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

## TL;DR

This paper introduces a new method to study how proteins interact with nucleosomes, revealing how different modifications on histones influence these interactions.

## Contribution

The novel contribution is the application of Nucleosome Mass Spectrometry (Nuc-MS) to directly analyze multivalent chromatin reader-nucleosome interactions.

## Key findings

- BPTF PHD-bromodomain synergistically binds to multiple PTMs on defined nucleosomes.
- BRD4 prefers di- and triacetylated H4 proteoforms, while DNMT3A-MPP8 and PtSHL recover hypermethylated H3 proteoforms.
- PtSHL identifies a new {H3K4me3K27me3} cis combinatorial signature in nucleosomes.

## 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
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 ([H3K4me3K9acK14acK18ac]2) 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.