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bioRxiv · 10.1101/2025.05.01.651740

Direct Readout of Multivalent Chromatin Reader-Nucleosome Interactions by Nucleosome Mass Spectrometry

Lee, A. S.·Fisher, N. P.·Marunde, M. R.·Su, P.·Khan, L. F.·Graham, B.·Taylor, H. F.·Onuoha, U. C.·Su, T.·Jooss, K.·Schachner, L. F.·Fuchs, H. A.·Noll, K.·Cheek, M. A.·Burg, J. M.·Sun, Z.-W.·Musselman, C. A.·Keogh, M. A.·Kelleher, N. L.
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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. O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/651740v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@8815e9org.highwire.dtl.DTLVardef@5685b5org.highwire.dtl.DTLVardef@643881org.highwire.dtl.DTLVardef@1b9bc20_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFor TOC only.C_FLOATNO Synopsis. Nuc-MS provides the ability to control the disassembly of chromatin-associated protein-nucleosome complexes (CAP-nuc) and delineate the histone proteoforms driving tandem reader domain binding. C_FIG

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Lee, A. S., Fisher, N. P., Marunde, M. R., Su, P., Khan, L. F., Graham, B., Taylor, H. F., Onuoha, U. C., Su, T., Jooss, K., Schachner, L. F., Fuchs, H. A., Noll, K., Cheek, M. A., Burg, J. M., Sun, Z.-W., Musselman, C. A., Keogh, M. A., Kelleher, N. L.. 2025-05-02. Direct Readout of Multivalent Chromatin Reader-Nucleosome Interactions by Nucleosome Mass Spectrometry. https://doi.org/10.1101/2025.05.01.651740

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