Fig. 6: ETV2 activates transcription through the ZRS region and antagonizes ETV4 and ETV5.

A–E ETV2 binds to and transcriptionally activates the ZRS region. A Luciferase reporter assay shows a dose-dependent activation of the ZRS in response to ETV2. *P-value of 0.0331, ***P-value 0.0004 and ****P-value < 0.0001 compared to the 0 ng Etv2. B The activation of ZRS-Luciferase is dependent on ETV2 but not ETS1 or ETS2 (black bars). Flt1 promoter-luciferase reporter was used as a positive control (white bars). ****P-value < 0.0001 compared to the (-). C Deletional constructs of the ZRS-luciferase reporter show that the ETV2 response is limited to region ii containing the ETS-binding motifs. ****P-value < 0.0001 for ii vs i and ii vs iii. D Titration with Etv4 expression vector shows antagonism of ETV2 activity by ETV4. ****P-value < 0.0001 compared to the 0 ng control. E Titration of Etv5 expression vector shows antagonism of ETV2 transcriptional activity by ETV5. Each bar represents transfections done in quadruplicate. ****P-value < 0.0001 compared to the 0 ng Etv5 vector only control. Statistical tests: A, B, D, E, one-way ANOVA with Dunnett’s multiple comparisons test; C, one-way ANOVA with Tukey’s multiple comparisons test. Each experiment was repeated at least three times with essentially the same results. Representative results are shown. Data in the graphs are presented as mean values + /− SEM. F–I EMSA using HOXD13, HAND2 and ETV2 reveal that these factors bind DNA independently of each other. F Diagram of the oligonucleotide (ETS#1-HOX-E) used for this study containing putative HOX, ETS and E-box binding sequences. G, H EMSA of ETV2, HOXD13 and HAND2/E47 reveal that each factor binds to the probe (ns refers to nonspecific bands; fp refers to free probe). G shows EMSA with ETV2 and HOXD13. H shows EMSA with HAND2 with a universal dimerization partner of basic helix-loop-helix proteins, E47. Note that ETV2, HOXD13 and HAND2/E47 each form a DNA-protein complex (indicated by red and blue dots in G, and yellow dots in H, respectively). HAND2 does not bind to DNA by itself, but does bind when complexed with E47 (H, yellow dots). In this reaction, a band corresponding to an E47 homodimer was also observed (H, green dots). The shifted bands of ETV2 and HOXD13 are supershifted by antibodies to respective proteins (red and blue asterisks in G), and the shift of HAND2 and E47 is blocked by the HAND2 antibody (H). h.i. indicates respective heat-inactivated antibodies used as controls. The band of E47 is blocked by an antibody to E47. Purple dots indicate bands containing both HOXD13 and ETV2 (G). I EMSA with combinations of ETV2, HOXD13 and HAND2/E47 show that DNA binding activity of one protein is not affected by the presence of additional factors. Note that shift originating from ETV2, HOXD13 and HAND/E47 separately (red, blue, and yellow dots) are unaffected by the presence of additional transcription factors. Each experiment was repeated at least three times with essentially the same results. Source data are provided as a Source Data file.