Abstract
BackgroundHistone post-translational modifications (PTMs) are key epigenetic regulators in chromatin-based processes. Increasing evidence suggests that vast combinations of PTMs exist within chromatin histones. These complex patterns, rather than individual PTMs, are thought to define functional chromatin states. However, the ability to interrogate combinatorial histone PTM patterns at the nucleosome level has been limited by the lack of direct molecular tools.ResultsHere we demonstrate an efficient, quantitative, antibody-free, chromatin immunoprecipitation-less (ChIP-less) method for interrogating diverse epigenetic states. At the heart of the workflow are recombinant chromatin reader domains, which target distinct chromatin states with combinatorial PTM patterns. Utilizing a newly designed combinatorial histone peptide microarray, we showed that three reader domains (ATRX-ADD, ING2-PHD and AIRE-PHD) displayed greater specificity towards combinatorial PTM patterns than corresponding commercial histone antibodies. Such specific recognitions were employed to develop a chromatin reader-based affinity enrichment platform (matrix-assisted reader chromatin capture, or MARCC). We successfully applied the reader-based platform to capture unique chromatin states, which were quantitatively profiled by mass spectrometry to reveal interconnections between nucleosomal histone PTMs. Specifically, a highly enriched signature that harbored H3K4me0, H3K9me2/3, H3K79me0 and H4K20me2/3 within the same nucleosome was identified from chromatin enriched by ATRX-ADD. This newly reported PTM combination was enriched in heterochromatin, as revealed by the associated DNA.ConclusionsOur results suggest the broad utility of recombinant reader domains as an enrichment tool specific to combinatorial PTM patterns, which are difficult to probe directly by antibody-based approaches. The reader affinity platform is compatible with several downstream analyses to investigate the physical coexistence of nucleosomal PTM states associated with specific genomic loci. Collectively, the reader-based workflow will greatly facilitate our understanding of how distinct chromatin states and reader domains function in gene regulatory mechanisms.
Highlights
Histone post-translational modifications (PTMs) are key epigenetic regulators in chromatin-based processes
Using three readers as proof of concept, we demonstrated a powerful workflow involving a combinatorial PTM histone peptide microarray and a reader-based affinity enrichment platform to capture and quantify chromatin states maintained on native nucleosomes
The chromatin affinity enrichment requires highly specific ‘baits’ that can distinguish different PTM states, making readers such as malignant brain tumor domains unsuitable for such application due to their insensitivity to residues surrounding the targeted methyl lysine group [26]
Summary
Histone post-translational modifications (PTMs) are key epigenetic regulators in chromatin-based processes. Increasing evidence suggests that vast combinations of PTMs exist within chromatin histones These complex patterns, rather than individual PTMs, are thought to define functional chromatin states. Current methodologies to define chromatin states rely almost exclusively on histone antibodies, which have been developed to recognize individual PTMs. As summarized, each of these approaches has its own limitations for analysis of the combinatorial histone PTM patterns. Lot-to-lot variability, high costs and engineering difficulties are critical concerns for the development and commercialization of histone antibodies [12,13]. All these have challenged the use of histone antibodies in accurately probing chromatin states. An antibody-free approach to directly enrich combinatorial PTM patterns that maintains native nucleosome structure and combinatorial PTM complexity for downstream quantitation and genome mapping of physical intranucleosomal PTM connections will greatly simplify attempts to characterize chromatin states
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