Reprogramming of H3K9bhb at Regulatory Elements is an Epigenetic Feature of Fasting in the Small Intestine

β-hydroxybutyrate (β-OHB) provides an essential metabolic energy source during fasting and functions as an epigenetic regulator by lysine β-hydroxybutyrylation (Kbhb) modification of core histones H3 and H4. We report that H3K9bhb, but not H4K12bhb, becomes highly enriched at proximal promoters of genes regulating lipolytic and ketogenic metabolic programs in small intestinal (SI) crypts during fasting and these pathways are transcriptionally activated in Lgr5 +  SI stem cells. Similar Kbhb enrichment is observed in Lgr5 +  stem cell-enriched epithelial spheroids treated with β-OHB in vitro. Combinatorial chromatin state analysis reveals that H3K9bhb enrichment is associated with active chromatin states and that fasting enriches for an H3K9bhb–H3K27ac signature at active metabolic gene promoters and distal DNA enhancer elements. Intestinal knockout of Hmgcs2 results in a marked loss of H3K9bhb associated fasting-induced gene programs suggesting that local production of β-OHB is responsible for epigenetic reprogramming. We conclude that specific modulation of H3K9bhb in SI crypts is a key epigenetic event in response to fasting.