Post-translational modifications of histone proteins create a "histone code" that regulates transcription. One such modification is the monoubiquitination of histone H2B (forming H2B-Ub), which is a marker for active transcription in chromatin and mediates several important processes in pre-initiation complex formation before it must be removed for transcript elongation. H2B-Ub also enhances transcription efficiency by recruiting FACT, a histone chaperone that disassembles the nucleosome ahead of RNA Polymerase II and reassembles it in its wake. The SAGA deubiquitinating module (DUBm) cleaves the isopeptide bond of H2B-Ub, removing ubiquitin, and thereby enhances transcription within a subset of SAGA-regulated genes. The dynamic state of the nucleosome during transcription suggests that H2B-Ub occurs in the context of the nucleosome and the H2A/H2B heterodimer, prompting us to wonder whether the DUBm may be acting preferentially against either complex. Our lab recently determined the crystal structure of the DUBm in the apo- and ubiquitin-bound states, allowing us to form hypotheses about how this complex specifically targets H2B-Ub. First, we will determine whether DUBm has a significant preference for H2B-Ub in the context of the nucleosome or H2A/H2B heterodimer using an approach that combines equilibrium binding assays with Michaelis-Menten kinetics. This goal is enabled by new techniques for the semisynthesis of natively linked H2B-Ub from intein-derivatized proteins using native chemical ligation (NCL). This work may reveal a novel functional linkage between SAGA and H2B-Ub, but will provide important information for the study of SAGA-DUBm regardless. In our second aim, we consider which parts of the DUBm complex governs specificity for H2B-Ub. Sgf11 is a DUBm component that is necessary for efficient cleavage of H2B-Ub and contains several conserved arginines on its zinc-finger (Sgf11-ZnF). We will mutate these residues and measure the effect on binding and catalysis to better understand their importance. The Sgf11-ZnF domain contacts Ubp8, the enzymatic subunit of DUBm, proximal to the active site residues. We also consider the possibility that upon recognition of substrates by Sgf11-ZnF residues, a small change at the interface between Ubp8 and Sgf11-ZnF drives a conformational change of a conserved loop that produces the reactive complex. We will explore this possibility with mutagenesis of the involved residues and crystallize mutants that show altered activity due to mutation. Finally, we will determine the structural basis of DUBm specificity for H2B-Ub by attempting to crystallize the DUBm bound to H2B-Ub in the context of the nucleosome, the H2A/H2B heterodimer, and free H2B-Ub. If crystals are not produced, we will use small angle X-ray scattering as an alternative method to determine how DUBm is oriented as it interacts with substrates. This work will answer important questions about DUBm catalysis and specificity, and will significantly improve our understanding of how SAGA interacts with chromatin.
The SAGA complex regulates gene activity with a sub-complex (DUBm) that allows it to cleave the bond linking ubiquitin to histone H2B. We will reveal how DUBm recognizes its target and performs its function using the tools of biochemistry and biophysics. Our findings will improve our understanding of gene regulation, which can lead to disease if disrupted.