Histone post-translational modifications (PTMs) play an important role in linking epigenetics and metabolism, significantly impacting human health and disease. A total of 8 short-chain lysine acylations such as β-hydroxybutyrylation, propionylation (Kpr), 2-hydroxyisobutyrylation, butyrylation (Kbu), crotonylation, and succinylation have been detected on histones with difference in regulatory functions. These histone PTMs are affected by cellular metabolism, as they depend on the acyl-CoA availability, which is derived from short-chain fatty acids (SCFAs). SCFAs such as propionate and butyrate are generally produced by gut microbiota. These serve as substrates for histone acylation and demonstrate anticancer characteristics by inhibiting enzymes called histone deacetylases (HDACs).
Michael P. Snyder et al. evaluate the regulatory role of specific short-chain acyl histone markers such as propionyl and butyryl H3K18 and H4K12 in colorectal cancer (CRC) and normal cells both in in-vitro and in-vivo studies. Moreover, explores the propionate and butyrate supplementation impact on chromatin accessibility and transcription. Additionally, assess global histone acetylation levels in response to propionate supplementation.
SW480, CCD841, and CT26 cell lines were included in this research study, where SW480 and CT26 were human and mouse CRC cells, respectively whereas CCD841 was a normal human colon cell. Different analysis methods like cell titer-blue cell viability assay, nano-capillary liquid chromatography triple quadrupole mass spectrometry (nLC-QqQ MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS), cleavage under targets and tagmentation (CUT&Tag) assay and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) used in this cell line study.
A dose-dependent relationship was observed between propionate supplementation and Kpr on H3 and H4. Specifically, at a 10 mM supplementation level, Kpr levels increased by 1.84-fold on H3K18 (P = 0.0043) and 1.75-fold on H4K12 (P = 0.0017) compared to the control. These results confirm that propionate is effectively taken up and incorporated into histone H3 and H4 as propionyl lysine marks. Furthermore, a dose-dependent increase in acyl-CoA levels was also observed. At 0.1 mM, 1 mM, and 10 mM propionate supplementations, propionyl-CoA rose from 0.08 ng (P < 0.0061) to 0.35 ng (P < 0.0001) and 0.59 ng (P < 0.0001) per 1 × 106 cells, respectively. Similarly, at 1 mM and 10 mM butyrate supplementation, butyryl-CoA also enhanced from 0.04 ng (P = 0.0220) to 0.08 ng (P = 0.0007) per 1 × 106 cells, respectively.
While investigating acetyl-CoA levels as a function of propionate and butyrate supplement, it was noted that propionate supplementation had no effect on acetyl-CoA levels, but the butyrate showed a 2.25-fold and 3.81-fold reduction in levels of acetyl-CoA at 1 mM (P < 0.0014) and 10 mM (P < 0.0002). Unlike butyrate, propionate did not alter histone acetylation or affect CRC cell viability except at 100mM.
The study demonstrated that 17,299 sites were identified with H3K18pr while 1,868 with H3K18ac (false discovery rate [FDR] < 0.05) among 19,167 sites after propionate supplementation. Kpr and kpu marks enhanced the chromatin accessibility more than the Kac mark. A total of 83% of differentially accessible regions exhibited positive fold change in the propionate-treated group than untreated group with FDR < 0.05 among 22,238 sites, which results in stronger binding affinity in chromatin. In contrast, 53% showed a negative fold change in butyrate treated group than the untreated group in 71,318 sites. Furthermore, there was no statistically significant difference observed between the 1 mM and 10 mM sodium chloride treated group compared to the control group (SW480 cell line).
Approximately 92% of sites showed higher differential binding affinity in normal cells compared to CRC cells (FDR < 0.05) in 89,340 H3K18bu sites. Likewise, 96% of sites exhibited higher binding affinity in normal cells compared to CRC cells in 64,886 H4K12bu sites. In addition, Butyrate treatment led to a 3-to-7-fold reduction in Kbu binding affinity with genes MYC and FOSL1(CRC-relevant genes) in normal cells compared to CRC cells. After butyrate treatment, 72% and 64% overlapping were observed in the comparison of mouse butyryl ATAC-seq with in-vivo H3K18bu and in-vivo H4K12bu, respectively.
“Our findings suggest that colonic SCFAs, propionate, and butyrate, enhance epithelial gene expression and inhibit carcinogenesis through histone PTMs. SCFA-induced chromatin acylation acts as a storage reservoir for the acylation process that is metabolized or recycled when SCFA levels are reduced because of antibiotics or fiber limitation. This emphasizes the significance of SCFA mechanisms for enhancing human health,” says Michael P. Snyder, Department of Genetics, Stanford University School of Medicine, USA.
Reference: Nshanian M, Gruber JJ, Geller BS, et al. Short-chain fatty acid metabolites propionate and butyrate are unique epigenetic regulatory elements linking diet, metabolism and gene expression. Nat Metab. 2025. doi:10.1038/s42255-024-01191-9
