Insights into the molecular mechanisms of function and regulation of histone crotonylation
Histone posttranslational modifications (HPTMs) are major regulators of chromatin dynamics and gene expression. Knowledge about the molecular machineries involved in the regulation and interpretation of a HPTM is crucial for understanding their functional significance. Histone crotonylation is a well-conserved PTM, structurally similar, but functionally distinct to the well-studied acetylation. Its levels are sensitive to changes in the availability of the physiologically-relevant microbiota-derived short-chain fatty acids (SCFA). This makes it a candidate that links the microbiome and cellular metabolism to genome regulation. This link is particularly relevant in the gut, where cells of the epithelium are in direct contact with a dynamic metabolic environment, including microbiota-produced metabolites. In this work, I demonstrate the availability of histone crotonylation in the intestinal epithelium, suggesting it is a physiologically relevant mark in this tissue. I explore its dynamics in colon-derived epithelial cells and unravel it is regulated in a cell-cycle dependent manner by an important family of enzymes with roles in multiple nuclear processes – the class I histone deacetylases (HDACs). I further characterise how its genome-wide localisation pattern is influenced by HDAC inhibition and compare it to acetylation. Pivotal for our understanding of histone crotonylation is the identification of 'reader' molecules, which are able to selectively recognise it and drive specific chromatin events. Thus far, an extensive screen of crotonyl-binding factors is missing and all known crotonylation readers were identified using target-based approaches. Therefore, I performed an unbiased screen for binding factors with specificity for crotonylation using peptide pull-downs combined with stable isotope labeling with amino acids in cells in culture (SILAC). This was complemented by a proteome array screen for direct crotonylation interactors. These two independent assays identified the histone chaperone DAXX, an important chromatin factor implicated in cancer processes, as a candidate crotonylated histone binder. Biochemical validations and characterisations of this interaction were performed in colon-derived epithelial cells, where knocking down DAXX altered the transcriptional response to crotonate, a SCFA known to promote histone crotonylation. In addition, DAXX knock-down affected the response of cells to crotonate by reducing the changes in chromatin-associated lysine crotonylation, suggesting a subset of the crotonate-dependent changes in histone crotonylation are reliant on DAXX. Altogether, my data support a model for the histone chaperone DAXX as a crotonylation interacting factor that contributes to its chromatin association. This and further validations of the functional link between DAXX and genome regulation through histone crotonylation would elucidate the role of crotonylation in intestinal biology in relation to gut microbiota and metabolism. Furthermore, they could highlight DAXX as an important protein mediator between microbiota-derived metabolites and gene control in the intestinal epithelium. This study is ultimately a valuable contribution to our understanding of the vast impacts the human commensal microbiome has on development, health and disease.