{"doi":"10.1101/2025.05.01.651740","title":"Direct Readout of Multivalent Chromatin Reader-Nucleosome Interactions by Nucleosome Mass Spectrometry","abstract":"Histone post-translational modifications (PTMs) often serve as distinct recognition sites for the recruitment of chromatin-associated proteins (CAPs) for epigenome regulation. While CAP-PTM interactions have been extensively studied using histone peptides, this cannot consider the regulatory potential of multi-site binding on intact nucleosomes. To overcome this limitation, we applied Nucleosome Mass Spectrometry (Nuc-MS), a native Top-Down MS approach that enables controlled disassembly of intact CAP:nucleosome (CAP:nuc) complexes to provide a direct readout of the contained histone proteoforms. As proof of principle, we show the BPTF PHD-BD native tandem reader requires coincident H3K4me3K9acK14acK18ac for effective nucleosome engagement. We extend our approach to explore how the BRD4 (native BD1-BD2), DNMT3A-MPP8 (chimeric PWWP-CD), and PtSHL (native BAH-BD) tandem readers interact with endogenous nucleosomes. Each reveals distinct enrichment profiles: BRD4 favoring di- and tri-acetylated histone H4 proteoforms, whereas DNMT3A-MPP8 and PtSHL preferentially interact with hypermethylated H3 proteoforms. Of note the latter enriches combinatorial {H3K4me3K27me3} on the same histone tail in HeLa chromatin, and thus expands the potential biology of this widely studied bivalent signature. By directly characterizing CAP:nuc complex composition with combinatorial PTM information in a single readout, Nuc-MS serves as a new approach to discover the modifications driving binding, and therefore primary candidates to explore for structural biology and genomic studies.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":555523,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":3,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9684,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1094458,"name":"Nickolas P. 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Schachner","orcid":"0000-0001-6157-0937","position":10,"is_corresponding":false},{"id":629647,"name":"Harrison A. Fuchs","orcid":"0000-0001-8314-3004","position":11,"is_corresponding":false},{"id":353039,"name":"Kelsey E. Noll","orcid":"0000-0002-3977-0083","position":12,"is_corresponding":false},{"id":275817,"name":"Marcus A. Cheek","orcid":"0009-0005-9814-3097","position":13,"is_corresponding":false},{"id":275704,"name":"Jonathan M. Burg","orcid":"0000-0003-1432-8915","position":14,"is_corresponding":false},{"id":685257,"name":"Zu‐Wen Sun","orcid":"0000-0001-8389-0843","position":15,"is_corresponding":false},{"id":398007,"name":"Catherine A. Musselman","orcid":"0000-0002-8356-7971","position":16,"is_corresponding":false},{"id":237417,"name":"Michael‐Christopher Keogh","orcid":"0000-0002-2219-8623","position":17,"is_corresponding":false},{"id":237416,"name":"Neil L. Kelleher","orcid":"0000-0002-8815-3372","position":18,"is_corresponding":false},{"id":651639,"name":"Alexander S. 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