Optimized Super-SILAC Normalization Enables Enhanced Profiling of Histone Post-Translational Modification Changes Associated with Microbiota and Acute Inflammation

The gut microbiota produces metabolites, such as short-chain fatty acids (SCFAs) that modulate histone acylation and serve as precursors for non-acetyl acyl marks, representing a mechanism in host-microbiome epigenetic crosstalk. However, the reliable quantification of these low-abundance modifications in complex tissues remains challenging. Here, we established an optimized, non-acetyl histone acylation-focused super-SILAC strategy for profiling histone post-translational modifications (HPTM) in the gut epithelium. We generated a functionally optimized internal standard from heavy-labeled colon cancer cells (HCT-116 and Caco-2) treated with propionate, butyrate, and crotonate, thereby enriching the standard in non-canonical acylations. This custom super-SILAC mixture enabled robust normalization and improved the quantification of low-abundance histone acylations in biological tissues. Our in vitro characterization revealed that SCFAs induced distinct and overlapping acylation profiles, with propionate leading to the most pronounced changes, including increased H3K14pr and H4K16pr. Applying this method to mouse models, we found that antibiotic-induced microbiota depletion led to the loss of marks associated with active chromatin (H3K14pr and H4K16pr) and a concurrent increase in heterochromatin-associated marks (H3K9me2/3). In contrast, Dextran Sodium Sulfate (DSS)-induced early-stage intestinal inflammation caused H4 hyperacetylation, increased H3K14pr, and reduced H3K27ac. In conclusion, the super-SILAC method enables robust identification and quantification of low-abundance histone acylations in cells and tissues under both physiological and disease conditions.