Metabolic Regulation of Gene Expression through Differential Histone Acylation: The Regulation and Function of Histone Crotonylation

Histone lysine acetylation (Kac) plays a critical role in gene regulation by affecting the accessibility of the DNA wrapped around histones and by recruiting effector complexes. Three major classes of proteins are associated with Kac, namely “writers,” enzymes that covalently modify specific lysine residues, “readers,” protein domains that specifically bind modified residues, and “erasers,” enzymes that catalyze the removal of the modification. While histone acetylation is well characterized within this paradigm, little is known about the regulation and function of an expanding list of histone lysine acylations. Lysine propionylation, butyrylation, and crotonylation were all discovered by proteomics-based approaches as I started in the Allis Lab. With reports suggesting that lysine crotonylation (Kcr) was the most functionally distinct from Kac I embarked to purify and identify the writers, readers, and erasers and thereby characterize the regulation and function of histone Kcr. To identify writers of Kcr I purified a histone crotonyltransferase (HCT) activity from nuclear extract by fractionation, which resulted in the purification of p300, a well-studied transcriptional co-activator and histone acetyltransferase (HAT). Together with colleagues in the Roeder lab, we established that p300’s HCT activity directly stimulates transcription to a greater degree than p300’s HAT activity. This work is discussed in Chapter 2. I developed several genetic and chemical approaches to manipulate the cellular concentrations of acetyl-CoA and crotonyl-CoA and established that acyl-CoA metabolism determines the state of differential histone acylation (Kac versus Kcr) thereby coupling the metabolic state to gene regulation. This work is discussed in Chapter 3. With these methods, I next studied the impact of Kcr in the macrophage inflammatory response, a classic model of signal-dependent gene activation. Through bioinformatics analysis of RNA-seq and ChIP-seq of macrophages in various conditions, I established that increased histone Kcr leads to enhanced expression of p300-regulated genes. This work is discussed in Chapter 4. These data suggested that there was a reader for Kcr with positive regulatory activity. In collaboration with colleagues in the Li lab, we identified the YEATS domain as a novel Kcr reader. Through a series of structural, biophysical, bioinformatic, and genetic studies we showed that AF9 and the YEATS-Kcr interaction is responsible for the enhanced expression of increased Kcr. This work is discussed in Chapter 5. As discussed in Chapter 6, I have also identified and characterized several decrotonylase (eraser) activities. The regulation of histone crotonylation or the functional consequence of a histone being acetylated versus crotonylated (differential acylation) has remained unclear since the discovery of the modification was reported. In my thesis work, I have demonstrated that the differential acylation state of histones is an integration of environmental and metabolic information, which serves a functional role in the regulation of gene expression