Products Cited in Publications Worldwide BPTF Target Engagement by Acetylated H2A. Z Photoaffinity Probes

BPTF Target Engagement by Acetylated H2A. Z Photoaffinity Probes

Peterson, Kerstin; Olson, Noelle; Dahlseid, Dani; Artymiuk, Jacklyn; Erber, Luke; de Luna Vitorino, Francisca; Landry, Joseph; Tretyakova, Natalia; Garcia, Benjamin

DOI:

Abstract

Histone variant H2A.Z has been increasingly associated with cancer progression, including cancers characterized by dysregulated function of the epigenetic reader protein BPTF (bromodomain and PHD finger containing transcription factor). Despite this association, a direct interaction between acetylated H2A.Z and BPTF has not been validated in a physiological context given the weak bromodomain-histone tail affinity. Here, we developed photoaffinity acetylated histone probes based on differentially acetylated H2A.Z isoforms I and II with an N-terminal diazirine and a C-terminal biotin tag for covalently capturing and enriching BPTF and new interactors. Photocrosslinking experiments followed by SDS-PAGE revealed that recombinant BPTF bromodomain (BPTF BD) directly interacts with both isoforms of H2A.Z in an affinity-dependent and acetyl-lysine binding pocket-specific manner. In nuclear lysates prepared from A549 non-small cell lung cancer cells, acetylated H2A.Z isoform probes enriched endogenous BPTF as a photocrosslinking target. Finally, to determine the natural acetylation patterns of H2A.Z in a human cancer cell line, we used bottom-up proteomics to quantify the levels and patterns of acetylation on H2A.Z from A549 cells. Mono- and diacetylation were the predominant acetylation patterns identified with patterns containing acetylation sites K4ac, K7ac, K11ac, and K15ac. These results indicate that our photoaffinity probes can capture transient epigenetic protein-protein interactions while optimization of conditions for interactome analysis of these transient interactions will be necessary for identifying additional epigenetic regulators of H2A.Z.

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