Dani Dahlseid

Discovering the Interactome of Histone Variant H2A.Z with Endogenous BPTF

Epigenetics is the study of how genetic information is accessed to produce specific phenotypes, and focuses on the molecular mechanisms involved in these processes. My focus this semester has surrounded the epigenetic regulatory protein bromodomain PHD finger transcription factor (BPTF) and how it interacts with histone variants, particularly the H2A.Z isoform 2 (H2A.Z II) protein, through a structured domain called a bromodomain. The Pomerantz lab has successfully characterized a specific interaction between BPTF and H2A.Z using short peptides and protein domains. My work extends this and helps evaluate the H2A.Z interaction with endogenous BPTF protein from cells. In this experiment, we seek to find the most efficient photo-crosslinker between H2A.Z II and BPTF. As histone bromodomain interactions tend to be weak, the purpose of photo-crosslinking is to capture this interaction and stabilize it for analysis using gel electrophoresis. The photo-crosslinking element is being synthesized at the N-terminus of our H2A.Z II peptide. Previously, we successfully photo-crosslinked H2A.Z II and BPTF using the photo-crosslinking diazirine group. Additionally, we successfully optimized our conditions. We are now exploring the photo-crosslinker benzoylphenylalanine (BPA). Our hypothesis is that BPA can be a better crosslinker because the reaction between BPA and UV light is reversible, as opposed to diazirines which are irreversible. This reversibility means BPA has more opportunities to crosslink, because it can relax to a stable state, and then be reactivated. We have discovered that BPA successfully crosslinks H2A.Z to BPTF, and now we intend to optimize these conditions for comparative analysis with diazirine.