Towards an Understanding of NatD Activity in the Context of Oncohistone Mutations

Abstract

NatD is an N-terminal acetyltransferase that has two known substrates: histones H2A and H4. In cancer cell models, inhibition or overexpression of NatD influences transcription, alters post-translational modification (PTM) deposition, and modulates histone crosstalk. Although previous research has characterized the role of NatD expression in cancer, how NatD activity is altered due to mutations in its histone substrates is not known. Oncohistone mutations are a class of cancer-associated point mutations in histone proteins. Canonical oncohistones such as H3.3K27M and H3.3K36M have been shown to alter PTM deposition and maintenance leading to tumorigenesis and cancer progression. Interestingly, the most prevalent oncohistone mutations on histones H2A and H4 reside in the N-terminal SGRG consensus residues required for NatD binding. The Muir lab has recently developed a 150-member DNA-barcoded oncohistone mononucleosome library, thus allowing for screening and interrogation of these mutations in a high throughput manner. The use of this library, however, is contingent upon appending an affinity handle to mononucleosomes in a NatD activity-dependent manner. A biotin-containing probe has been developed by the Muir lab to append biotin to substrates that can accept a crotonyl group from crotonyl-CoA, thus allowing for enrichment via streptavidin pulldown. Given the similarities between acetyl-CoA and crotonyl-CoA, I hypothesized NatD can utilize crotonyl-CoA as a cofactor, thus allowing for the use of the probe to evaluate NatD activity. In this thesis, I first demonstrate that NatD can utilize crotonyl-CoA as a cofactor on peptides and mononucleosomes. Then, utilizing the probe, I employ the library to unbiasedly investigate which oncohistone mutations impact NatD acetylation. Given the role both NatD and oncohistones play in advancing cancer, understanding their interplay is of great interest.