Project Summary: The post-translational modification (PTM) of core histones is integral to the regulation of transcription. The SAGA (Spt-ADA-GCN5-acetyltransferase) complex, capable of H3 acetylation and H2B deubiquitination, is a transcriptional co-activator involved in nearly all Pol II mediated transcription. This 1.8mDa complex, composed of 19 subunits in yeast, contains a core complex and two enzymatic complexes: the histone acetyltransferase (HAT) module and deubiquitinating (DUB) module. The heterotetrameric HAT module, composed of the acetyltransferase GCN5, ada2, ada3, and sgf29, contains multiple reader domains that bind chromatin to acetylate histone tails. Increasingly, the SAGA HAT module is being recognized as a therapeutic target in a multitude of c-myc driven cancers. While recent cryo-EM structures of yeast and human SAGA have been reported, neither the DUB nor HAT modules could be resolved, presumably due to their high mobility. In addition, none of these studies explored the structural engagement of these modules to chromatin. Interestingly, the distance between the DUB and HAT modules strongly suggests that these modules could act in concert on neighboring nucleosomes, thereby acetylating one while deubiquitinating the other. Despite this, the vast majority of research on SAGA has been done on peptides and, in a more limited way, on single nucleosomes. In Aim 1 of my studies, I plan to study the acetylation activity of SAGA complex on a library of mono- and di-nucleosomes with biologically relevant PTMs to probe how these modules act in concert to exert their enzymatic activities. More broadly, this will inform our understanding of how SAGA utilizes its reader, writer, and eraser functions to regulate transcription. Additionally, no structure exists to date of the SAGA HAT module, limiting our mechanistic understanding of how SAGA acts to promote active transcription. In Aim 2 of my studies, I plan to solve the cryo-EM structure of the SAGA HAT module bound to both unmodified and H3K4me3 modified nucleosomes. From biochemical data and other chromatin modifying enzymes, I hypothesize that the PTM H3K4me3, recognized by sgf29, limits the conformational landscape of the HAT module on a nucleosome for the processive acetylation of H3 histone tails. Altogether, our studies will provide insight into SAGA complex regulation in the context of histone crosstalk and establish a new paradigm for studying chromatin modifying enzymes. Furthermore, our research will provide the field with a mechanistic understanding of how the SAGA HAT promotes active transcription and, feasibly, will provide new therapeutic targets in c-myc driven cancers.