translationally modified histone H3.1. We bacterially express an IPTG-inducible truncated histone precursor containing a modified TEV-cleavage site (ENLYFQ↓C) followed by the core sequence of histone H3.1 starting from glycine 33. The plasmid also contains TEV-protease under the control of the AraC/P_BAD-promoter. TEV-protease accepts a cysteine instead of glycine or serine as the P1′-residue of its recognition site, and upon arabinose induction it processes the precursor histone into the truncated form (H3.1Δ1-31 T32C) which is purified and ligated to modified thioester peptides spanning the N-terminal residues 1 to 31 of histone H3.1. All ligated histones contain the desired modification and a T32C mutation. (C) Summary of the modified histone octamers. The upper panel shows 1 μg of each octamer separated by SDS-PAGE and stained with Coomassie. For the bottom panel octamers were dot blotted on PVDF-membranes and probed with modification-specific antibodies as indicated. The anti-H3K27me3 antibody shows slight cross-reactivity with H3K4me3 and H3K9me3. (D) Functional test of the nucleosome affinity matrix. R₁₀K₈-labelled nuclear extract was incubated with immobilised modified nucleosomes as indicated. Binding of PHF8, HP1α, and SUZ12 was detected by immunoblot. Equal loading was confirmed by silver and Coomassie staining. Modification of histone H3 was verified by immunoblot against H3 tri-methyl lysine marks. All three antibodies show slight cross-reactivity with the other histone marks. See also Figure S1.

(A) Experimental design of the SILAC nucleosome affinity purifications. Nuclear extracts are prepared from HeLaS3 cells grown in conventional “light” medium or medium containing stable isotope-labelled “heavy” amino acids. The resulting “light”- and “heavy”-labelled proteins can be distinguished and quantified by MS. Immobilised unmodified or modified nucleosomes are separately incubated with light or heavy extracts, respectively. Both pulldown reactions are pooled and eluted proteins are separated by SDS-PAGE. After in-gel trypsin digestion, peptides are analysed by high resolution MS. (B) Results of SNAP performed with H3K9me3-modified nucleosomes containing unmethylated 601-DNA. Shown are the Log₂-values of the SILAC ratios (ratio H/L) of each identified protein for the forward (X-axis) and the reverse (Y-axis) experiments. The identities of several interacting proteins are indicated. Subunits of the MBD2/NuRD-complex are labelled in orange. (C) Results of SNAP performed with H3K9me3-modified nucleosomes containing CpG-methylated 601-DNA. For additional SNAP results see Figure S2 and Table S1. (D) Differential recognition of nucleosomes. The graphs show the forward SILAC enrichment values (Ratio H/L forward) of MeCP2, L3MBTL3, USF2, and the TFIIIC subunit GTF3C5 on CpG-methylated DNAs and modified nucleosomes. Binding to the modified nucleosomes or DNAs is indicated in red, exclusion is indicated in blue. If proteins were not detected (n.d.) no value is assigned. (E) Crosstalk between DNA and histone methylation. The graphs show the SILAC enrichment values of the proteins KDM2A, UHRF1, the PRC2 subunit EED, and the ORC subunit Orc2 as described in (D). (F) Immobilised modified nucleosomes were incubated with an independently prepared R₀K₀-nuclear extract as indicated. Binding of KDM2A, UHRF1, Orc2, and the PRC2 subunit SUZ12 was detected by immunoblot. Equal loading and modification of histone H3 were verified as in Figure 1D. The asterisk marks a cross-reactive band recognised by the KDM2A antibody.

Agglomerative hierarchical clustering was performed on the SILAC enrichment values of proteins regulated by DNA and histone methylation to identify proteins with related binding profiles. This analysis includes proteins based on an enrichment/exclusion of at least 1.5 fold in both directions in one of the nucleosome pulldown experiments and excludes factors that were found solely in the DNA pulldowns. Log₂(ratio_for/ratio_rev) is the log2 ratio between the SILAC values (ratio H/L) of the forward and reverse experiments. Enrichment by modifications is indicated in red, exclusion is indicated in blue. Grey bars indicate if proteins were not detected (n.d.) in particular experiments. These incidences were not included in the cluster analysis. Clusters of several known protein complexes and their respective subunits are indicated on the right. For values see Table S2.

(A) LRWD1 co-localises with Orc2. IF staining of MCF7 cells with LWRD1 (2527) and Orc2 antibodies following pre-extraction shows co-localisation at distinct nuclear foci. (B) LRWD1 and ORC co-immunoprecipitate. LRWD1 and Orc2 were immunoprecipitated from MCF7 whole cell extracts and interacting proteins were detected by immunoblot as indicated. LRWD1 was immunoprecipitated using anti-LRWD1 (A301-867A) and detected using anti-LRWD1 (2527) antibodies. Anti-FLAG and anti-GFP antibodies were used as IgG negative controls. Asterisks mark bands derived from antibody heavy chains. (C) FLAG-tagged full length and truncated versions of LRWD1 were over-expressed in 293T cells and immunoprecipitated using an anti-FLAG antibody. 1 % of the input and 10% of the IP were separated by SDS-PAGE and Orc1, Orc2 and the FLAG fusions were detected by immunoblot. The asterisks mark bands derived from the anti-FLAG IP antibody. (D) Identities of the LRWD1 truncation constructs. Only deletions containing the WD40 repeats interact with ORC. (E) LRWD1 expression is Orc2-dependent. Expression levels of LRWD1 and ORC proteins in MCF7 cells were detected by immunoblot after transfection with siRNAs against LRWD1 and Orc2 as indicated. Cells were reverse-transfected twice, 56 h and 28h before harvesting. GAPDH serves as a loading control. The asterisk marks a cross-reactive band detected by the anti-LRWD1 (2527) antibody. See also Figure S3.

(A) In vitro binding of KDM2A to modified nucleosomes. Whole cell extracts prepared from transiently transfected 293T cells over-expressing FLAG-tagged KDM2A were incubated with immobilised modified nucleosomes or modified H3-peptides as indicated. Binding reactions were supplemented with recombinant purified HP1α or GST as a control. Binding was detected by immunoblot against the FLAG-tag or HP1α. Equal loading of the nucleosomes and peptides, and modification of histone H3 were verified as in Figure 1D. (B) KDM2A binding to H3K9me3-Nucleosomes is mediated by HP1α, β, and γ. Unmodified or H3K9me3-modified nucleosomes were immobilised on streptavidin beads and incubated with 293T whole cell extracts over-expressing FLAG-tagged KDM2A. Pulldown reactions were supplemented with recombinant purified HP1α, β, or γ or GST as indicated. Binding of KDM2A was detected by immunoblot against the FLAG-tag. (C) Recruitment of KDM2A to the rDNA locus is augmented by HP1α. MCF7 cells were transfected with HP1α-specific siRNAs and analysed for the enrichment of the H13 region of the rDNA locus by ChIP using antibodies against KDM2A, HP1α and histone H3K9me3. Shown are the mean ± SD of the signals normalised to input of three independent experiments. KDM2A shows only little enrichment at the GAPDH locus. (D) Analysis of KDM2A and HP1α expression in siRNA-treated MCF7 cells by immunoblot. GAPDH serves as loading control. See also Figure S4.