Evaluation of histone post-translational acylations landscape caused by short-chain fatty acids in the gut

Abstract

The short-chain fatty acids (SCFA) produced by the microbiota act in several ways in the gut epithelial cells to regulate gene expression and have been linked to the metabolic state of cells. Histone post-translational modifications (hPTM) have gained prominence with the development of mass spectrometry (MS)-based technologies and the discovery of non-classical acylations such as crotonylation, butyrylation, and propionylation. However, a comprehensive and detailed determination of how these hPTMs in the gut are linked to SCFAs is lacking. This project proposes the use of organoid culture from the cecum and colon using stable isotope labeling by amino acids in cell culture (SILAC) for identification and quantitation of complex patterns of histones post-translational acylations in gut epithelial cells treated with crotonate and butyrate using MS. Furthermore, to increase the accuracy of in vivo hPTMs identification and quantification by the super-SILAC method, the cells obtained from the organoid culture will be used as a heavy spike-in internal standard for the colon and cecum tissues, thus obtaining a background of acylation similar to the baseline found in gut epithelial cell of each organ as a control. Beyond that, the animals will be treated with antibiotics for acute depletion of the microbiota and the landscape of acylations in vivo will be characterized. Based on the MS data, it is expected that histone modifications are associated with specific functions in epithelial cells that can be validated by chromatin immunoprecipitation (ChIP). Finally, these data may provide the mechanistic basis for the control of gene expression by the microbiota via SCFA in gut epithelial cells and its role in homeostasis. (AU)