Introduction: Epigenetic changes associated with aging gain importance in the development and regulation of cardiovascular diseases (CVD) and in identifying patients at risk of these diseases. In addition to DNA methylation, post-translational modifications of histones present an additional layer of epigenetic control over gene expression. In current research, predominantly methylation, acetylation, and ubiquitination are analyzed and considered modifications of interest. Since histones are among the longest-lived proteins in cells, they are also prone to accumulating non-enzymatic modifications. The addition of glucose moieties to lysines (K) and arginines (R), which are the classical sites of enzymatic epigenetic modifications, leads to covalent glycation and, through a cascade of reactions, to formation of advanced glycation end-products (AGEs). We have identified endogenous AGEs on the core histones H2A, H2B, H3, and H4 in young and senescent primary cells and atrial appendages of heart surgery patients using HPLC-MS/MS analyses. Insights into the mechanistic roles of single AGE modifications and their potential as epigenetic regulators are currently missing.
Methods: We expressed wild-type histones and mutated forms in E. coli, in which lysine was exchanged for glutamine (Q) and arginine for tyrosine (Y) to mimic the modification by the AGEs CML or MG-H1, respectively. Following purification of the histones (> 90% purity) by FPLC, histone super-structures, including dimers (H2A and H2B), tetramers (H3 and H4), octamers, and using the synthetic 601 DNA, also nucleosome core particles were assembled in vitro. All structures contained either a single amino acid exchange H2AK96Q, H2BK44Q, H3R43Y, H4K32Q or the exchanged H2A and H2B or H3 and H4 in combination. To assess the stability of these structures, a qPCR-based thermal shift assay was performed, which reported the melting dynamics.. To identify the molecular changes in binding, an in silico mutation analysis was done.
Results: Of the 32 identified AGE-sites in cells or heart tissue, more than 85% were previously found to contain enzymatic modifications that facilitate epigenetic regulation. From these, H2AK96, H2BK44, H3R43, and H4K32 were chosen and produced recombinantly as AGE mimetics for further evaluation. The AGE modification at H2A did not affect the stability of octamers. In H2B and H4 AGE-mimics, the Tm was lowered, indicating reduced stability. In contrast, nucleosomes showed increased Tm / stability for the H2A mutation and decreased Tm /stability for the H2B and H3 mutations compared to the wild-types. The in silico analyses confirmed that in octamers, losses of π-cation interactions, and in nucleosomes of high numbers of salt bridges coincide with the respective changes in Tm.
Conclusion: The changes in Tm and interactions indicate that single, naturally occurring AGE modifications at histones can alter the stability of nucleosomes, thereby facilitating epigenetic regulations comparable to those of classical enzymatic modifications. Thus, we provide the first evidence for the epigenetic regulatory potential of AGE modifications.
