ZNF198 Stabilizes the LSD1–CoREST–HDAC1 Complex on Chromatin through Its MYM-Type Zinc Fingers
Figure 3
ZNF198 binds preferentially to the intact LSD1–CoREST–HDAC1 (LCH) ternary complex.
(A) Recombinant His6-ZNF198 purified from Sf9 cells was treated with TEV protease and analyzed by SDS-PAGE followed by Coomassie staining. TEV protease digestion removed the His6-tag and caused the protein to migrate faster, thus confirming the identity of the band as His6-ZNF198. (B) Recombinant His6-ZNF198 (6 µg) was added to glutathione-agarose beads that had been preincubated with 1 µg GST-CoREST, 2 µg HDAC1-FLAG, or 3 µg His-LSD1 as indicated. When indicated, TCP or TSA were present for the entire procedure. After washing, bound proteins were detected by western blotting with the indicated antibodies. (C) GST pull-downs assays were performed as in (B), except that bound proteins were stained with Coomassie blue. The bands belonging to His6-ZNF198, His6-LSD1, GST-CoREST, and HDAC1-FLAG are labeled. (D) ZNF198 competes with REST for binding to the LCH complex. HDAC1-FLAG (1 µg) and the His6-LSD1–His6-CoREST binary complex (3 µg) were preincubated with anti-FLAG M2 agarose in the indicated combinations. After washing, 35S-REST was added to each binding reaction in the presence or absence of His6-ZNF198 (10 µg). Bound REST was detected using a phosphoimager (upper panel). The intensities of REST bands in each reaction were quantified and normalized to lane 5. The values were averages of two experiments. Proteins bound to the anti-FLAG beads were also analyzed by SDS-PAGE and stained with Coomassie blue (lower panel). Note that His6-CoREST and HDAC1-FLAG migrate at the same position on the gel.