Sp100 interacts with ETS-1 and stimulates its transcriptional activity - PubMed (original) (raw)

FIG. 1.

FIG. 1.

Protein structures. (A) Human ETS-1 and the baits. ETS-1 can be subdivided into domains A to F; the last amino acid in each domain is indicated (21, 54, 70, 73). E is the highly conserved ETS DBD, D and F inhibit DNA binding, C is the transcriptional activation domain (AD), B is the Pointed domain which is conserved in a number of Ets proteins, and A has no known function. The baits are fusion proteins between LexA and ETS-1 deletion mutants. (B) Human Sp100, preys, and constructs. Sp100 is the first variant described (67). Sp100.L contains additional amino acids at the C terminus that were found in one of the preys and are common to the three Sp100 variants: Sp100B, Sp100-HMG, and Sp100C (14, 19, 57, 58, 77). HSR is involved in ND targeting and homodimerization (46, 65) and is the only region conserved in murine Sp100-rs that is encoded by an amplified gene that forms a homogeneously staining region (16, 49). The HP-1 region interacts with HP-1 (33, 58). AD is a transcription activation domain (77). NLS (positions 444 to 450) is a nuclear localization signal (65). The preys isolated by the yeast two-hybrid screen, EIF11 and EIF25, are fusion proteins between the Gal4 AD and the C-terminal parts of Sp100.L and Sp100, respectively. The constructs, Sp100.N and Sp100.C, are deletion mutants retaining sequences from 1 to 332 and from 333 to 480, respectively.

FIG. 2.

FIG. 2.

Interaction of ETS-1 and Sp100 in yeast. (A) Histidine prototrophy test. Yeast transformants were streaked on histidine selection plates containing 25 mM 3-aminotriazole (an inhibitor of the histidine pathway) and scored after a 4-day incubation at 30°C. Growth is indicated as “±” to “+++++” based on the size of the colonies. The control baits were LexA, LexA-Lamin, LexA-Myc-Cterm, LexA-Max, LexA-Bicoid, LexA-Cdc2 (79), and LexA-cyclin C (69). Control inserts were transferred to pBTM116, in order to express the control and test ETS-1 baits from the same vector. The control preys were Gal4 AD and Gal4 AD-HLA-DRγ. (B). Liquid β-Gal assays. β-Gal activity was measured with the substrate ONPG (_o_-nitrophenyl-β-

d

-galactopyranoside) and is expressed in arbitrary units. Yeast extracts were adjusted for protein determined by the Bradford assay.

FIG. 3.

FIG. 3.

Interaction of ETS-1 and Sp100 in mammalian cells. (A) Dual-hybrid assay with Sp100. HeLa cells were transfected with the expression vectors pGal4 (0, 0.5, and 2 μg), pGal4-ΔC (0, 0.5, and 2 μg), pVP16-Sp100 (0, 0.1, 0.5, and 3 μg), the reporter UAS-TK-Luc, and the internal control pSG5-LacZ (0.5 μg). Values from three independent experiments with at least two DNA preparations are plotted as the fold stimulation relative to the samples without VP16-Sp100. (B) Dual-hybrid assay with Sp100.C. The transfections were as in panel A except that pGal4 (0, 0.1, 0.3, and 3 μg), pGal4-ΔC (0, 0.1, 0.3, and 3 μg), and pVP16-Sp100.C (0, 0.1, 0.5, and 3 μg) were used. (C) In vivo GST pull-down assay. COS-7 cells were transfected with the expression vectors pSG5-Flag-ΔC (5 μg, lanes 1 to 4), pBC-Sp100 (10 μg, lanes 2 and 4), and pBC (5 μg, lanes 1 and 3). Cell extracts were incubated with glutathione-Sepharose beads, and immobilized proteins were analyzed by SDS-PAGE and Western blotting with antibodies against the Flag tag of ΔC (2Fl1B11 mouse monoclonal, upper panels) and GST (1D10, lower panels). The input (lanes 1 and 2) was 10% of the amount used for the solid-phase immunoprecipitation (sIP, lanes 3 and 4). (D) Coimmunoprecipitation. COS-7 cells were transfected with the expression vectors pSG5-ETS-1 (5 μg) and pSG5-Flag-Sp100 (5 μg). Cell extracts were incubated with immobilized Flag antibodies (anti-Flag M2 affinity gel, sIP, lanes 5 and 6) or Flag antibodies (2Fl1B11), followed by protein G (IP, lanes 7 and 8). The input (lanes 1 to 4) was 10% of the amount used for the immunoprecipitations. Western blots were probed with rabbit antibodies against Ets-1 (TEBU, N276, and sc-111), followed by protein A-peroxidase and with mouse anti-Flag monoclonal antibody (2Fl1B11), followed by goat anti-mouse κ-light chain peroxidase (Southern Biotechnology).

FIG. 4.

FIG. 4.

ETS-1 levels alter the nuclear distributions of Sp100 (A and D) and PML (B). (A and B) HeLa cells were transfected with the following expression vectors: pSG5-Flag-ETS-1 (1 μg, columns 2 and 5), pSG5-Flag-ΔC (0.5 μg, columns 3 and 6), pGal4-AB (0.5 μg, column 7), and pSG5-Sp100 (0.5 μg, columns 4 to 7) (A) and pSG5-Flag-Sp100 (0.5 μg, columns 1 and 2) and pSG5-ETS-1 (0.5 μg, column 2) (B). The cells were fixed with acetone-methanol and treated with rabbit anti-Sp100 (α SP26, columns 1 to 7) and mouse anti-Flag (2Fl1B11, α-ETS-1, columns 1 to 6) or mouse anti-Gal4 (2GV3 plus 3GV2, column 7), followed by donkey anti-rabbit antibody coupled to Texas Red (α−Sp100) and donkey anti-mouse coupled to FITC (α-ETS-1) (A) or with mouse anti-Flag (2Fl1B11, columns 1 to 2) and rabbit anti-PML (494, columns 1 to 2), followed by donkey anti-mouse antibody coupled to Texas Red (α-Sp100) and donkey anti-rabbit antibody coupled to FITC (α−PML) (B). Nuclei were visualized with Hoechst stain. The cells were examined by confocal microscopy. (C) Expression levels in HeLa cells. The cells were transfected with the expression vectors pSG5-Flag-ETS-1 (1 μg, lanes 2, 5, and 6) and pSG5-Flag-Sp100 (0.5 μg, lanes 3 and 5; 1 μg, lanes 4 and 6). Cell extracts were analyzed by SDS-PAGE and Western blotting with mouse anti-Flag monoclonal antibodies (2Fl1B11), followed by goat anti-mouse peroxidase. (D) HepG2 cells in six-well plates were transfected with antisense or sense Ets-1 phosphorothionate oligonucleotides (10 μM), an expression vector for GFP (0.5 μg of pXJ41-GFP-C1) and carrier DNA (3 μg of pBluescript). After 24 h they were fixed, permeabilized, stained with rabbit anti-Sp100 (α SP26) and mouse anti-ETS-1 (MAb94) antibodies, followed by donkey anti-rabbit antibody coupled to Texas Red and donkey anti-mouse antibody coupled to FITC. Nuclei were visualized with Hoechst stain. The cells were examined by confocal microscopy. Endogenous ETS-1, detected with FITC (green), is shown for cells that had not been subjected to transfection (normal). The GFP fluorescence of transfected cells is shown for the antisense- and sense-transfected cells. (E) Expression levels in HepG2. The cells in six-well plates were transfected with antisense or sense Ets-1 phosphorothionate oligonucleotides (10 μM) and 3 μg of pHook (Invitrogen). Tranfected cells were purified with the Capture-Tec kit (Invitrogen). Extracts were analyzed by SDS-8% PAGE and Western blotting with rat anti-Sp100 antibody (gift from H. Will), rabbit anti-ETS-1 antibody (TEBU Ets1 N276), and mouse anti-Myc tag (monoclonal antibody 9E10.2, to detect the Hook protein as an efficiency control). “Normal” refers to cells that had not been subjected to transfection.

FIG. 5.

FIG. 5.

Stimulation of ETS-1 and ETS-2 transcriptional activity by Sp100. HeLa cells were transfected with expression vectors, reporters and the internal control pSG5-LacZ (0.5 μg). Corrected luciferase (A to D and F) or CAT (E) activities were used to calculate the fold activation relative to transfections with empty expression vectors. The values are from three independent experiments with at least two DNA preparations. (A, B, and F) Reporters with the natural ets-reponsive collagenase promoter (A, 1 μg) and the ets-specific promoter (B and F; 1 μg). The tranfections contained pSG5-ETS-1 (A and B) or pSG5-ETS-2 (F) (0 μg, bars 1 and 5 to 7; 0.1 μg, bars 2 and 8 to 10; 0.5 μg, bars 3 and 11 to 13; 2 μg, bar 4) and pSG5-Sp100 (0 μg, bars 1 to 4; 0.1 μg, bars 5, 8, and 11; 1 μg, bars 6, 9, and 12; 2 μg, bars 7, 10, and 13). (C, D, and E) Sp100 does not stimulate the activity of weak (C, Gal4), strong (D, Gal4-VP16), and related (E, Fli-1) transcription activators. The transfections contained pSG5-Gal4 (C, 0.5 μg, bars 2 to 5), pSG5-Gal4-VP16 (D, 0.1 μg, bars 2 and 4 to 6; 0.5 μg, bar 3), pSG5-Sp100 (0.1 μg, bar 3 [C] and bar 4 [D]; 0.5 μg, bar 4 [C] and bar 5 [D]; 2 μg, bar 5 [C] and bar 6 [D]; 4 μg [E]), UAS-TK-Luc (1 μg [C and D]), PAL-CAT (4 μg [E]) and pBL-CAT4 (4 μg [E]), pSG5-ETS-1 (4 μg [E]) and pSG5-Fli-1 (4 μg [E]).

FIG. 6.

FIG. 6.

The ETS-1 activation domain C is required for efficient stimulation of ETS-1 by Sp100. (A) Scheme of the ETS-1 mutants. (B) HeLa cells were transfected with the PALx8-Luc reporter (1 μg), the internal control pSG5-LacZ (0.5 μg), pSG5-ETS-1 (0.1 μg, bar 2; 0.5 μg, bars 3 and 8 to 11; 2 μg, bar 4), pSG5-Flag-ΔC (0.5 μg, bars 12 to 15), pSG5-Flag-CDEF (0.5 μg, bars 16 to 19), pSG5-Flag-DEF (0.5 μg, bars 20 to 23), and pSG5-Sp100 (0.1 μg, bars 5, 9, 13, 17, and 21; 0.5 μg, bars 6, 10, 14, 18, and 22; 1 μg, bars 7, 11, 15, 19, and 23). Corrected luciferase activities were used to calculate the fold activation relative to transfections lacking Sp100 (open bars). The values are from at least three independent experiments with at least two DNA preparations.

FIG. 7.

FIG. 7.

The ETS-1 activation domain C is required for efficient activation of Gal4-ETS-1 fusion proteins by Sp100 (A to C) and transcription regulation by Sp100 mutants through ETS-1 domains ABCD (D) and directly (E). (A) Scheme of the Gal4-ETS-1 fusion proteins. (B) HeLa cells were transfected with the UAS-TK-Luc reporter (1 μg), expression vectors for the indicated Gal4 fusion proteins (0.5 μg), pSG5-Sp100 (0.1 μg, bars 2, 5, 8, 11, and 14; 0.5 μg, bars 3, 6, 9, 12, and 15), pSG5-VP16-Sp100 (0.1 μg, bars 17 and 21; 0.5 μg, bars 18 and 22; 1 μg, bars 19 and 23), and the internal control pSG5-LacZ (all bars). Corrected luciferase activities were used to calculate the fold activation relative to transfections lacking Sp100. The values are from at least three independent experiments with at least two DNA preparations. (C) Expression of Gal4-ETS-1 proteins. A total of 2 μg of pSG5-Gal4, pSG5-Gal4-ABCD, pSG5-Gal4-ABC, pSG5-Gal4-AB, and pSG5-Gal4-DEF was cotransfected without (−) or with (+) 4 μg of the Sp100 expression vector in COS-7 cells, and whole-cell extracts were prepared. Comparable amounts of extracts, based on protein concentration, were analyzed by SDS-PAGE and Western blotting with a mixture of monoclonal antibodies against Gal4 (2GV3 and 3GV2) at a 1:1,000 dilution. The sizes of the marker bands are indicated on the left (molecular weight [10−3]). (D and E) HeLa cells were transfected with the reporter UAS-TK-Luc (1 μg), the internal control pSG5-LacZ (0.5 μg), and the expression vectors pSG5-Sp100 (D, 0.1 μg, bar 3; 0.5 μg, bar 4), pSG5-Sp100.C (D, 0.1 μg, bar 5; 0.5 μg, bar 6), pSG5-Sp100.N (D, 0.1 μg, bar 7; 0.5 μg, bar 8), pGal4 (pG4MpolyII; D and E, 0.5 μg, bars 1), pGal4-ABCD (D, 0.5 μg, bars 2 to 8), pGal4-Sp100 (E, 0.1 μg, bar 2; 0.5 μg, bar 3; 2 μg, bar 4), pGal4-Sp100.C (E, 0.1 μg, bar 5; 0.5 μg, bar 6; 2 μg, bar 7), and pGal4-Sp100.N (E, 0.1 μg, bar 8, 0.5 μg, bar 9, 2 μg, bar 10). Corrected luciferase activities were used to calculate the fold activation relative to the controls (D and E, bars 1). The values are from at least three independent experiments with at least two DNA preparations.

FIG. 8.

FIG. 8.

Working model of Sp100 activation of ETS-1. Promoter bound ETS-1 is inactive, possibly due to the activation domain C being masked or incorrectly folded. Sp100 is mainly confined to NBs. Cellular events that increase the availability of Sp100 (e.g., release from NBs) result in complex formation between Sp100 and promoter-bound ETS-1. A conformation change in ETS-1 leads to the interaction of the activation domain with the transcription machinery and transcription activation.