The physiological state of the human genome is chromatin, a nucleo-protein complex consisting of approximately 50% protein. Histone proteins package the genome into nucleosomes, help regulate gene expression and coordinate genome maintenance. These functions are achieved by the use of multiple sequence variants of histone proteins carrying complex single molecule combinations of post-translational modifications (PTMs) or more succinctly ‘histone proteoforms’. It is increasingly clear that histones store and transduce signals through the interplay of the multiple attributes (PTMs, sequence variation) rather than these attributes operating independently. This proteoform-based system is most effectively addressed by quantitative top down proteomics. We have developed high throughput quantitative top down (undigested and intact) proteomics methods to directly and rigorously quantitate hundreds of histone proteoforms as low as 10 ppm abundance starting from limited cells or tissues.<br/>We demonstrate here several unique dimensions of histone proteoform function in multiple contexts, including transcription regulation, genome maintenance, neurodegeneration, and aging. Histone proteoforms exhibit hierarchical patterns of PTMs that while universally constrained by common rules, vary greatly in different biological contexts. Histone PTMs are essential to persistent or epigenetic regulation of the genome and cell identity. However, surprisingly large dynamic changes are often observed very quickly in response to pharmacological perturbation. The persistence of epigenetic mechanisms is not due to slow chemical reaction rates but rather through a network of proteoform specific enzymatically mediated reactions. The crucial histone H3K4me3 signal that initiates transcription requires hyperacetylation in cis to disengage the positively charge histone tail from the DNA and allow access to the methyltransferase. Spatially, proteoforms also vary, such as through different sections of the mouse brain as well as by cell type. The abundance of histone proteoforms change both during development and in aging. Our intact proteoform analysis reveals previously unknown roles for proteolysis in modulating these histone-mediated signals. We observe truncated versions of specific histone variants that lack crucial sites of modification and appear to suppress DNA damage checkpoints and proliferation regulators.

Quantitation of histone proteoforms reveal single molecule mechanisms of chromatin regulation

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ACS Fall 2022

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