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 are extensively studied using histone peptides, this cannot represent the regulatory potential of multisite binding on intact nucleosomes. To overcome this limitation, we applied Nucleosome Mass Spectrometry (Nuc-MS), a native Top-Down MS approach that enables the controlled disassembly and proteoform analysis of CAP: nucleosome (CAP: nuc) complexes. As proof of principle, we show the BPTF plant homeodomain (PHD) -bromodomain (BD) native tandem reader binds synergistically to both PTM classes in fully defined (H3K4me3K9acK14acK18ac2) nucleosomes. We then extend to explore the engagement of BRD4 (native BD1-BD2), DNMT3A-MPP8 (chimeric PWWP-CD), and Populus trichocarpa Short Half Life (PtSHL) (native bromodomain-adjacent homology (BAH-PHD) tandem readers with endogenous HeLa nucleosomes. In the resulting enrichment profiles, BRD4 favors di- and triacetylated histone H4 proteoforms, whereas DNMT3A-MPP8 and PtSHL recover distinct hypermethylated H3 proteoforms. Of note, PtSHL enriches a potential H3K4me3K27me3 cis combinatorial that expands the biology of this bivalent signature previously only described in trans. By directly characterizing CAP: nuc complex composition, Nuc-MS informs on the nucleoforms driving binding and thus identifies primary candidates for direct biochemical, structural, and genomic studies.
Lee et al. (Thu,) studied this question.