Figure 1.
ZNF198-like proteins are not required for the repression of REST-responsive genes.
(A) LSD1, CoREST, and HDAC1/2 are major binding partners of ZNF198 in 293 cells. IP of ZNF198 from HEK293 whole cell lysates was separated on SDS-PAGE and stained with Colloidal Blue. Bands were excised and identified by mass spectrometry. (B) HeLa cells were transfected with siRNAs against luciferase (siLuc) or ZNF198, ZNF261, and ZNF262 (siMYM). Cell lysates were blotted with the indicated antibodies. (C & D) U2OS cells were transfected with the indicated siRNAs for three days, followed by quantitative RT-PCR using the indicated primer sets. Cycling-time values were normalized to the housekeeping gene cyclophilin B. Each PCR reaction was performed in triplicate, and error bars indicate the standard deviation of three separate experiments.
Figure 2.
ZNF198-like proteins are required for the repression of E-cadherin.
(A) ChIP analysis reveals that REST binds to the promoter of keratin 17 (KRT17). (B) LSD1 is required for the repression of KRT17. U2OS cells were transfected with the indicated siRNAs for three days, followed by quantitative RT-PCR using KRT17 primers. (C) U2OS cells were transfected with the indicated siRNAs for three days, followed by quantitative RT-PCR using the E-cadherin primer set. Cycling-time values were normalized to the housekeeping gene cyclophilin B. Each PCR reaction was performed in triplicate, and error bars indicate the standard deviation of three separate experiments.
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.
Figure 4.
ZNF198 binds to chromatin through its P/V-rich domain.
(A) HeLa Tet-on cells were transfected with the indicated Myc-tagged ZNF198 constructs along with GFP-MCM7. Cells were either fixed directly (–Extraction) or extracted prior to fixation (+Extraction) and stained with anti-Myc antibody (red) and DAPI (blue). GFP is shown in green. (B) Summary of the staining data described in (A). The ZNF198 fragments are shown on the left while the percentages of GFP-positive cells that were also Myc-positive after extraction are shown on the right. More than 30 GFP-positive cells from 10 random fields were counted for each fragment. The boundaries of ZNF198 fragments are indicated by triangles.
Figure 5.
ZNF198-like proteins regulate the chromatin association of LSD1.
(A) HeLa Tet-on cells were transfected with the indicated siRNAs: Luc (firefly luciferase), MYM (ZNF198, ZNF261, and ZNF262), or LSD1. Lysates from these RNAi cells were immunoprecipitated with either anti-ZNF198 (top panel) or anti-LSD1 (middle panel). The IPs and the lysates (bottom panel) were blotted with the indicated antibodies. (B) After RNAi of the indicated proteins, nuclear pellets were generated by subjecting HeLa Tet-on cells to hypotonic lysis followed by centrifugation. Normalized samples from each step were subjected to SDS-PAGE and blotted with the indicated antibodies. (C) Nuclear pellets in lanes 7–9 in (B) were subjected to extraction with a high-salt buffer. Normalized samples from each step were blotted with the indicated antibodies. (D) HeLa cells transfected with siLuc were subjected to fractionation as in B. The nuclear pellet in lane 7 in (B) was digested with micrococcal nuclease (MNase) followed by extraction with 2 mM EDTA. Supernatants (S) and pellets (P) were blotted with the indicated antibodies.
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.
Figure 7.
MYM-type zinc fingers of ZNF198 mediate its binding to the LCH complex and HDAC1-SUMO2.
(A) 35S-labeled ZNF198 fragments and mutants were incubated with anti-FLAG beads that had been preincubated with HDAC1-SUMO2, HDAC1, or the LSD1–CoREST–HDAC1 complex. After washing, the bound proteins were visualized by autoradiography and Coomassie blue staining (bottom panel). Three putative SUMO-interacting motifs (SIMs) are indicated. The SIM3 mutant contains V483A, L484A, and V485A mutations. (B) HeLa Tet-on cells were transfected with the indicated constructs for 24 hours. Lysates and the Myc IPs of the transfected cells were blotted with the indicated antibodies. (C) Ribbon drawings of three Krüppel-like zinc fingers bound to DNA (PDB ID: 1ZAA; left) and a MYM-type zinc finger from ZNF237 (PDB ID: 2DAS; right). Zinc ions are shown as spheres while zinc-binding ligands are shown in sticks.
Figure 8.
Proposed mechanisms by which ZNF198 regulates the LSD1–CoREST–HDAC1 complex.
See DISCUSSION for details.