Decoding chromatin states by proteomic profiling of nucleosome readers

DNA and histone modifications combine into characteristic patterns that demarcate functional regions of the genome1,2. While many ‘readers’ of individual modifications have been described3–5, how chromatin states comprising composite modification signatures, histone variants and internucleosomal linker DNA are interpreted is a major open question. Here we use a multidimensional proteomics strategy to systematically examine the interaction of around 2,000 nuclear proteins with over 80 modified dinucleosomes representing promoter, enhancer and heterochromatin states. By deconvoluting complex nucleosome-binding profiles into networks of co-regulated proteins and distinct nucleosomal features driving protein recruitment or exclusion, we show comprehensively how chromatin states are decoded by chromatin readers. We find highly distinctive binding responses to different features, many factors that recognize multiple features, and that nucleosomal modifications and linker DNA operate largely independently in regulating protein binding to chromatin. Our online resource, the Modification Atlas of Regulation by Chromatin States (MARCS), provides in-depth analysis tools to engage with our results and advance the discovery of fundamental principles of genome regulation by chromatin states.

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Data Policy information about availability of data
All manuscripts must include a data availability statement.This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Gel source raw data for western blots shown in Figure 5e, Extended Data Figure 2b, and Extended Data Figures 5g,h,j is provided in Supplementary Figure 1.
The mass spectrometry data that was generated for this study has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (https:// www.ebi.ac.uk/pride/) with the following identifiers:

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Life sciences study design
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Sample size
The nucleosomes tested in SILAC nucleosome affinity purification experiments were selected based on the prior reports of biologically relevant modification signatures of chromatin states.It was attempted to achieve a good coverage of modification signatures representing enhancer, promoter, and different heterochromatin states, while balancing this with the requirements for single modification controls and the limited availability of nuclear extracts.Studies that were used as information sources for the general design of the library of modified nucleosomes include: Young et al Label-free nucleosome affinity purifications using di-nucleosomes incorporating different DNA linkers followed two design strategies: (1) to test the effect of DNA linker length on protein binding to heterochromatin-like chromatin states (Trojer & Reinberg, 2007, Mol Cell 28: 1-13; Saksouk et al., 2015, Epigenet Chromatin 8: 3) di-nucleosomes incorporating H3K9me3 or H3K27me3 modifications (markers of constitutive and facultative heterochromatin, respectively) were assembled with a series of short DNA linkers increasing in length from 35-55 bp in 5bp increments, since these are the most frequently found linker lengths in mammalian cells (Voong et al., 2016, Cell 167: 1555-1570.e15);(2) to test the effects of the presence of a nucleosome-depleted region on protein binding to enhancer-and promoter-like chromatin states, dinucleosomes incorporating either H3K4me1 or H3K4me1K27ac modifications and a 200bp DNA linker containing the functional elements of the SV40 early enhancer (enhancer chromatin state), or H3K4me3K9acK14acK18acK23acK27ac and H4K5acK8acK12acK16acK20me2 modifications and the histone variant H2A.Z together with a 200bp DNA linker containing the SV40 early core promoter (active promoter chromatin state) were assembled and compared to the respective unmodified di-nucleosomes and a library of di-nucleosomes containing 100 million different 200 bp DNA linkers with random sequences.The viral SV40 promoter and enhancer sequences were chosen since they constitute very well characterised enhancer and promoter sequences (Banerji et al., 1981, Cell 27: 299-308;Schirm et al., 1987, Genes Dev 1: 65-74;Keiser et al., 2015, J Gen Virol 96: 601-606), and both are around 200 bp in length, enabling the assembly of di-nucleosomes with 200 bp nucleosome-depleted regions resembling enhancer-and promoter-like chromatin states (Haberle & Lenhard, 2016, Semin Cell Dev Biol 57: 11-23;Haberle & Stark, 2019, Nat Rev Mol Cell Biol 19: 621-637).Detailed information about the design of modified di-nucleosomes incorporating various different DNA linkers is provided in the Supplementary Information.
Data exclusions • One di-nucleosome in the SILAC nucleosome affinity purification experiments in which we profiled H3K4me3-5ac/H4-4ac/H2A.Z in combination with methylated DNA failed our quality checks for the nucleosome assembly described in the supplementary document.This nucleosome was therefore not included in our experiments and analyses.
• One measurement in the label-free di-nucleosome affinity purification experiments in which we profiled H3K27me3 in combination with 35 bp linker DNA failed our mass spectrometry data quality checks.This measurement was therefore excluded from our statistical analyses.
• A series of label-free di-nucleosome affinity purifications in which we tested combinations of H3K27ac with 50bp, 200bp scrambled, and 200bp SV40 enhancer linkers was carried out and analysed together with the affinity purifications testing combinations of H3K4me1 and H3K4me1K27ac with 50bp, 200bp scrambled, and 200bp SV40 enhancer linkers.Since the H3K27ac affinity purifications did not add any additional valuable information, they were not included in the final figures in order to reduce the complexity of the displayed data.

Replication
• For the SNAP experiments the two isotopically labelled batches of nuclear extracts allow each of the experiments to be performed as a biological replicate (in forward and reverse settings, see methods).The extracts were mixtures of three independently prepared extracts to level out differences in individual extracts.Multiple nucleosomes with similar modification patterns serve as internal controls.
• Label-free nucleosome affinity purifications, IP-MS, and X-ChIP-MS experiments were carried out in triplicates to allow statistical analyses.
• Native ChIP-MS of INO80-bound nucleosomes was performed in two independent replicates with similar results in each replicate.
• Nucleosome affinity purifications followed by western blot detection to validate the nucleosome binding characteristics of INO80 (Figure 5e) were carried out in three independent experimental replicates with similar results in each replicate.
• Nucleosome affinity purifications followed by western blot detection to validate the nucleosome binding characteristics of CBX4 and CBX8 (Extended Data Figure 2b) were carried out in two experimental replicates with similar results in both replicates.
Randomization Samples were not randomised.However, being a high-throughput proteomics study, many samples were handled at any given time and for nature portfolio | reporting summary

April 2023
Randomization each set of experiments with no particular preference or bias towards specific samples.During the experiments care was taken to treat batches of samples evenly by rotating the order of the samples during repeated processing steps.During the mass spectrometric measurements the liquid chromatography columns were cleaned in regular intervals between runs, and replicate samples belonging to the same set of experiments were injected in random order to avoid any measurement biases.Batch effects and experimental biases were also minimised during the downstream computational analyses by normalisation and cross-validation of all measurements of a given dataset.

Blinding
Investigators were not blinded.Blinding was not required since the outcome of the high-throughput proteomics measurements and western blot readouts are unknown to the experimenter at the time of performing the experiment and carrying out the data acquisition.The results can therefore not be affected by personal bias or knowledge of the identity of the sample at the time of acquiring the data.
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Materials
Further information about databases used in this study is provided in the Key Resources Table in Supplementary Table10and the Supplementary Information.A detailed list of ENCODE datasets used for the integration of MARCS with ChIP-seq data, including ENCODE accession numbers, is provided in Supplementary Table4.