Decoding the protein composition of whole nucleosomes with Nuc-MS

Abstract

Nuc-MS characterizes histone modifications and variants directly from intact endogenous nucleosomes. Preserving whole nucleosome particles enables precise interrogation of their protein content, as for H3.3-containing nucleosomes which had 6fold co-enrichment of variant H2A.Z over bulk chromatin. Nuc-MS, validated by ChIPseq, showed co-occurrence of oncogenic H3.3K27M with euchromatic marks (e.g., H4K16ac and >15-fold enrichment of H3K79me2). By capturing the entire epigenetic landscape, Nuc-MS provides a new, quantitative readout of nucleosome-level biology. MainThe post-translational modifications (PTMs) decorating the four core histones in an intact nucleosome encode information for nuclear effectors that trigger defined cellular events critical to health and disease. [1][2][3][4] For decades, affinity reagents, mass spectrometry and proteomics have played key roles in characterizing the many histone isoforms and PTMs involved in epigenetic regulation. [5][6][7] However, the standard practice of relying on digestion 8 and/or denaturation 5 removes the linkage between modifications and their nucleosomes of origin (Fig. 1a, at left). By digesting histone mixtures into small peptides, correlations among PTMs are forfeited, precluding strong assertions about the original composition of the intact histone. Similarly, when a nucleosome population is denatured, information about co-localization of histone isoforms and PTMs within the same nucleosome particle is lost. This inference problem diminishes our understanding of the organization and impact of co-occurring modifications, isoforms and mutations in epigenetics and disease pathogenesis. 9,10

Single-cell epigenetic analysis reveals principles of chromatin states in H3.3-K27M gliomas

“…We identified upregulation of H4K16ac as one of the most prominent effects of H3-K27M expression, similar to the well-established upregulation of H3K27ac 29 . Schachner et al recently applied mass spectrometry to intact nucleosomes and showed high levels of H4K16ac on H3-K27M-mutant nucleosomes 55 , supporting a cis effect of the mutation on this specific H4 acetylation. The global increase observed in our data strongly suggests a trans effect as well, similar to H3K27ac.…”

Decoding Post-Translational Modification Crosstalk With Proteomics

“…This allows the analysis of protein complexes and their PTMs, thereby enabling the study of inter-protein PTM crosstalk ( 65 ). Indeed, a recent study applied native top-down mass spectrometry to quantify the relative abundance of histone isoforms and their respective PTM-proteoforms from endogenous nucleosome preparations in a single mass spectrum ( 66 ). While limited by low throughput, low proteome coverage, and protein-specific sample preparation considerations, top-down methods are highly complementary to bottom-up approaches and allow for complex patterns of intra- and inter-protein PTM co-occurrence to be measured for proteins and complexes of interest.…”

The H2A.Z-nucleosome code in mammals: emerging functions

“…Cracking the H2A.Z nucleosome code and deciphering H2A.Z nucleosome dynamics during specific molecular processes is key to further understanding H2A.Z differential functions. With implementation of single-molecule imaging in living cells [113], as well as Nuc-MS to study nucleosomes in their native form [36], MNase-X-ChIP [22] to interrogate unwrapping nucleosomes, and ChIP-re-ChIP [114] to investigate histone co-occurrence in the chromatin, we envision that studies in the next 5 years will detail the dynamics and composition of H2A.Z nucleosomes during transcription and molecular processes, such as replication and DNA repair.…”

“…A recently developed method, nucleosome mass spectrometry (Nuc-MS), that captures the protein composition of synthetic or endogenous nucleosomes in their native form, confirmed that H3.3 nucleosomes have a sixfold enrichment of H2A.Z over bulk chromatin [36]. According to Nuc-MS, around 13% of H3.3 nucleosomes also contain H2A.Z, in line with the 20% overlap between H2A.Z and H3.3 ChIP-seq peaks found at promoter and intronic regions.…”