MassIVE MSV000085717

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Chemical footprinting of Polyhomeotic-proximal1289-1577

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Description

The Polycomb protein Polyhomeotic (Ph) is implicated in organizing chromatin to regulate gene expression. This activity depends on the sterile alpha motif in Ph, which can form helical polymers. This polymerization activity is believed to create macromolecular assemblies of Polycomb proteins and chromatin in cells. To understand how this may occur, and the possible role of phase separation in the process, we analyzed a truncated version of Ph (aa1289-1577, Mini-Ph) in vitro. We find that Mini-Ph forms phase separated condensates with DNA or chromatin in vitro. To understand how Ph SAM polymerization contributes to this activity, and how Mini-Ph conformation might change on binding DNA and phase separating, we used a protein footprinting method based on chemical acetylation of accessible lysine residues. Mini-Ph or a polymerization mutant, alone, or with DNA, was treated with sulfo-NHS-acetate to acetylate accessible lysines. After protein denaturation, the protein was treated with propionic acid to propionylate unacetylated lysines. Samples were analyzed by MS, and the ratio of acetylated/propionylated lysines quantified at each position using MaxQuant. [doi:10.25345/C5017H] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: phase separation, polycomb, chemical footprinting

Contact

Principal Investigators:
(in alphabetical order) 		Nicole Francis, IRCM, Canada
Submitting User: nicolejfrancis

Publications

Seif, E., Kang, J-J., Sasseville, C., Senkovich, O., Kaltashov, A., Boulier, E.L., Kapur, I., Kim, C.A., Francis, N.J.
Phase separation by the Polyhomeotic Sterile Alpha Motif of compartmentalizes Polycomb Group proteins and enhances their activity.
Nature Communications, accepted.

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