Semisynthesis of Isomerized Histone H4 Reveals Robustness and Vulnerability of Chromatin toward Molecular Aging

Proteins are subject to aging in the form of spontaneous, nonenzymatic post-translational modifications (PTMs). One such PTM is the formation of the β-linked isomer l-isoaspartic acid (isoAsp) from aspartic acid (Asp) or asparagine residues, which tends to occur in long-lived proteins. Histones can exhibit half-lives on the order of 100 days, and unsurprisingly, isoAsp formation has been observed in nearly every histone family. Delineating the molecular consequences of isoAsp formation in histones is challenging due to the multitude of processes that occur on such time scales. To isolate the effects of a specific isoAsp modification thus necessitates precise <i>in vitro</i> characterization with well-defined substrates. Here, we adapt a protein semisynthesis approach to generate full-length variants of histone H4 in which the canonical Asp at position 24 is replaced by its isoAsp isomer (H4isoD24). This variant was incorporated into chromatin templates, and the resulting constructs were used to interrogate key parameters of chromatin integrity and maintenance <i>in vitro</i>: compaction, nucleosome remodeling, and methylation of H4 lysine 20 (H4K20). Remarkably, despite its disruptive changes to the backbone's spacing and direction, isoD24 did not dramatically disrupt Mg²⁺-mediated chromatin self-association or nucleosome repositioning by the remodeler Chd1. In contrast, H4isoD24 significantly inhibited both Set8- and Suv4-20h1-catalyzed methylation at H4K20. These results suggest that H4isoD24 gives rise to a complex reorganization of the chromatin functional landscape, in which macroscopic processes show robustness and local mechanisms exhibit vulnerability to the presence of this mark.