A subset of Ets proteins cooperatively binds DNA with Fos-Jun. Gel shift assays were performed in the presence or absence of Fos-Jun with ERG (amino acids 283 to 462), ETV1 (amino acids 324 to 477), Ets2 (amino acids 354 to 468), and PU.1 (amino acids 156 to 264); the probe contains the tandem sites in the UPP promoter. In each case, the concentrations of the Ets proteins were 35.3, 141, and 565 nM. Site selection studies have shown that PU.1 can bind the sequence 5′-AGTA-3′, which is also present in the UPP probe, accounting for the binding of two molecules of PU.1 on the UPP probe at high concentrations. Note that the binding of PU.1 and AP-1 is actually somewhat anticooperative, while Ets2 the binding of Ets2 and Fos-Jun is noncooperative.

Spatial flexibility in cooperative DNA binding by Fli1 and Fos-Jun. (A) Alignment of the tandem Ets and AP-1 sites from the indicated promoters. In each case, the Ets site is placed 5′ to facilitate the alignment, although in some cases this represents the anticoding strand. (B) Gel shift assays performed with probes corresponding to the tandem elements with the indicated spacings. The 1-nucleotide spacing was from the UPP promoter, the 6-nucleotide spacing was a 5-nucleotide deletion from the human collagenase promoter, the 11-nucleotide spacing was from the human collagenase promoter, the 16-nucleotide spacing was a 5-nucleotide insertion into the human collagenase promoter, and the 22-nucleotide spacing was from the mouse collagenase promoter. (C) The concentration dependence of Fli1 binding to the different probes in the presence and absence of Fos-Jun is plotted. Details of the plots are as described in the legend to Fig. 3C.

EWS-Fli1 activates gene expression from sites that support cooperative DNA binding with AP-1. (A) Schematic diagrams of the reporter genes. Two copies of the tandem Ets and AP-1 sites from the indicated genes were placed upstream of a minimal promoter to drive luciferase expression. As controls, either the two Ets sites (muEts [mutant Ets]) or the two AP-1 sites (muAP-1) in the reporter genes were mutated. WT, wild type. (B) The indicated reporters were then cotransfected into 3T3 cells along with either pBabepuro or pBabepuro-EWS-Fli1 and a Renilla luciferase control plasmid. Forty hours later, luciferase activity was determined. The results are the average of at least three independent experiments performed in triplicate. RLU, relative light units.

The C terminus of Fli1 is required for cooperative DNA binding. (A) Schematic diagrams of the indicated Ets proteins. (B) The isolated Fli1 Ets domain does not cooperatively bind DNA. Shown is a gel shift assay with Fli1 (amino acids 270 to 371) and Fos-Jun on the tandem elements of the human collagenase promoter. The concentrations of Fli1 were 35.3, 141, and 565 nM. (C) An Ets2-Fli1 fusion protein binds DNA cooperatively with Fos-Jun. A fusion protein containing the Ets domain of Ets2 and the C-terminal sequences of Fli1 was used in gel shift assays with the tandem elements of the human collagenase promoter as a probe. Protein concentrations were as described above. (D) Schematic diagrams of the indicated EWS-Ets fusion proteins. (E) The indicated EWS-Ets fusion proteins were cotransfected with reporter genes harboring the indicated tandem elements into 3T3 cells. At 40 h after transfection, luciferase activity was determined. RLU, relative light units.

EWS-Fli1 and Fos-Jun cooperatively bind to the tandem Ets-AP-1 element in the UPP promoter. Gel shift reactions were performed with 3T3 cell nuclear extract (NE) as a source of Fos-Jun, Flag-EWS-Fli1 purified from baculovirus-infected insect cells, and radiolabeled oligonucleotides corresponding to the indicated tandem Ets and AP-1 sites as probes. In lanes 1 to 6, 3T3 cell nuclear extract was used with the tandem Ets and AP-1 sites of the UPP promoter as probes and the indicated competitors or antibodies. Note that the AP-1 and UPP competitors block the formation of the Fos-Jun complex, while the E74 (Ets site) competitor does not. In lanes 7 to 16, nuclear extract and recombinant EWS-Fli1 (10.5, 31.5, and 94 nM, respectively, for lanes 7 to 12 and 31.5 nM for lanes 13 to 16) were used with the wild-type UPP probe and the indicated competitor or antibody. In lanes 17 to 20, the Ets site in the UPP probe was mutated, and in lanes 21 to 24, the AP-1 site in the UPP probe was mutated; the specific mutations are described in Materials and Methods. The concentration of EWS-Fli1 used in lanes 21 to 24 was 376 nM. In lanes 25 to 28, the probe was the tandem Ets and AP-1 sites from the human collagenase (Coll) promoter; the concentration of EWS-Fli1 was 94 nM in lanes 25 and 26 and 24 nM in lane 28.

A mutant form of EWS-Fli1 that cannot cooperatively bind DNA shows loss of biologic activity. (A) 3T3 cells were infected with recombinant retroviruses directing the expression of Flag-tagged EWS-Fli1 or EWS-Fli1ΔC, and infected cells were selected with puromycin. Shown is an immunoblot assay with anti-Flag; note the similar levels of expression of EWS-Fli1 and EWS-Fli1ΔC. (B) RNA was isolated from 3T3 cells expressing EWS-Fli1, EWS-Fli1ΔC, and activated Ras. The RNA was separated, and expression of the UPP mRNA was analyzed by Northern blotting. In the bottom panels, chromatin was prepared from formaldehyde-cross-linked samples, sheared to a size of 0.5 to 1.5 kb, and immunoprecipitated with anti-Flag agarose. The bound DNA was eluted, the cross-links were reversed, and the presence of UPP promoter DNA was detected by PCR. Reverse transcription-PCR with SYBR Green showed that EWS-Fli1 samples contained 12 times more UPP promoter DNA than EWS-Fli1ΔC samples. EtBr, ethidium bromide. (C) 3T3 cells or cells expressing EWS-Fli1 or EWS-Fli1ΔC were seeded at 5 × 10³/6-cm dish in soft agar. At 18 days later, the colonies greater than 0.6 mm in diameter were counted. Shown also are photomicrographs of the indicated cells.

Fli1 and Fos-Jun cooperatively bind many different spatial arrangements of Ets and AP-1 sites with high affinity. (A) EWS sequences are dispensable for cooperative DNA binding with Fos-Jun. Gel shift reactions were performed as described above, with the tandem Ets and AP-1 sites from the UPP promoter (left side) or the HB-EGF promoter (right side). Protein concentrations were 35.3, 141, and 565 nM, respectively. Note that the Ets-Fos-Jun-DNA complex does not form on probes in which the Ets site has been mutated. (B) Binding of Fli1 and Fos-Jun to the human collagenase tandem elements. Shown is a gel shift assay with Fos-Jun (96 nM) and Fli1 (amino acids 270 to 452) at concentrations of 35.3, 70.6, 141.3, 282.5, 565, and 1,130 nM. In control experiments, the Fli1-Fos-Jun-DNA complex did not form on a human collagenase probe in which the Ets site had been mutated (data not shown). Also shown is a plot of the concentration dependence of Fli1 DNA binding either in the cooperative complex or to DNA alone. Squares indicate the binding of Fli1 in the presence of Fos-Jun, while circles indicate binding in the absence of Fos-Jun. Because nearly all of the probe is bound by Fos-Jun, the binding reaction for the higher-order complex can be simplified to P-D/D_t = Fli1-Fos-Jun-DNA/(Fli1-Fos-Jun-DNA + Fos-Jun-DNA), where P-D is the amount of Fli1-Fos-Jun-DNA complex and D_t is the total amount of DNA. The plot was obtained by fitting the data to a sigmoidal function with Delta Graph. (C) Concentration dependence of Fli1 DNA binding. The dotted lines indicate binding of Fli1 to the human collagenase probe in the presence and absence of Fos-Jun. Filled squares indicate binding of Fli1 to the probe in the presence of Fos-Jun, and open squares indicate binding in the absence of Fos-Jun. Note that while the binding of Fli1 alone changes with different probes, the cooperative binding is very similar to that observed with the human collagenase probe.

EWS and Fli1 interact on DNA but not in solution. (A) HA-c-Fos, c-Jun, and Flag-EWS-Fli1 were coexpressed by transient transfection of 293 cells. Immunoprecipitations (IP) were performed with antibody against either the HA or the Flag tag. The immunoprecipitates were then analyzed by immunoblotting (IB) with antibodies directed against c-Fos, c-Jun, or Fli1; the Fli1 antibody recognizes a C-terminal epitope present in EWS-Fli1. WCE, whole-cell extract. (B) Extracts from 3T3 cells or 3T3 cells transformed by stable expression of Flag-EWS-Fli1 were immunoprecipitated with Flag antibody. The resulting extracts were then analyzed for the presence of either c-Jun or EWS-Fli1 by immunoblotting. (C) Recombinant EWS-Fli1 was used in gel shift reactions with the human collagenase probe. The concentrations of EWS-Fli1 were 35, 70, and 140 nM. The two complexes originate from a second low-affinity Ets site in the human collagenase probe. Fli1 and Flag antibodies (Ab) were added to reaction mixtures with 140 nM EWS-Fli1 in lanes 5 and 6, respectively. (D) Gel shift reactions were set up as in Fig. 1 with the human collagenase probe. At time zero, a 200-fold excess of unlabeled competitor was added, and aliquots of the reaction mixture were analyzed by gel shift assay at the indicated times. In the first lane of the left half, no EWS-Fli1 was added. The amount of residual complex was determined by PhosphorImager analysis, and a plot of P-D/P-D₀ versus time is shown, where P-D is the amount of the indicated protein-DNA complex at the indicated time, and P-D₀ is the amount of the same complex at time zero.