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Experimental procedures

Table of Contents

Preparation of recombinant canonical histones

Recombinant human canonical histone proteins were expressed in Escherichia coli BL21(DE3)-CodonPlus-RIL cells (Agilent Technologies) from pET21b(+) (Novagen) vectors and purified by denaturing gel filtration and ion-exchange chromatography as previously described25,51.

Preparation of recombinant histone H2A.Z

A codon-optimized sequence encoding human H2A.Z (H2AFZ, UniProtKB: P0C0S5) was purchased from GenScript and cloned into the NdeI/XhoI sites of the pET24a(+) vector (Novagen). H2A.Z was then expressed in E. coli BL21(DE3)-CodonPlus-RIL cells (Agilent Technologies) and purified as previously described for canonical H2A25.

Preparation of truncated histones for native chemical ligations

Truncated human H3Δ1–31T32C protein for ligations of modified histone H3 was expressed in E. coli BL21(DE3)-CodonPlus-RIL cells (Agilent Technologies) and purified as previously described52. Truncated human H4Δ1–28I29C protein for ligations of modified histone H4 was expressed from pET24b(+) vectors (Novagen) in E. coli BL21(DE3)-CodonPlus-RIL cells (Agilent Technologies). The insoluble protein was extracted from inclusion bodies with unfolding buffer (20 mM Tris (pH 7.5), 7 M guanidine hydrochloride, and 100 mM dithiothreitol (DTT)) for 1 h at room temperature, and the cleared supernatant was loaded onto a Sephacryl S-200 gel filtration column (Cytiva) in SAU-1000 buffer (20 mM sodium acetate (pH 5.2), 7 M urea, 1 M NaCl, and 1 mM ethylenediaminetetraacetic acid (EDTA)) without any reducing agents. Positive fractions were combined and further purified by reversed-phase chromatography. Truncated H3Δ1–31T32C was purified over a Resource RPC column (Cytiva) using a gradient of 0–65% B (buffer A: 0.1% trifluoroacetic acid in water; B: 90% acetonitrile, 0.1% trifluoroacetic acid) over 20 column volumes. Truncated H4Δ1–28I29C was purified over a PerkinElmer Aquapore RP-300 (C8) column (250 mm × 4.6 mm inner diameter) using a gradient of 0–65% B (buffer A: 0.1% trifluoroacetic acid in water; B: 90% acetonitrile, 0.1% trifluoroacetic acid) over 20 column volumes. The fractions containing pure H3Δ1–31T32C or H4Δ1–28I29C were pooled and lyophilized.

Preparation of modified histone H3 and histone H4 by native chemical ligation

For the preparation of modified histone H3, N-terminal H3 peptides (amino acids 1–31) were ligated to truncated H3Δ1–31T32C and, for the preparation of modified histone H4, N-terminal H4 peptides (amino acids 1–28) were ligated to truncated H4Δ1–28I29C using native chemical ligation. All peptides contained a C-terminal benzyl thioester. All histone H4 peptides were N-terminally acetylated. Ligations were performed in 550 μl of degassed ligation buffer (200 mM KPO4, 2 mM EDTA, 6 M guanidine hydrochloride) containing 1 mg of modified/unmodified histone tail thioester peptide (purchased from Cambridge Peptides or Almac Sciences), 4 mg of truncated histone, 20 mg 4-mercaptophenylacetic acid and 25 mg Tris(2-carboxyethyl)phosphine as reducing agent at a pH of 7.5. The reactions were incubated overnight at 40 °C and quenched by addition of 60 μl 1 M DTT and 700 μl 0.5% acetic acid. After precipitation clearance by centrifugation, the ligation reactions were directly loaded and purified onto a reversed-phase chromatography column (PerkinElmer Aquapore RP-300 (C8) 250 mm × 4.6 mm inner diameter). Modified histone H3 was purified using a gradient of 45–55% B (buffer A: 0.1% trifluoroacetic acid in water; B: 90% acetonitrile, 0.1% trifluoroacetic acid) over 10 column volumes. Modified histone H4 was purified using a gradient of 35–45% B (buffer A: 0.1% trifluoroacetic acid in water; B: 90% acetonitrile, 0.1% trifluoroacetic acid) over 10 column volumes. Positive fractions containing ligated full-length histone H3 or histone H4 were then combined and lyophilized.

Nucleosome assembly

Histone octamers were refolded from the purified histones and assembled into nucleosomes with biotinylated DNA through salt deposition dialysis as previously described25,51. Biotinylated nucleosomal DNAs containing either one (mononucleosomes) or two 601 nucleosome-positioning sequences47 separated by a 50-base-pair (bp) linker (dinucleosomes), or four 601 nucleosome-positioning sequences (tetranucleosomes), were prepared as described previously25. CpG-methylated DNA was prepared using the M.SssI methyltransferase and complete methylation was confirmed by restriction digest (Supplementary Information). Dinucleosomes and tetranucleosomes were assembled in the presence of mouse mammary tumour virus A (MMTVA) competitor DNA (prepared in the same way as 601 DNA) and a slight excess of octamers as described for longer chromatin arrays to ensure saturation of the 601 repeats53. The reconstituted nucleosomes were then immobilized on streptavidin Sepharose High Performance beads (Cytiva) through the biotinylated DNA, washed to remove MMTVA competitor DNA and MMTVA nucleosomes (in the case of dinucleosomes and tetranucleosomes), and directly used for SILAC or label-free nucleosome affinity purifications. Correct assembly and immobilization of nucleosomes was verified by native polyacrylamide gel electrophoresis (Supplementary Information). Nucleosomes for pull-downs in which only modifications on histone H3 were tested were assembled with octamers containing recombinant histone H4 purified from E. coli instead of ligated H4. Likewise, nucleosomes for pull-downs in which only modifications on histone H4 were tested contained recombinant H3 and not ligated histone H3. Matched unmodified control nucleosomes were assembled with unmodified ligated H3 and recombinant H4, or recombinant H3 and unmodified ligated H4 accordingly. Nucleosomes containing only CpG methylation (H27M) were assembled with ligated unmodified H3 and recombinant H4, and nucleosomes containing only H2A.Z (H36) and no other modifications were assembled with recombinant (and therefore unmodified) H3 and H4 produced in E. coli.

Generation of 601 dinucleosomes incorporating different linker DNAs

Plasmid constructs for the preparation of biotinylated 601 dinucleosome DNAs containing different linker lengths (35 bp, 40 bp, 45 bp, 50 bp and 55 bp linkers) between the two 601 nucleosome-positioning sequences were generated by annealing forward and reverse primers of corresponding length and ligating them into pUC19-di601_NcoI/NheI_5xGal4 (pTB891, gene synthesis by Genscript) digested with NcoI and NheI restriction enzymes (Thermo Fisher Scientific), thereby exchanging the ‘5×Gal4 linker’ against the different linker fragments. Plasmid constructs for the preparation of biotinylated 601 dinucleosome DNAs containing 200 bp linkers consisting of either the SV40 enhancer or the SV40 promoter were generated by PCR amplification of the SV40 enhancer and promoter sequences from pGL3-control (Promega) and cloning the resulting fragments into the vector backbone of pUC19-di601_NcoI/NheI_5xGal4 through NcoI and NheI, thereby exchanging the ‘5×Gal4 linker’ against the 200 bp SV40 enhancer or promoter sequences. For all of the constructs, the dinucleosome sequences were then amplified from one copy to eight copies per plasmid as described previously25,51.

The biotinylated 601 dinucleosome DNAs containing 200 bp linkers with randomized DNA sequences were generated from a library of single-stranded 200 bp scrambled linker oligonucleotides (custom synthesis by Biolegio) containing 192 bp of randomized DNA sequence flanked by 5′ NcoI and 3′ NheI restriction sites and 5′ bGHR and 3′ pCIfor primer-binding sites. The single-stranded oligo was converted to double-stranded DNA by annealing it to the pCIfor primer (Sigma-Aldrich) and performing a primer extension of pCIfor. The primer extensions were performed using Taq DNA polymerase in a 96-well plate format with 96 × 50 µl reactions. Each 50 µl reaction contained 1 µg of the 200 bp scrambled linker oligonucleotide (250 nM), 340 ng pCIfor primer (1 µM, fourfold molar excess over the 200 bp scrambled linker oligonucleotide), 200 µM dNTPs and 2.5 U Taq polymerase (New England Biolabs) in 1× ThermoPol buffer (New England Biolabs). Using a thermocycler, the oligonucleotides were denatured for 5 min at 95 °C, annealed for 1 min at 58 °C and the primer extension reaction was then allowed to proceed for 5 min at 68 °C. The reactions were pooled and the remaining single-stranded DNA was removed by direct addition of 2,000 U of exonuclease I (New England Biolabs) per ml reaction volume and incubation for 30 min at 37 °C. The resulting double-stranded DNA was purified using the QIAquick PCR purification kit (Qiagen) according to the manufacturer’s instructions (20× columns, total yield of 75 µg in 1 ml buffer EB). The double-stranded 200 bp scrambled linker DNAs were digested with NcoI and NheI (Thermo Fisher Scientific) using 5 µl of FastDigest enzyme per µg DNA, concentrated using the QIAquick PCR purification kit (10× columns, total elution volume of 500 µl buffer EB) and separated by 2.5% agarose gel electrophoresis. The 200 bp band containing the scrambled linker fragments was excised from the gel and purified using the QIAquick gel extraction kit (Qiagen) according to the manufacturer’s instructions (eight columns, total yield of 11.64 µg in 300 µl buffer EB). The purified NcoI/NheI-digested 200 bp scrambled linker fragments were subsequently ligated into the NcoI/NheI-digested, dephosphorylated (Quick CIP, New England Biolabs) and agarose-gel-purified vector backbone of pUC19-di601_NcoI/NheI_5×Gal4, thereby exchanging the ‘5×Gal4 linker’ against the library of 200 bp scrambled linker fragments. Ligations were assembled using 50 µg of NcoI/NheI-linearized pUC19-di601 vector backbone, 11.64 µg of NcoI/NheI-digested 200 bp scrambled linker inserts (approximately 3.5-fold molar excess of inserts over the 3 kb vector backbone) and 200 µl (400,000 cohesive end units) of T4 DNA Ligase (New England Biolabs) in a total volume of 4 ml of 1× T4 DNA ligase reaction buffer, and incubated overnight at 16 °C. After the ligation, ATP was added to the reaction to a final concentration of 1 mM and unligated linear DNA was digested by addition of 1,000 U of exonuclease V (New England Biolabs) and incubation for 50 min at 37 °C. Circular plasmid DNA that was protected from the exonuclease V digestion was then purified and concentrated using the QIAquick PCR purification kit (10 columns, elution in 30 µl buffer EB per column). The total yield of ligated circular plasmid DNA was 6.5 µg in 280 µl. The ligated plasmids represent a library of pUC19 vectors in which each vector contains one copy of a 601 dinucleosome DNA each incorporating a different 200 bp linker of random sequence between the two 601 nucleosome-positioning sequences. The plasmid library was amplified by electroporation into 10-beta electrocompetent E. coli cells (New England Biolabs) according to the manufacturer’s instructions using 2 µl (47 ng) of library DNA and 25 µl of competent cells per electroporation. Cells were recovered in 1 ml of outgrowth medium and selected on 24.5 cm2 BioAssay LBAmp-agar plates (Corning). Serial dilutions were plated to determine the transformation efficiency and complexity of the library. In total, >108 independent clones were obtained from 24 electroporations. The colonies were gently scraped off the plates in liquid LB medium and plasmid DNA was isolated using the NucleoBond PC 10000 Giga-prep kit (Macherey-Nagel). The total yield of plasmid DNA from 24 plates was 16 mg. In total, 20 clones were picked from a high-dilution plate and sequenced to verify the correct length and random composition of the 200 bp linker sequences.

For preparing the different biotinylated dinucleosome DNAs the pUC19 601 dinucleosome plasmid constructs were first digested with EcoRV, ethanol-precipitated and then further digested with EcoRI (New England Biolabs) to liberate the dinucleosome DNAs. After another ethanol precipitation, the EcoRI overhangs were filled in with dATP and biotin-11-dUTP (Yorkshire Bioscience) using Klenow (3′→5′ exo) polymerase (New England Biolabs). The biotinylated dinucleosome DNAs were again concentrated by ethanol precipitation, separated from the pUC19 vector DNA by preparative agarose gel electrophoresis and then purified from the excised gel slices using the NucleoSpin gel extraction Maxi kit (Macherey-Nagel). Biotinylation and the purity of the dinucleosome DNAs were verified by depletion with streptavidin Sepharose High Performance beads (Cytiva) and agarose gel electrophoresis of the inputs and supernatants (Supplementary Information). Dinucleosomes were then assembled in the presence of MMTVA competitor DNA as described above.

Eukaryotic tissue culture

HeLa S3 cells (ATCC, CCL-2.2) cells were obtained from the Cancer Research UK Clare Hall Laboratories Cell Services Facility and maintained in suspension culture at 37 °C under 5% CO2 in RPMI 1640 medium. HeLa S3 cells were authenticated by morphology on the basis of their ability to grow both in suspension culture and as round spherical cells in adhesion culture. A HeLa Kyoto BAC cell line expressing the C-terminal localization and affinity purification (LAP)-tagged INO80 subunit ACTR548 was a gift from M. Mann (Max Planck Institute of Biochemistry). Cells were cultured at 37 °C under 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 mg ml−1 glucose, 10% fetal calf serum, 1% penicillin–streptomycin and 1% l-glutamine and validated by immunoprecipitation and immunoblotting against the tagged ACTR5. MCF-7 cells (ATCC, HTB-22) were obtained from the Cell Services Facility of the IGBMC. Cells were cultured at 37 °C under 5% CO2 in DMEM containing 4.5 mg ml−1 glucose, 10% fetal calf serum, 1 mM sodium pyruvate, 1% penicillin–streptomycin and 1% l-glutamine and authenticated by morphology and by regularly testing the induction of oestrogen-responsive genes by quantitative PCR with gene-specific primers or global RNA-sequencing after 17β-estradiol treatment. IMR90 human fibroblasts were purchased directly from ATCC (CCL-186) and cultured at 37 °C under 5% CO2 in DMEM containing 4.5 mg ml−1 glucose, 10% fetal calf serum, 1 mM sodium pyruvate, 1% penicillin–streptomycin and 1% l-glutamine. Cells were authenticated by morphology and only maintained for a limited number of passages. All of the cell lines were tested and were mycoplasma free.

SNAP

SILAC-labelled nuclear extracts were prepared from HeLa S3 cells as previously described25. The isotopically light (R0K0) or heavy (R10K8) nuclear extracts were mixes of three independently prepared nuclear extracts. For each pull-down, nucleosomes corresponding to 12.5 μg of octamer were immobilized on 10 μl streptavidin Sepharose High Performance beads (Cytiva) in the final reconstitution buffer (10 mM Tris (pH 7.5), 250 mM KCl, 1 mM EDTA and 1 mM DTT; supplemented with 0.1% NP-40) and then rotated with 0.5 mg HeLa S3 SILAC-labelled nuclear extract in 1 ml of SNAP buffer (20 mM HEPES (pH 7.9), 150 mM NaCl, 0.2 mM EDTA, 10% glycerol) supplemented with 0.1% NP-40, 1 mM DTT and protease inhibitor cocktail (Roche) for 4 h at 4 °C. Nucleosome pull-downs with acetylated histones and the corresponding unmodified control pull-downs were supplemented with HDAC inhibitors (5 mM sodium butyrate (Sigma-Aldrich, B5887) and 250 nM TSA (Sigma-Aldrich, T1952)) to prevent removal of the acetyl modifications. After two washes with 1 ml SNAP buffer + 0.1% NP-40 and then two washes with 1 ml SNAP buffer without NP-40, the beads from both SILAC pull-downs (modified and unmodified control nucleosome) were pooled. The supernatant was completely removed, and bound proteins were eluted by on-bead digestion (see below).

Label-free nucleosome affinity purifications

Nuclear extracts were prepared from HeLa S3 cells as previously described25 except that cells were cultured with 10% regular fetal calf serum and no isotopically labelled amino acids were used. Unlabelled nuclear extracts were a mix of three independently prepared nuclear extracts. Nucleosome pull-downs were performed in the same manner as described above for SNAP, except for the bead washing and protein elution steps, which were performed as follows: after incubation with nuclear extracts, beads with immobilized nucleosomes were washed three times with 1 ml SNAP buffer + 0.1% NP-40, the supernatant was completely removed and bound proteins were eluted by boiling the beads in 50 µl Laemmli sample buffer containing 1% SDS at 95 °C for 5 min. A 20 µl protein aliquot was then digested with trypsin using a filter-aided sample preparation (FASP) protocol and analysed using liquid chromatography–mass spectrometry (LC–MS) as described below.

Cross-linking ChIP for MS analysis

IMR90 human fibroblasts were cultured as described above. Cells were washed three times with PBS and cross-linked on the plate with 1.25 µM ethylene glycol bis(succinimidyl succinate) (EGS) and 0.75 µM disuccinimidyl glutarate in PBS for 30 min at room temperature. After the first cross-linking reaction, cells were washed twice with PBS and cross-linked with 1% formaldehyde in PBS at room temperature for 10 min. Cross-linking reactions were quenched by the addition of glycine solution in PBS to a final concentration of 125 mM and incubation at room temperature for 5 min. Cells were then washed three times with ice-cold PBS, collected by scraping and pelleted by centrifugation (1,000g, 5 min, 4 °C). Cells were lysed in a hypotonic buffer (10 mM Tris (pH 7.6), 5 mM NaCl, 1.5 mM MgCl2) supplemented with 0.1% NP-40, protease inhibitor cocktail (Roche), 10 mM sodium butyrate and 1 mM DTT using a Dounce homogenizer as described previously25. Nuclei were pelleted by centrifugation (3,000g, 5 min, 4 °C), washed in hypotonic buffer supplemented with 300 mM NaCl and pelleted again (3,000g, 5 min, 4 °C). Nuclei were resuspended in nuclear lysis buffer (15 mM Tris (pH 7.6), 10% glycerol, 1% SDS) and incubated for 5 min on ice. Chromatin was pelleted by centrifugation (5,000g, 5 min, 4 °C), washed in chromatin wash buffer (15 mM Tris (pH 7.6), 300 mM NaCl, 1.5 mM MgCl2, 0.5% NP-40, 0.5% Triton X-100), pelleted again (5,000g, 5 min, 4 °C) and resuspended in ChIP buffer (20 mM Tris (pH 7.6), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.01% SDS) supplemented with protease inhibitor cocktail (Roche) and 10 mM sodium butyrate. DNA was fragmented to an average size of 150–300 bp by sonication (Qsonica, Q800R2, 70% amp, 10 s off, 10 s on, 40 min active sonication time, 4 °C). Chromatin debris was pelleted by centrifugation (16,000g, 10 min, 4 °C). Then, 25 µl of supernatant was used for DNA purification to check the average DNA fragment size and another 25 µl supernatant aliquot was transferred to a fresh tube, de-cross-linked as described below, and stored at 4 °C until it was later used as the input sample for histone PTM analysis to define the average levels of core histone PTMs in bulk chromatin. For DNA purification, the sample was mixed 1:1 with 2× de-cross-linking buffer (20 mM Tris (pH 7.6), 600 mM NaCl, 2% SDS) and incubated at 65 °C overnight. The next day, proteinase K was added and the mixture was incubated at 37 °C for 2 h. DNA was purified using the QIAquick PCR purification kit (Qiagen) and eluted in RNase/DNase-free water. RNase A was added and the mixture was incubated at 37 °C for 1 h. DNA was resolved on an agarose gel and visualized with ethidium bromide. Approximately 0.2 mg chromatin (as measured by DNA content) was used for each ChIP reaction with the following antibodies: anti-H3K4me1 (Abcam, ab8895), anti-H3K4me3 (Millipore, 17-614), anti-H3 (Active motif, 39163), anti-H4 (Abcam, ab31830). For H3K4me3 ChIP reactions, 0.6 mg chromatin was used. To boost the identification of H3K4 methylation-state-specific protein interactors, H3 and H4 ChIPs were performed using chromatin inputs partially depleted in H3K4me1- and H3K4me3-modified nucleosomes and co-bound protein factors. Specifically, H3K4me1 and H3K4me3 ChIPs were performed first, then the chromatin inputs used for the H3K4me1 and H3K4me3 ChIPs were combined and subsequently used for H3 and H4 ChIPs. This aimed to shift the composition of the bulk chromatin-associated proteome measured in H3 and H4 control ChIPs towards regions devoid of H3K4me1 and H3K4me3. The antibody–chromatin mixture was incubated overnight on a rotation wheel (25 rpm) at 4 °C. Antibodies were captured using a 1:1 mixture of protein A and protein G Dynabeads (Thermo Fisher Scientific) for 2 h at 4 °C while rotating on a rotation wheel (25 rpm); 40 µl of bead mixture was used per ChIP sample. Beads were washed three times with ice-cold ChIP buffer and twice with ice-cold ChIP buffer supplemented with NaCl to a final concentration of 500 mM. Antibodies and co-bound chromatin were eluted by boiling the beads in 30 µl of Laemmli sample buffer containing 1% SDS and supplemented with 300 mM NaCl for 10 min at 95 °C. The eluate was transferred to a fresh tube and incubated in a thermomixer at 65 °C and 500 rpm for 12 h. For the histone PTM proteomic analysis, eluted proteins as well as the input samples (see above) were resolved on a 4–20% polyacrylamide gel (Novex WedgeWell Tris-Glycin-Minigel, Invitrogen), histone bands were excised, in-gel derivatized, digested with trypsin and processed for LC–MS analysis as described below. For the identification and quantification of co-purified chromatin proteins, a 10 µl aliquot of the eluted proteins in Laemmli sample buffer was processed for trypsin digestion using a FASP protocol and analysed using LC–MS as described below.

Native chromatin immunoprecipitations for MS analysis

The HeLa Kyoto BAC cell line expressing the C-terminal LAP-tagged INO80 subunit ACTR548 was cultured as described above. Cells were collected by trypsinization and were washed three times with ice-cold PBS. Nuclei were isolated using a Dounce homogenizer under hypotonic conditions in the presence of 0.1% NP-40 as described previously25. Nuclei were resuspended in ice-cold MNase digestion buffer (10 mM Tris (pH 7.6), 15 mM NaCl, 60 mM KCl, 0.1% NP-40) supplemented with protease inhibitor cocktail (Roche) and 10 mM sodium butyrate, and MNase was added at a proportion of 150 U per approximately 20 × 106 nuclei. The nucleus suspension was transferred to a thermomixer and, after 2 min incubation at 37 °C and 400 rpm, CaCl2 was added to a final concentration of 1.5 mM and the mixture was incubated at 37 °C for another 6 min. The MNase digestion was stopped by the addition of EDTA to a final concentration of 10 mM. The mixture was then diluted 1:1 with ice-cold 2× SNAP buffer (30 mM HEPES (pH 7.8), 300 mM NaCl, 0.1% NP-40, 20% glycerol, 0.4 mM EDTA) supplemented with protease inhibitor cocktail (Roche) and 10 mM sodium butyrate. The samples were rotated on a rotation wheel for 45 min at 4 °C and further incubated in a thermomixer at 4 °C and 1,000 rpm for another 15 min. Nuclear debris was pelleted by centrifugation (16,000g, 10 min, 4 °C). The resulting supernatants were transferred to fresh 1.5 ml low-protein-binding Eppendorf tubes and used for the purification of nucleosomes bound to the INO80 complex as described below. To determine the efficiency of the MNase digestion, the pellets containing the insoluble chromatin fraction were resuspended in 1× supernatant volume of SNAP buffer, supplemented with proteinase K, and incubated at 37 °C overnight. In parallel, 25 µl aliquots of the supernatants were transferred to fresh tubes, supplemented with proteinase K and incubated at 37 °C overnight. After proteins were digested with proteinase K, DNA was extracted using the QIAquick PCR purification kit (Qiagen) and eluted in RNase/DNase-free water. RNase A was added, and the mixtures were incubated at 37 °C for 1 h. The DNA was then resolved on an agarose gel and visualized with ethidium bromide. For each sample, another 25 µl aliquot of the supernatant was transferred to a fresh tube and subsequently used as the input sample to define average histone modification levels on bulk chromatin. For the purification of nucleosomes bound to the INO80 complex, 25 µl of GFP-Trap Agarose beads (ChromoTek) were added to MNase-digested supernatants and the mixture was incubated on a rotation wheel (25 rpm) overnight at 4 °C. The beads were pelleted by centrifugation (250g, 3 min, 4 °C), followed by two washes with ice-cold SNAP buffer and one wash with SNAP buffer supplemented with NaCl to the final concentration of 200 mM. The supernatant was completely discarded and the beads were resuspended in 40 µl of SNAP buffer supplemented with 1 µg of 3C protease (Sigma-Aldrich). The mixture was then incubated for 8 h at 4 °C. The beads were pelleted by centrifugation, and the supernatant was transferred to a fresh tube, mixed with Laemmli sample buffer and boiled at 95 °C for 5 min. To identify histone PTMs of INO80-bound nucleosomes the immunopurified proteins and input samples were resolved on a 4–20% polyacrylamide gel (Novex WedgeWell Tris-Glycin-Minigel, Invitrogen), histone bands were excised, in-gel derivatized, digested with trypsin and analysed using LC–MS as described below.

CRISPR–Cas9-mediated endogenous protein tagging

The core INO80 complex subunit INO80B was endogenously tagged at its C-terminus with a V5 epitope in the MCF-7 cell line using the tagging strategy described previously54. Specifically, 1 day before transfection, MCF-7 cells were seeded onto 24-well plates at approximately 1.0 × 105 cells per well in 500 µl of low-glucose DMEM medium supplemented with 10% FBS, 1 mM glutamine and 100 μg ml−1 penicillin–streptomycin. On the day of transfection, 25 µl of Opti-MEM medium was added to a 1.5 ml sterile Eppendorf tube, followed by the addition of 1,250 ng of TrueCut Cas9 Protein v2 nuclease (Invitrogen) and 240 ng of two-piece gRNA (crRNA:tracrRNA duplex) generated by annealing crRNA (IDT) and tracrRNA (IDT) according to the manufacturer’s instructions. After mixing briefly by vortexing, 1 µl Cas9 Plus reagent was added to the solution containing Cas9 protein and gRNA. The mixture was incubated at 25 °C for 5 min to allow the formation of Cas9 ribonucleoprotein particles (RNPs). For co-delivery of homology donor DNA, 800 ng of single-stranded DNA oligonucleotide (IDT) was added to the Cas9 RNPs at this point. Meanwhile, 25 µl Opti-MEM medium was added to a separate sterile Eppendorf tube, followed by the addition of 1.5 µl of Lipofectamine CRISPRMAX. After briefly vortexing, the Lipofectamine CRISPRMAX solution was incubated at 25 °C for approximately 5 min. After incubation, the Cas9 RNPs were then added to the Lipofectamine CRISPRMAX solution. The mixture was incubated at 25 °C for 10–15 min to form Cas9 RNPs and Lipofectamine CRISPRMAX complexes and then added to the cells. At 48 h after transfection, the cells were collected by trypsination and seeded in 96-well plates at 1 cell per well. After reaching 60–80% confluency, the cells were trypsinized and split 1:1 into two 96-well plates where the first plate was used for immunofluorescence screening with monoclonal mouse anti-V5 primary antibodies (eBioscience, TCM5 14-6796-82, 1:250) and Alexa-Fluor-488-coupled anti-mouse IgGs as secondary antibodies (Jackson ImmunoResearch Laboratories, 715-545-150, 1:333), and the second plate was used for the subsequent expansion and further testing of V5-positive clones. The immunofluorescence screen for V5-positive clones was performed as previously described54.

Co-IP

Approximately 1.0 × 107 MCF-7 WT or INO80B-V5 cells were used for nuclear extract preparations as described previously25. The nuclear extract was diluted with IP buffer (20 mM HEPES (pH 7.9), 50 mM NaCl, 0.2 mM EDTA, 5% glycerol, 0.1% NP-40, 1 mM DTT and protease inhibitor cocktail (Roche)) to a final protein concentration of around 1 µg µl−1 and a NaCl concentration of 160 mM and subsequently cleared by centrifugation at 20,000g for 10 min at 4 °C. Then, 1 ml of cleared nuclear extract was mixed with 5 μl of anti-V5 antibodies (Abcam, ab15828) and incubated on a rotating wheel over night at 4 °C. The next day, 20 µl of a 1:1 mixture of protein A and protein G Dynabeads (Invitrogen) were added to the sample followed by 1 h incubation on a rotation wheel at 4 °C. Magnetic beads were washed three times with the IP buffer containing 150 mM NaCl. Co-immunoprecipitated proteins were eluted from the beads by boiling in 20 µl of Laemmli sample buffer for 5 min at 95 °C. Eluted proteins were subsequently used for immunoblotting and LC–MS experiments (IP–MS). For LC–MS analysis, proteins were digested with trypsin using a FASP protocol as described below.

Protein detection by immunoblotting

Proteins were separated by SDS–PAGE and blotted onto nitrocellulose membranes (0.45 µm, Thermo Fisher Scientific) using a Bio-Rad PROTEAN mini-gel and blotting system. Antibodies were diluted in TBST + 5% milk (25 mM Tris (pH 7.5), 137 mM NaCl, 2.7 mM KCl, 0.2% Tween-20, 5% non-fat dry milk). The following primary antibodies were used for immunoblots: anti-V5 tag (eBioscience, TCM5 14-6796-82, 1:1,000), anti-INO80 (Abcam, ab118787, 1:2,000), anti-INO80B (Santa Cruz (E-3), sc-390009, 1:1,000), anti-ACTR5 (GeneTex, GTX80453, 1:1,000), anti-TBRG1 (Santa Cruz (D-9), sc-515620, 1:1,000), anti-H3K4me3 (Millipore, 17-614, 1:2,000), anti-H4 (Abcam, ab31830, 1 µg ml−1), anti-H4ac (pan-acetyl) (Active Motif, 39967, 1:1,000), anti-CBX4 (Cell Signaling Technology, E6L7X 30559, 1:1,000), anti-CBX8 (Santa Cruz (C-3), sc-374332, 1:1,000), anti-H2B (Abcam, ab1790, 1:1,000), anti-H2A.Z (Abcam, ab4174, 1:1,000). Immunoblot images were acquired by CCD camera using the Bio-Rad ChemiDoc Touch Imaging System running Image Lab Touch Software (v.2.3.0.07).

MS methods

Sample preparation for MS

On-bead digestion and peptide purification for SNAP samples

The beads were resuspended in 50 μl of elution buffer (2 M urea, 100 mM Tris (pH 7.5), 10 mM DTT) and incubated on a shaker (1,000 rpm) at 25 °C for 20 min. Iodoacetamide (Sigma-Aldrich, I1149) was added to a final concentration of 50 mM and the sample was incubated on a shaker (1,000 rpm) at 25 °C in the dark for 10 min. After digestion with 0.3 μg trypsin (Promega V5113) for 2 h on a thermo shaker (1,000 rpm) at 25 °C, the supernatant was transferred to a new tube and was further digested with 0.1 μg trypsin overnight at 25 °C. The digestion was stopped by adding 5.5 μl of 10% trifluoroacetic acid. Eluted peptides were purified on C18 stage-tips (Glygen 10-200 μl TopTips) according to the manufacturer’s instructions and dried using a SpeedVac.

FASP of label-free proteomics samples

Filter-aided sample preparation was performed as described previously52. In brief, 10–20 µl aliquots of protein mixtures in 1% SDS Laemmli sample buffer were diluted with 200 µl of 100 mM triethylammonium bicarbonate buffer (TEAB; pH 8.5). For protein reduction, 1 µl of 1 M DTT was added to each sample and the samples were incubated at 60 °C for 30 min. After cooling the samples to room temperature, 300 µl of freshly prepared UA buffer (8 M urea in 100 mM TEAB (pH 8.5)) was added to each sample. Proteins were alkylated by the addition of 10 µl of 300 mM iodacetamide solution and subsequent incubation for 30 min at room temperature in the dark. The samples were then concentrated to dryness in a 30 kDa cut-off centrifugal spin filter unit (Millipore), and washed three times with 200 µl UA buffer and twice with 200 µl of 50 mM TEAB (pH 8.5). Then, 40 µl of a 50 ng µl−1 trypsin solution in 50 mM TEAB (pH 8.5) was added to each sample and protein digestion was performed overnight at 37 °C. Peptides were centrifuged through the filter, and the collected flow through was acidified by the addition of trifluoroacetic acid to a final concentration of 0.5% (v/v). About 300 ng of the tryptic peptide mixtures was then used for LC–MS analysis as described below.

Histone sample preparation for proteomics analysis

Histone proteins were prepared for LC–MS analysis using a hybrid chemical derivatization protocol adopted for in-gel sample preparation. In brief, proteins were resolved on 4–20% polyacrylamide gels (Novex WedgeWell Tris-Glycin-Minigel, Invitrogen) followed by Coomassie staining. Histone protein bands were excised from the gel and destained in a destaining buffer (100 mM triethylammonium bicarbonate in 50% acetonitrile). After destaining, the gel pieces were dehydrated with 200 μl of 100% acetonitrile for 10 min at room temperature after which acetonitrile was discarded. Propionylation solution was prepared by mixing 50 mM TEAB (pH 8.5) and freshly prepared 1% (v/v) propionic anhydride solution in water at a 100:1 ratio. Immediately after preparation, 100 µl of propionylation solution was added to the dehydrated gel pieces followed by 10 min incubation at room temperature. The propionylation reaction was quenched by the addition of 10 μl of 80 mM hydroxylamine and subsequent incubation for 20 min at room temperature. The propionylation solution was discarded and gel pieces were dehydrated with 200 μl of 100% acetonitrile for 10 min at room temperature. After this, the acetonitrile solution was discarded and 20 μl of 50 ng µl−1 trypsin solution in 100 mM TEAB (pH 8.5) was added. Trypsin digestion was performed overnight at 37 °C. The next day, 50 μl of 100 mM TEAB (pH 8.5) solution was added to each sample followed by 30 min incubation in a thermo shaker (37 °C, 1,500 rpm). A 1% (v/v) solution of phenyl isocyanate in acetonitrile was freshly prepared and 15 μl added to each sample and incubated for 60 min at 37 °C. The samples were acidified by the addition of 24 μl 1% trifluoroacetic acid. Peptides were desalted with C18 spin columns (Thermo Fisher Scientific) according to the manufacturer’s instructions, dried in a speed-vac, resuspended in 50 μl 0.1% trifluoroacetic acid and subsequently used for LC–MS analysis.

LC–MS-based proteomics measurements

MS analysis of SNAP samples

SNAP samples were processed and analysed by LC–MS on a Q-Exactive mass spectrometer (Thermo Fisher Scientific) as described previously55. In brief, the samples were loaded at 8 μl min−1 onto a trap column (Thermo Fisher Scientific, Acclaim PepMap 100; 100 μm internal diameter, 2 cm length, C18 reversed-phase material, 5 μm diameter beads and 100 Å pore size) in 2% acetonitrile and 0.1% trifluoroacetic acid. Each of the samples was loaded twice, providing two technical replicates. Peptides were eluted on line to an analytical column (Thermo Fisher Scientific, Acclaim PepMap RSLC; 75 μm internal diameter, 25 cm length, C18 reversed-phase material, 2 μm diameter beads and 100 Å pore size) and separated using a flow rate of 250 nl min−1 and the following gradient conditions: initial 5 min with 4% buffer B; a 90 min gradient of 4–25% B; a 30 min gradient of 25–45% B; a 1 min gradient 45–90% B; and finally 15 min isocratic at 100% B before returning to the starting conditions for a 15 min equilibration (buffer A: 2% acetonitrile and 0.1% formic acid in water; B: 80% acetonitrile and 0.1% formic acid). The Q-Exactive instrument acquired full-scan survey spectra (m/z 300–1,650) at 70,000 resolution. An automatic gain control target value of 3 × 106 and a maximum injection time of 20 ms were used. The top 10 most abundant multiply charged ions were selected in a data-dependent manner, fragmented by higher-energy collision-induced dissociation, and data were collected over the range 200–2,000 m/z at 17,500 resolution. An automatic gain control target value of 1 × 105 with a maximum injection time of 120 ms was used. A dynamic exclusion time of 30 s was enabled.

MS analysis of label-free proteomics samples

LC–MS/MS analysis of label-free nucleosome pull-downs and ChIP–MS proteomics samples was performed on the Q-Exactive HF mass spectrometer (Thermo Fisher Scientific) coupled in-line to a nanoEasy LC (Thermo Fisher Scientific). The samples were loaded in solvent A (0.1% formic acid) on a two-column set-up consisting of a 3.5 cm, 100 µm inner diameter pre-column packed with Reprosil-Pur 120 C18-AQ (5 µm; Dr. Maisch) and an 18 cm, 75 µm inner diameter analytical column packed with Reprosil-Pur 120 C18-AQ (3 µm; Dr. Maisch). A gradient of solvent B (95% acetonitrile, 0.1% formic acid) was applied at a flow rate of 250 nl min−1 as follows: 3% to 25% B in 90 min; 25% to 45% B in 30 min; 45% to 100% B in 3 min; and 100% B in 8 min. MS was obtained at a resolution of 120,000 and MS/MS as top 15 at a resolution of 15,000 and with a dynamic exclusion of 30 s. The maximum injection time was set to 100 ms for both MS and MS/MS and only peptides of charge state 2, 3 and 4 were selected for MS/MS.

LC–MS/MS analysis of INO80-V5 IP–MS samples was performed on the Q-Exactive HF mass spectrometer (Thermo Fisher Scientific) coupled to a nano-RSLC (Ultimate 3000, Dionex). In brief, the samples were automatically loaded onto a nano trap column (300 µm inner diameter × 5 mm, packed with Acclaim PepMap100 C18, 5 µm, 100 Å; LC Packings) before separation by reversed-phase chromatography (HSS-T3 M-class column, 25 cm, Waters) in a 95 min nonlinear gradient from 3 to 40% acetonitrile in 0.1% formic acid at a flow rate of 250 nl min−1. Eluted peptides were analysed using the Q-Exactive HF mass spectrometer equipped with a nano-flex ionization source. Full scan MS spectra (m/z 300–1,500) and MS/MS fragment spectra were acquired in the Orbitrap with a resolution of 60,000 or 15,000, respectively, with maximum injection times of 50 ms each. Up to ten most intense ions were selected for higher-energy collisional dissociation fragmentation depending on signal intensity. Dynamic exclusion was set for 30 s.

MS analysis of histone samples

For LC–MS analysis of modified histone proteins, the acidified histone peptide digests were analysed on the Q-Exactive HF mass spectrometer (Thermo Fisher Scientific) coupled in-line to a nanoEasy LC (Thermo Fisher Scientific). In brief, the samples were automatically loaded onto an in-house packed 2 cm 100 µm inner diameter C18 pre-column with buffer A (0.1% formic acid) and then eluted and separated on an in-house packed Reprosil-Pur 120 C18-AQ (3 µm; Dr. Maisch) analytical column (20 cm × 75 µm inner diameter) using a 35 min linear gradient from 0% to 40% buffer B (90% acetonitrile, 0.1% formic acid). Full scan MS spectra (m/z 300–1,000) and MS/MS fragment spectra were acquired in the Orbitrap with a resolution of 120,000 or 15,000, respectively, with maximum injection times of 50 ms each. Up to the 20 most intense ions were selected for higher-energy collisional dissociation fragmentation depending on signal intensity. Dynamic exclusion was disabled.

MS RAW data search and quantification

Analysis of SNAP MS data

Protein abundances were quantified from the Q-Exactive raw data files using MaxQuant (v.1.5.2.8)56 against the UniProt UP000005640 canonical proteome (downloaded in September 2016) using 2-plex labelling (Arg0/Lys0 and Arg10/Lys8). The search was performed allowing for fixed carbamidomethyl modification of cysteine residues and variable oxidation of methionine residues and acetylation of amino termini. The minimum peptide length was set to 7. All raw files resulting from the forward and reverse pull-downs, including technical replicates for each nucleosome tested, were processed together using the ‘match between runs’ feature. H/L ratios were computed in advanced ratio computation mode, with the minimal ratio and peptide count set to 1. The corresponding mqpar.xml file is deposited along with the proteomics data. Initial trial experiments with mono-, di- and tetra-nucleosomes (Supplementary Information) were quantified separately by MaxQuant v.1.5.1.0 against the December 2015 version of UniProt proteome with more stringent settings requiring at least two peptides for ratio estimation.

Analysis of label-free MS data

Protein identification and quantification was performed using Proteome Discoverer v.2.5 (Thermo Fisher Scientific). Data were searched against the human Swiss-Prot database using Mascot57 as the search engine, with a precursor mass tolerance of 5 ppm and a fragment mass tolerance of 0.05 Da. Two missed cleavages were allowed for trypsin and carbamidomethylation of cysteine was set as a static modification, while oxidation of methionine was set as dynamic. Label-free quantification was achieved as match between runs by using the Minora Feature Detector, the Feature Mapper and the Precursor Ions Quantifier. The maximum retention time shift for chromatographic alignment was set to 2 min and the retention time tolerance for mapping features was set to 1 min. Peptide quantification was performed as the peak area normalized to the total peptide amount and protein quantification as the average of the top three unique peptides.

Analysis of histone MS data

For the identification and quantification of histone PTMs in ChIP–MS samples and the quality control of recombinantly produced modified histone proteins, MS raw data files were manually analysed using Skyline (v.20.1.0.31)58. In brief, a list of unmodified as well as differentially modified histone H3 and H4 peptides was manually compiled and used to evaluate the modification status of histones in each sample. All lysine residues not bearing acetylation or methylation were considered to be propionylated and all peptide N termini were considered to be modified with phenyl isocyanate. MS1 filtering was set to include 3 isotope peaks and the MS1 resolving power was set at 120,000. MS2 resolving power was set at 15,000. For each modified histone peptide, the relative abundance was estimated by dividing its peak area by the sum of the areas corresponding to all of the observed forms of that peptide (that is, all peptides sharing the same amino acid sequence). The relative abundance of histone variant H2A.Z was estimated by dividing the sum of peak areas of unique H2A.Z peptides (that is, only present in H2A.Z but not in any other H2A variants) by the sum of peak areas of all unique peptides corresponding to histones H2A, H2B and H2A.Z.

Data postprocessing and bioinformatic analyses

Data postprocessing

Postprocessing of SNAP MS data

MaxQuant proteinGroups entries marked as ‘potential contaminant’, ‘reverse’ or ‘only identified by site’ were removed from the datasets analysed. The SILAC H/L ratios for each of the remaining entries were converted to a log2 scale. In initial trial experiments (Supplementary Information), the median and first and third quartiles log2[H/L ratio] values were estimated in all experiments individually, treating forward and reverse experiments separately. Proteins were assumed to be significantly enriched if they fell 1.5× the interquartile range away from first and third quartiles for both forward and reverse experiments, matching the box plots. The data for the main set of experiments were additionally annotated with up to date (as of 30 July 2019) metadata that were downloaded from the mygene.info API service59 based on the IDs in the ‘Majority Protein ID’ column. Protein identifiers were assigned readable counterparts on the basis of the associated gene names. Duplicate entries were enumerated in parentheses (for example, SMARCA (1) and SMARCA (2)), assigning lower numbers to entries with a higher MaxQuant score. Common prefixes of the gene names were collapsed (for example, SMAD[2,3,9]) for brevity. The principal direction of the data spread (that is, the direction of enrichment) in each of the pull-downs was estimated by determining the first principal component of the data in the top-left and bottom-right quadrants of the forward and reverse log2[H/L ratio] plot. The estimate was adjusted by re-evaluating the principal direction after removing outlier points ±2 s.d. away from the median in the second principal direction. Protein-specific variation in the second principal direction across pull-down experiments was adjusted to zero to correct systemic heavy and light cell population batch effects resulting from different abundances of proteins in the nuclear extracts from the H/L cell populations or different labelling efficiencies of proteins with the heavy-labelled amino acids. In cases in which either the forward or the reverse H/L ratio was measured for the protein (9.13% of ratio pairs), but not both, the missing ratio was imputed by projecting the measured ratio to the estimated principal enrichment line. In six cases (0.01%) in which the estimated H/L ratio was infinite as protein intensity could have been measured in the modified nucleosome, but not in the unmodified nucleosome, the ratio was imputed to the maximum ratio identified in the particular SNAP experiment. All other missing H/L ratios were imputed to zero (24.27%). Five proteins of which the forward and reverse H/L ratios were equal to zero in all of the experiments were removed. The resulting data for each of the pull-down experiments were then further rotated so the estimated principal direction of variation lays exactly on the ideal 45° diagonal, so the reverse ratio on average equals the negative of the forward one. For visualizations and computational analyses, the sign of the reverse experiment was flipped to be on the same scale as the forward one.

Postprocessing of cross-linked H3K4me1 and H3K4me3 ChIP–MS data

Protein abundances obtained from H3K4me1 and H3K4me3 cross-linking-ChIP–MS experiments were converted to log2 scale, treating zero abundances as missing data. The data were normalized to ten histone proteins observed in the data: H2AC20, H2AC21, H2AW, H2AZ2, H2BC4, H2BU1, H3-2, H4C1, MACROH2A1 and MACROH2A2. Specifically, we calculated the average log2-transformed abundance for the histone proteins in each of the experiments, and calculated the residuals (that is, log2-transformed abundances minus the average (M value)) for the histone proteins. The data were normalized by subtracting the median of these residuals for each of the samples, so that the median M value of the normalized data for the histone proteins remains approximately zero across experiments. The normalized data were then further filtered to include only proteins that were detected in at least two replicates of at least one experiment.

We used limma60 to estimate the log2[FC] values between H3K4me3 and controls (H3 and H4), H3K4me1 and controls, and H3K4me3 and H3K4me1. Specifically, we used a zero-intercept means model encoding one parameter for each experiment (H3, H4, H3K4me1, H3K4me3), and analysed the contrasts between protein abundance in H3K4me1/3 experiments and the average abundance of H3 and H4 (for example, (H3 + H4)/2), as well as a contrast between H3K4me3 and H3K4me1. The analysis was run using the default parameters of limma (v.3.50.1), with the addition of ‘robust=True’ in the ‘eBayes’ step, hypothesis testing was performed using the default settings, assuming zero log2[FC] under the null hypothesis. P values were corrected using the Benjamini–Hochberg procedure, and significance was assumed at an FDR of 0.05.

In some cases, the contrasts could not be estimated due to missing data. This frequently happened when proteins were detected in one of the experiments, but not in controls (or vice versa). In these cases, we imputed such log2[FC] estimates with infinities (positive and negative). Moreover, whenever it was possible to estimate the H3 or H4 controls, but not both, we imputed the log2[FC] estimates using one of such controls only. The imputed estimates are clearly flagged in the data and figures. Estimates based only on single data points (that is, an observed abundance in one of the three replicates only) are flagged as well.

To be able to link the ChIP–MS data with MARCS feature effect estimates, we mapped the ChIP–MS proteins to their MARCS counterparts through their accession numbers and gene names. The cases in which one ChIP–MS protein mapped to multiple proteins in the MARCS dataset were resolved by assigning the feature effect estimate with the lowest P-value estimate across all of the matched identifiers.

To obtain association statistics, we performed a Mann–Whitney U-test, comparing the imputed ChIP–MS log2[FC] estimates of proteins strongly recruited to or excluded by a MARCS feature to the imputed log2[FC] estimates of other proteins detected in both MARCS and ChIP–MS data. Only the groups with at least five proteins were tested. For visualization purposes, we computed the mean log2[FC] estimates in each of the groups, and their respective differences. For this purpose, we assumed the infinities to be equal to the maximum finite log2[FC] plus a small number.

Postprocessing of variable-linker nucleosome pull-down data

Label-free MS quantification datasets for the short linker nucleosome, long linker SV40 promoter nucleosome and long linker SV40 enhancer nucleosome affinity-purification experiments were analysed independently. The protein abundances were converted to a log2 scale, treating zero intensities as missing values. The data were normalized using the abundances of HIST1H4A and HIST2H2BF histones (short linkers) or H4C1 and H2BC12 histones (long linkers) as described in the H3K4me1/3 cross-linking-ChIP–MS methods.

For each set of experiments, we used a zero-intercept means model in limma and hand-crafted contrasts to measure two types of effects on protein binding to dinucleosomes: (1) modification-specific effects, that is, the log2-transformed FC in protein abundance between modified nucleosome and unmodified nucleosome, given a specific linker of certain length, for example, log2[H3K27me3 with 50 bp linker] versus log2[unmodified with 50 bp linker], as well as (2) linker-specific effects, that is, the log2-transformed FC in protein abundance between two different linkers, given a certain nucleosome modification, for example, log2[H3K27me3 with 55 bp linker] versus log2[H3K27me3 with 50 bp linker]. Owing to the large number of missing values, the second replicate of the H3K27me3 experiment with 35 bp linker was excluded from the analysis. Only proteins that had at least two values in at least one condition were analysed.

The analysis was run using the default parameters of limma (v.3.50.1), using the ‘robust=True’ parameter in the ‘eBayes’ step. P values were corrected using the Benjamini–Hochberg procedure, assuming significance at an FDR of 0.05. In addition to this, significant estimates were considered to be ‘strong’ if the absolute log2[FC] was greater than 1.

As in the H3K4me1/3 cross-linking-ChIP–MS experiment, we imputed contrasts that could not be estimated from the data using the following heuristics: proteins detected in one of the conditions, but not the other, received either infinite enrichments or infinite depletions. Such imputed estimates were flagged in the data, together with estimates based on single data points.

To aid the data visualization, we divided the proteins into three groups on the basis of the effects of the modifications and linkers on dinucleosome binding in the different analyses: (1) modification-responsive proteins, that is, proteins that have a significant and strong response to a modification signature in at least one of the linkers visualized; (2) linker-responsive proteins, that is, proteins with a significant and strong response to the linker in either modified or unmodified nucleosomes; and (3) proteins that respond to both, that is, satisfy conditions (1) and (2) simultaneously.

Postprocessing of endogenous INO80B-V5 IP–MS data

For analysis of INO80B-V5 IP-MS data, only proteins identified based on three or more unique peptides were considered. The quantified MS1 protein abundances were normalized to the IGHG1 abundance. Differential enrichment analysis was performed using a two-tailed t-test. P values were adjusted for multiple comparisons using the Benjamini–Hochberg method. The protein stoichiometry was determined using MS1-based label-free quantification61. Specifically, protein abundances were calculated as the mean of MS1 intensities of all unique peptides identified for the protein. To assess the stoichiometry of INO80 complex subunits, the abundance of each subunit (mean of unique MS1 peptide intensities) was divided by the abundance of INO80B (mean of unique INO80B MS1 peptide intensities) used as a bait in co-IP complex purification experiments.

Decoupling of the effects of individual modification features (SNAP dataset)

Pairs of nucleosomes differing by a single modification only were identified by arranging the nucleosomes into a directed graph of which the edges track the difference by one modification, including self-informative nucleosomes that contain only one chromatin feature (for example, H3K4me3). H3K9acK14ac, full acetylation on histone H3 (H3K9acK14acK18acK23acK27ac), H4K5acK12ac and fully acetylated H4 (H4K5acK8acK12acK16ac) were treated as single modification. Only chromatin features that have two or more informative nucleosome pairs, and therefore an independent experimental replicate, were analysed. As each pull-down consists of a forward and reverse experiment, this results in at least four experimental measurements, enabling a robust statistical analysis. Moreover, a feature effect estimate was computed only for proteins that have at least one nucleosome pair with no imputed data.

The relationship between nucleosomes was modelled in limma using the following formula: ‘~ 0 + edge + ptm’. Here the ‘edge’ parameter tracks edges in the directional graph and ptm captures the direction of the edge and is set to one at the endpoint that contains the target feature and zero at the other. This expression allows the baseline effect of a nucleosome pair to be captured by the ‘edge’ parameter allowing the ‘ptm’ parameter to measure the change of the effect caused by the modification feature (that is, a PTM, histone H2A.Z or DNA methylation). Self-evident purifications were assigned no edge coefficient. Limma was run with robust empirical Bayes, with weights set to number of unique peptides detected plus one. Significance was assumed at Benjamini–Hochberg-adjusted FDR of 0.01.

Significant responses were additionally labelled as strong if their parameter estimates were greater than or equal to 1. For the proteins that respond strongly to at least one feature, the collective modification response profiles across all features were clustered. The clustering was performed using protoclust62 (v.1.6.3) under cosine distance. The dendrogram corresponds to Minimax Hierarchical Linkage. In cases in which no estimate for the effect could be made, for clustering purposes the values were imputed using three nearest neighbours (bnstruct package63). The resulting dendrogram was divided into 40 flat clusters that were annotated with their respective prototype protein in Fig. 2e and Supplementary Table 5.

The joint response of protein complexes to chromatin features was analysed using CAMERA64. Only complexes with 3–40 members (inclusive) were analysed. Significance was assumed at a Benjamini–Hochberg-adjusted FDR of 0.01. Whenever possible, the enrichment of both the whole protein complex, and the enrichment of only the exclusive subunits of the complex, not including subunits shared with other complexes, was tested. The median effect of chromatin features on protein complexes was estimated from 100,000 random samples from the effect distributions of individual subunits. The median, as well as the empirical 95% confidence interval (CI) is reported.

Network inference (SNAP dataset)

We used the network inference algorithms ARACNE, MRNET and CLR implemented in the minet package36 to infer the protein–protein interaction networks in an unsupervised manner, using only the 1,915 × 110 matrix of processed log2-transformed heavy/light ratios of identified proteins as the input. The algorithms were configured to use Miller-Madow (mi.mm) estimator for MI and the equal width discretization strategy with the bin number set to 10. In addition to the algorithms above, the performance of the MI metric on its own (without subjecting it to network algorithms) was also evaluated (network RAW-MI).

In addition to the MI-based methods above, we have benchmarked the networks defined by the interprotein correlation matrix computed both naively (CORR) or using Ledoit–Wolf shrinkage (CORR-LW)65. These networks were built by assuming the adjacency between the nodes to be equal to the corresponding entry in the correlation matrix. Negative values in the correlation matrix were avoided by adding one to each of the entries and dividing the result by two.

The inferred networks were evaluated against the BioGRID database34 (release 3.5.174) after training. BioGRID entries were linked with our identifiers through Entrez identifiers downloaded previously through the mygene.info API service59. Networks were evaluated by computing their precision (fraction of predicted edges in the network that were also in the BioGRID database) and recall (fraction of edges in BioGRID database that were predicted by the network) at multiple stringency levels. We used the scaled truncated area under precision and recall curve (auPRC) statistic66, which combines the multiple precision/recall estimates into a single score as our primary metric. As we did not anticipate a full recovery of BioGRID interactions by our networks and therefore wanted to trade higher precision for lower recall, we did not consider any threshold settings with a recall of greater than 0.2 for the evaluation of the algorithms. Interactions with histone proteins, as well as self-interactions (either homodimers in BioGRID or interactions between two proteins with the same gene name) were excluded from the evaluation.

To produce the inferred networks described in the paper, we noted that the scores of the CLR algorithm can be converted to P values by noting that for the CLR scoring function \(s(i,j)=\sqrt{\max {(0,{z}_{i})}^{2}+\max {(0,{z}_{j})}^{2}}\) where zi and zj are assumed to follow standard normal distribution under the null hypothesis35,36, the P values under null can be expressed as \(P(s(i,j)\ge x > 0)=\frac{1}{4}(2\times \text{erfc}(\frac{x}{\sqrt{2}})+{e}^{-{x}^{2}/2})\). Where erfc is the complementary error function. Adjusting those computed P values for multiple hypothesis testing using the Benjamini–Hochberg procedure (that is, converting them to a q value) enabled us to pick a set of intuitive thresholds to produce the networks presented in the paper.

Networks at different adjusted q-value thresholds were drawn using the Force Atlas 2 algorithm in gephi67 and adjusted manually. Only proteins with at least five non-zero values were drawn. Isolated nodes (connected components with size of 1) were not drawn. Network nodes were either coloured by communities (Louvain algorithm68 implemented in the Python-Louvain package) or overlaid by the colour-coded chromatin response estimates (see the ‘Decoupling of the effects of individual modification features (SNAP dataset)’ section above). In the network projection plots, the names of protein complexes were annotated manually on the basis of protein complexes that were significantly regulated by the chromatin modification (as reported by the CAMERA procedure), and had empirically estimated median effects of at least 0.3. Expert judgement was used to disambiguate complexes with a high number of shared subunits, as well as to determine which labels to exclude to reduce crowding. Protein complexes that did not form tight clusters in the network were not annotated.

An additional high confidence network was generated for protein interaction predictions by selecting a network threshold at which 70% precision was achieved. BioGRID interactions that were not predicted by the algorithm (false negatives) were added to the network plot. The network was visualized using cytoscape69. Network node labels and annotations were added to the network manually. Both high-confidence and standard network interaction predictions are provided in Supplementary Table 7.

Curation of protein complex list (SNAP dataset)

A curated protein complex list was seeded with complexes downloaded from the EBI complex portal version 19 July 2019 (ref. 38) and the EpiFactors database (obtained on 29 July 2019)37. Protein members of the complexes that were not detected in our experiments were filtered out. Only complexes with at least two protein subunits left after filtering were retained, merging protein complexes that became indistinguishable (that is, had the same subunits) after filtering. Protein complex annotations from the databases that were substantially similar (for example, variants of protein complexes defined by redundant adapter proteins) were merged together based on manual review. Missing annotations from the databases were added manually based on the review of the inferred protein network and corresponding literature. In some cases, the entries were also augmented with data from CORUM70 and UniProt71. Where possible, protein complexes were renamed manually to match the canonical designations. All sources of annotations were recorded and are available in Supplementary Table 8.

Integration of MARCS with ChIP–seq data

For joint MARCS and ChIP–seq analysis, the relevant ENCODE30 ChIP–seq, DNase-seq and ATAC–seq datasets for the K562 cell line were downloaded together with the chromatin state predictions from ROADMAP1. We next divided the hg38 reference genome, excluding blacklisted regions72 and chromosome Y, into a set of non-overlapping 1,000-bp-wide bins and marked the bins containing peaks from each of the NGS datasets. We have assumed each of the genomic bins to be independent and identically distributed and therefore modelled the presence or absence of a given peak as a Bernoulli event. For a given pair of NGS datasets, we therefore computed their joint distribution by counting the bins for which both datasets are co-present, co-absent and mutually exclusive (both ways). A pseudocount of 100 was added to avoid zeroes and to smooth the probability estimates. This joint distribution enabled us to compute the MI between two NGS datasets, which is equivalent to the Kullback–Leibler divergence from the joint distribution under independence. To obtain an interpretable statistic that measures the fraction of information about A that can be predicted by knowing B, the MI was divided by the Shannon entropy (H) of one of the two datasets: U(A,B) = MI(A,B)/H(A). We frequently refer to this ratio as fraction of entropy of A explained by B or, simply, the normalized MI. As a convention, we use this to measure the fractional entropy of a protein (for example, PHF8) NGS experiment that the knowledge of a chromatin feature (such as H3K4me3) NGS experiment provides, for example, U(PHF8, H3K4me3) = MI(PHF8, H3K4me3)/H(PHF8) (Extended Data Fig. 4a).

We next compared these normalized MI estimates for each of the MARCS-identified proteins for which ENCODE ChIP–seq data were available in K562 cells. For each of the MARCS chromatin features, and for each of the ChIP–seq chromatin features, we measured whether the proteins predicted to be strongly recruited or strongly excluded by MARCS feature had significantly higher or lower uncertainty coefficients, when compared to proteins neither strongly recruited nor strongly excluded, or proteins identified in MARCS for which we had no MARCS feature effect estimates at all. For these comparisons, we used a Mann–Whitney U-test (two-sided) and Benjamini–Hochberg correction. For the benefit of visualization we also computed the differences between mean log2-normalized MI estimates for MARCS-feature-associated proteins and others. In cases in which proteins had multiple ChIP–seq replicates, we used the harmonic average of their normalized MI coefficients for the analysis. We treated replicates of chromatin feature ChIP–seq analyses independently. In cases in which one ChIP–seq protein mapped to multiple MARCS proteins, we used the chromatin feature effect estimates from the proteins with the lowest P value.

As an additional similarity metric to the normalized MI statistic described above, we computed the Kendall correlation between the peak heights (as defined by the column 7 signalValue in the ‘narrowPeak’ and ‘broadPeak’ file formats) for genomic bins for which the peaks were co-present. This metric is used in Extended Data Fig. 4e–j.

For verification of the network analysis results in Extended Data Fig. 7f, we divided each pair of proteins for which ChIP–seq data were available into groups based on the confidence of inferred interactions from the MARCS-based network analysis. In the case of multiple mappings to MARCS, the highest-confidence outcome was chosen. For each such pairs, we computed the symmetric variant of normalized MI statistic: Usym(A,B) = (2MI(A,B))/(H(A) + H(B)), based on their ChIP–seq datasets. The statistics of replicate experiments were averaged harmonically. A one-sided Mann–Whitney U-test was used to test whether the distribution of symmetric normalized MI coefficients is statistically different across the MARCS confidence levels (Bonferroni correction).

Statistics

The details of quantification and statistical analyses are described in detail in the Methods. Where appropriate, the necessary information is also described in the figure legend.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.



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