Mapping H2A.Z Nucleosome Exchange

Nuclei were isolated from mouse BM cells using the Nuclei isolating Kit (NUC101, Sigma-Aldrich) according to the manufacturer’s protocol, followed by micrococool MNase digestion. Gel filtration chromatography was performed by Superdex 200 10/300 GL (GE Healthcare) according to the manufacturer’s instruction to isolate stripped nucleosome fractions. SRCAP was immunoprecipitated by anti-SRCAP antibody and protein A/G beads from nuclear extracts of Pcid2^+/+ or Pcid2^−/− MPPs. His-tagged H2A.Z and H2B protein were purified from E. coli and assembled to H2A.Z/H2B dimer in vitro. Purified His-tagged PCID2 or GST-tagged ZNHIT1 protein (4 μg for each protein) was directly added into the exchange reaction buffer (70 mM NaCl, 10 mM Tris-HCl (pH8.0), 5 mM MgCl₂, 0.1 mg/ml BSA, 2 mM ATP and 1 mM DTT). Isolated mononucleosomes, immunoprecipitated SRCAP complex, purified proteins of PCID2 or ZNHIT1 and recombinant His-H2A.Z-H2B dimers were incubated at 37 °C for 30 min and resolved by native gel. His-tagged H2A.Z exchanging into nucleosomes was probed by immunoblotting^{59 (link)}.