Biomolecular tracking by FIRESCAPE reveals distinct modes of clearance, damage induction and cellular uptake for extracellular histone H3
The study of chromatin has mostly been restricted to defining its function in the nucleus, where histone proteins fulfil vital roles in packaging genomic DNA and regulating transcription. However, chromatin components are often released into the extracellular space, either intentionally via cellular secretion or during disease or damage-induced cell death. These extracellular chromatin components, depending on the context, can consist of free histones, free DNA, intact nucleosomes (histone octamers wrapped by DNA), or heterogeneous, higher order structures such as neutrophil extracellular traps (NETs). They have been associated with diverse pathologies such as inflammation, cancer, and sepsis, and distinct toxic effects, most notably the damaging effects of free histones over intact nucleosomes. Extracellular chromatin components are emerging, suggested biomarkers for various associated diseases, yet there is a widely acknowledged lack of methods that distinguish between them and their unique functions in circulation. Here, we explore the fate and effects of extracellular free histone H3 by utilizing FIRESCAPE ([18F]-Fluorine Isotopic Radiolabeling Enabling Scanning of Clearance After Proteolytic Events), a novel radiolabeling concept that leverages the unique, high-sensitivity properties of the radioisotope 18F and residue-specific protein editing chemistry. By installing true 18F-containing protein sidechain mimics site-specifically into histone H3, FIRESCAPE enabled in vivo scanning of the half-lives, proteolytic susceptibility, and clearance of single residues in a protein of interest, at microdoses far below toxic levels (low nanomole). This now precisely reveals the remarkably distinct distribution, half-lives, damage-inducing abilities, and accumulation of free extracellular histones in cellulo and in vivo, comparing them to other chromatin states such as intact nucleosomes where appropriate. Free extracellular histones are rapidly cleared from circulation, first mediated by proteolysis of the histone tail. However, direct injection of free histones versus nucleosomes into tissue (brain) that is unprotected by such proteolysis provokes a starkly different response: free histones exhibited limited diffusion and swiftly promoted damage both in cell culture and in vivo, in contrast to the largely passive and benign nature of intact nucleosomes. Synthetic extracellular histone H3 uptake was observed and characterized with biochemical, genomic, and proteomic methodologies, revealing apparent deposition into chromatin indistinguishable from native H3 in localization and post-translational modification (PTM) accumulation, yet paired with cellular and tissue damage. These exploratory studies now provide much-needed clarity to the distinct fates and effects of extracellular histones versus nucleosomes, in particular the strongly damaging effects of free histones, their rapid uptake into cells, and an associated histone-specific proteolysis pathway via the removal of the histone tail. Overall design: This study tested whether extracellular histones could penetrate cells and incorporate into chromatin in an in vivo context. To do this, histone H3 (containing a C-terminal dual FLAG-HA epitope tag) was intracranially injected into rats, followed by brain excision, crosslinking, and ChIP-seq against the HA mark, endogenous histone H3 mark, or input control. This study found widespread chromatin incorporation of the injected HA tagged histone H3, with similar occupancy throughout the genome to endogenous histone H3, only in the conditions in which the histone was injected, and not a PBS mock injection.