DOI: 10.25442/hku.28352069.v1
title: Supporting data for Histone crotonylation is a novel epigenetic regulation and a therapeutic vulnerability for liver cancer treatment

description: Hepatocellular carcinoma (HCC) is a highly aggressive cancer with limited treatment options, necessitating the exploration of novel therapeutic strategies. Recent studies underscore the importance of epigenetic modifications in cancer development and progression. Among these, histone lysine crotonylation (Kcr), a newly discovered histone modification, has been implicated in various cellular processes, including gene expression regulation. However, its specific role in HCC and chromatin remodeling remains largely unexplored.<br>In this study, we first demonstrated that the enzymes responsible for producing crotonyl-CoA, the source of histone Kcr, are downregulated in HCC tissues, with lower expression correlating with poor patient prognosis. We further showed that crotonate supplementation, which elevates histone Kcr levels, effectively suppresses liver cancer cell proliferation <i>in vitro</i> and reduces tumor growth <i>in vivo</i>, with minimal toxicity. Mechanistically, RNA-seq analysis revealed that crotonate induces cell cycle arrest and restores hepatic identity, thereby reducing cancer stemness.<br>To delve deeper into the molecular mechanisms of histone Kcr in HCC, we employed CUT&Tag sequencing to generate genome-wide profiles of histone Kcr and 13 well-characterized epigenetic markers. Using the ChromHMM machine learning algorithm, we annotated chromatin states based on distinct combinations of these markers. This analysis revealed that histone Kcr is predominantly enriched at active promoters and enhancers, suggesting its involvement in transcriptional regulation.<br>Building on these findings, we used the crotonate treatment model to explore how histone Kcr is regulated and its functions across different genomic elements. Through a detailed time-course genomic profile of histone Kcr during crotonate treatment, we identified FOXA1/2, pioneer transcription factors linked to liver development, as key transcription regulators associated with histone Kcr. A comprehensive analysis of the epigenetic landscape across gene loci demonstrated that histone Kcr levels at promoters were closely correlated with gene expression. Crotonate treatment induced epigenetic reprogramming, during which we observed that changes in histone Kcr levels at promoters were highly correlated with gene expression levels, promoting developmental pathways. Additionally, crotonate treatment suppressed cell cycle-related pathways, and through detailed analysis of RNA Polymerase II dynamics, we found that this suppression was mediated by regulating RNA Polymerase II pause-release.<br>Finally, using Hi-C technology, we discovered that crotonate treatment leads to the formation of numerous new chromatin loops, implicating histone Kcr in the establishment of enhancer-promoter interactions that activate target gene expression. Importantly, this chromatin remodeling did not significantly alter compartments or TADs, indicating that Kcr’s role is more localized to specific regulatory elements.<br>In conclusion, our study provides a comprehensive understanding of histone Kcr’s role in HCC, highlighting its critical functions in regulating gene expression, chromatin architecture, cell cycle progression, cancer stemness, and hepatic identity. These findings offer promising avenues for the development of novel therapeutic strategies targeting histone Kcr and emphasize the need for further investigation into its role in cancer biology.