ZNF198 Stabilizes the LSD1–CoREST–HDAC1 Complex on Chromatin through Its MYM-Type Zinc Fingers (original) (raw)
Figure 6
Sumoylation of HDAC1 weakens its interaction with CoREST, but enhances its binding to ZNF198.
(A) HDAC1-FLAG (40 ng) was incubated with SUMO2 and sumoylation enzymes with or without ATP. The reaction mixtures were then added to glutathione-agarose beads that had been preincubated with buffer or GST-CoREST (1 µg). After washing, bound (top panel) and unbound (bottom panel) proteins were blotted with anti-FLAG. The sumoylated and un-sumoylated HDAC1 bands are labeled. (B) The indicated 35S-labeled in vitro translated proteins were incubated with glutathione-agarose beads bound with 10 µg of GST, GST-SUMO1, or GST-SUMO2. The bound proteins were separated by SDS-PAGE, stained with Coomassie blue (bottom panel, a representative image), and analyzed using a phosphoimager (top panel). (C) HDAC1-FLAG (1 µg) bound to anti-FLAG M2 agarose beads was incubated with SUMO2 and sumoylation enzymes with or without ATP. After washing, either His6-ZNF198 (6 µg) or GST-CoREST (2 µg) was added to the beads. The bound proteins were blotted with the indicated antibodies.