EID-1 binding to pRB. (A) The indicated immobilized GST–EID-1 proteins were incubated with ³⁵S-radiolabeled wild-type RB (lanes 2 to 5) or a tumor-derived pRB mutant (Δexon22) in vitro translate (lanes 7 to 10). Specifically bound proteins were resolved by SDS-polyacrylamide gel electrophoresis and detected by fluorography. Twenty percent of the input proteins was loaded directly in lanes 1 and 6. (B) U-2OS cells were transfected to produce HA-tagged pRB (lanes 1 and 2), T7 epitope-tagged EID-1 (lanes 3 and 4), or both (lanes 5 and 6). Cell extracts were prepared and immunoprecipitated with anti-T7 antibody (lanes 2, 4, and 6) or loaded directly (lanes 1, 3, and 5) prior to anti-HA immunoblot analysis. (C) SAOS-2 cells were transiently transfected with a β-Gal control reporter plasmid, a luciferase reporter plasmid containing TetR binding sites, and plasmids encoding the indicated TetR–EID-1 and VP16-RB fusion proteins in the amounts shown at the bottom of the graph (in micrograms). Cell extracts were prepared, and luciferase activity, corrected for β-Gal activity, was measured. Corrected luciferase values were expressed as fold activation relative to that of TetR–EID-1 and TetR–EID-1(1-177) alone. (D) U-2OS cells were transfected so as to produce T7–EID-1 alone (lane 1), HA-RB alone (lane 2), or HA-RB with the indicated EID-1 mutants (lanes 3 through 10). Cell extracts were prepared and immunoblotted with anti-HA (top). In parallel, an aliquot of each extract was immunoprecipitated with anti-T7 antibody prior to immunoblot analysis with anti-HA (middle) or anti-T7 (bottom). Note that the Δ53Δ92 EID-1 mutant comigrates with the antibody light chain (asterisk).

EID-1 blocks differentiation but does not override a pRB-induced G₁/S block. (A) SAOS-2 cells were transfected with a plasmid encoding wild-type RB and plasmids encoding either E2F1 or EID-1 in the amounts shown at the bottom of the graph. The percentage of transfected cells in G₁ phase was determined by FACS and was expressed as the absolute increase in the percentage of cells in G₁ phase relative to that of mock transfectants. (B) SAOS-2 cells were transfected with a neomycin resistance plasmid encoding pRB and increasing amounts (10 ng, 100 ng, 1 μg, 3 μg, and 5 μg) of plasmids encoding the indicated EID-1 proteins depicted by the triangles. After 14 days of G418 selection, the number of flat cells per 10 high-powered fields was determined and expressed relative to the number of flat cells observed with pRB alone. (C) Stable murine C2C12 subclones producing T7–EID-1, T7–EID-1(1-157Δ53Δ92), and an empty vector transfectant, were grown under conditions that do (2% horse serum) or do not (20% FBS) promote differentiation. Cell extracts were prepared and immunoblotted for the indicated proteins. (D) Anti-EID-1 and anti-α tubulin immunoblot analysis of the indicated cell lines. The anti-EID-1 antibody does not react with murine EID-1.

EID-1 inhibits p300 and CBP HAT activity. (A) The indicated EID-1 proteins were produced in E. coli as His fusion proteins and purified by nickel chromatography. Aliquots were resolved by SDS-polyacrylamide gel electrophoresis and detected by Coomassie blue staining. (B and C) In vitro HAT assays were performed with p300 immunoprecipitated from cells (B) or recombinant CBP (C) in the presence of increasing amounts of the indicated EID-1 proteins. Acetylated histones H3 and H4 were resolved by SDS-polyacrylamide gel electrophoresis and detected by autoradiography.

Downregulation of EID-1 upon cell cycle exit. (A) WI38 fibroblasts grown in the presence (lane 1) or absence (lane 2) of serum were lysed and immunoblotted with anti-RB (top) or anti-EID-1 (bottom) antibodies. Both lanes contained comparable amounts of protein as determined by the Bradford method and confirmed by anti-MDM2 immunoblot (middle). (B) Cells grown as described in the legend to panel A were stained with propidium iodide, and cell cycle distribution was determined by FACS.

EID-1 turnover blocked by a pRB mutant that cannot bind to MDM2 (A) U-2OS cells were transfected with a plasmid encoding HA-RB, HA-RB(1-792), or empty vector, as indicated, along with a plasmid encoding T7–EID-1. At the indicated time points following the addition of cycloheximide, cell extracts were prepared and immunoblotted with anti-HA or anti-T7 antibody. Densitometry data for the anti-T7 blot is shown. (B) U-2OS cells were transfected with a plasmid encoding HA-RB, HA-RB(1-792), or empty vector, as indicated, along with a plasmid encoding CD19. Transfected cells were captured on anti-CD19 magnetic beads, lysed, and immunoblotted with anti-HA, anti-p53, or anti-EID-1 antibody.

Expression of EID-1. (A) Conceptual open reading frame of EID-1 cDNA. The pRB-binding motif (LXCXE) is underlined in black. Two acidic clusters are underlined with a dotted line. (B) Northern blot of RNA from indicated tissues with radiolabeled EID-1 cDNA probe. (C) Schematic of EID-1 mutants used in this study.

EID-1 contains a potential transactivation domain and binds to p300. (A) SAOS-2 cells were transiently transfected with a β-Gal control reporter plasmid, a luciferase reporter plasmid containing TetR binding sites, and plasmids encoding the indicated TetR–EID-1 fusion proteins or TetR alone in the amounts shown along the abscissa (in micrograms). Cell extracts were prepared, and luciferase activity, corrected for β-Gal activity, was expressed as fold activation relative to that of cells producing TetR alone. (B) The indicated ³⁵S-radiolabeled EID-1 in vitro translates were incubated with immobilized GST, GST fused to the CH1 domain of p300, or GST fused to the CH3 domain of p300. Specifically bound proteins were resolved by SDS-polyacrylamide gel electrophoresis and detected by fluorography. In parallel, 20% of the input proteins were resolved by SDS-polyacrylamide gel electrophoresis and detected by fluorography (top).

EID-1 inhibits transcription factors that utilize p300 as a coactivator. (A) C2C12 myoblasts were transfected with a β-Gal control reporter plasmid, a luciferase reporter plasmid containing the MCK promoter, a plasmid encoding MyoD, and plasmids encoding the indicated EID-1 proteins in the amounts shown at the bottom of the graph. (B) SAOS-2 cells were transfected with a β-Gal control reporter plasmid, a luciferase reporter plasmid containing GREs, a plasmid encoding GRα, and the indicated EID-1 proteins in the amounts shown at the bottom of the graph. (C) U-2OS cells were transfected with a β-Gal control reporter plasmid, a luciferase reporter plasmid containing Gal4 DNA-binding sites, a plasmid encoding Gal4-p300, and plasmids encoding the indicated EID-1 proteins in the amounts shown (in micrograms) along the abscissa. (D) SAOS-2 cells were transfected with a β-Gal control reporter plasmid, a luciferase reporter plasmid containing GREs, a plasmid encoding GRα, and plasmids encoding EID-1 and the indicated pRB proteins in the amounts shown (in micrograms) at the bottom of the graph. Cell extracts were prepared and luciferase activity, corrected for β-Gal activity, was measured and expressed as fold activation relative to that of cells that did not ectopically produce EID-1.

EID-1 is degraded during differentiation. U937 leukemia cells were induced to differentiate by the addition of TPA. At the indicated time points, protein and mRNA were isolated and subjected to immunoblot (A) and Northern blot (B) analysis with the indicated antibodies and cDNA probes. EID-1 mRNA abundance was normalized to β-actin in the graph shown in panel B. (C) Undifferentiated (lanes 1 to 6) and differentiated (lanes 7 to 12) U937 cells were treated with 20 μg of cycloheximide per ml. At the indicated time points thereafter, cell extracts were immunoblotted with anti-RB and anti-EID-1 antibodies. All lanes contained comparable amounts of protein as determined by the Bradford method.

Degradation of EID-1 occurs via ubiquitin-dependent proteolysis and correlates with MDM2 binding. (A) U-2OS cells were transfected to produce Myc epitope-tagged ubiquitin (lanes 1 and 2), T7 epitope-tagged EID-1 (lanes 3 and 4), or both (lanes 5 and 6). Cell extracts were prepared and immunoprecipitated with anti-Myc antibody (lanes 2, 4, and 6) or loaded directly (lanes 1, 3, and 5) prior to anti-T7 immunoblot analysis. (B) U-2OS cells were transfected to produce Myc-ubiquitin alone (lane 1), T7–EID-1 alone (lane 2), or Myc-ubiquitin with the indicated EID-1 proteins (lanes 3 to 10). Cell extracts were prepared and immunoblotted with anti-T7 antibody. (C) U-2OS cells were transfected to produce MDM2 (lane 1), T7–EID-1 alone (lane 2), or MDM2 with the indicated EID-1 proteins (lanes 3 to 10). Cell extracts were prepared and immunoblotted with anti-MDM2 antibody (top) or with anti-T7 antibody (bottom). In parallel, an aliquot of each extract was immunoprecipitated with anti-T7 antibody prior to immunoblot analysis with anti-MDM2 (middle). (D) Undifferentiated U937 cells (lanes 1 to 6) and U937 cells treated with TPA for 2 days (lanes 7 to 12) were treated with N-acetyl-Leu-Leu-norleucinal. At the indicated time points thereafter, cell extracts were immunoblotted with anti-RB and anti-EID-1 antibodies. All lanes contained comparable amounts of protein as determined by the Bradford method.