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Khan, L. F.

Publications and source records attributed to Khan, L. F..

2 recordsLinked to original sources
bioRxiv2025

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

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

biochemistry↗
bioRxiv2025

Nucleosome context regulates chromatin reader preference

Chromatin is more than a simple genome packaging system, and instead locally distinguished by histone post-translational modifications (PTMs) that can directly change nucleosome structure and / or be "read" by chromatin-associated proteins to mediate downstream events. An accurate understanding of histone PTM binding preference is vital to explain normal function and pathogenesis, and has revealed multiple therapeutic opportunities. Such studies most often use histone peptides, even though these cannot represent the full regulatory potential of nucleosome context. Here we apply a range of complementary and easily adoptable biochemical and genomic approaches to interrogate fully defined peptide and nucleosome targets with a diversity of mono or multivalent chromatin readers. In the resulting data, nucleosome context consistently refined reader binding, and multivalent engagement was more often regulatory than simply additive. This included abrogating the binding of the Polycomb group L3MBTL1 MBT to histone tails with lower methyl states (me1 or me2 at H3K4, H3K9, H3K27, H3K36 or H4K20); and confirmation that the CBX7 chromodomain and AT-hook-like motif (CD-ATL) tandem act as a functional unit to confer specificity for H3K27me3. Further, in vitro nucleosome preferences were confirmed by in vivo reader-CUT&RUN genomic mapping. Such data confirms that more representative chromatin substrates provide greater insight to biological mechanism and its disorder in human disease.

biochemistry↗