Characterization of –5.5HS enhancer function. (A-B) One hundred percent –5.5HS enhancer activity was defined as the difference in normalized reporter gene activity measured in EA.hy926 cells transfected with either a construct containing wild-type –5.5HS upstream of SV40P (–5.5HS) or the same construct without –5.5HS (control). Normalized enhancer activity of –5.5HS constructs in which (A) the Ets, GATA, or Tal1 binding sites in Figure 3 were disrupted or (B) the spacing between the GATAand Tal1 motifs was altered (by the number of bp indicated) are shown graphically. Data are presented ± SEM (n = 6). (C) Chromatin immunoprecipitation (ChIP) experiments were performed on EA.hy926 and HepG2 cells using primers that amplified the –5.5HSCR. The fold enrichments in immunoprecipitates obtained with anti–acetyl H3 and control IgG antibodies are shown ± SEM. (D) ChIP experiments were performed from the murine endothelial cell line, MS1, using anti-Ets1, Ets2, Elf1, Fli1, Erg, GATA2, and Tal1 antibodies, or control IgG antibodies. The primers used amplified the murine region homologous to the –5.5HSCR (located –8.3 kb from mEPCR). The fold enrichment in immunoprecipitates obtained with antitranscription factor antibodies relative to control IgG antibodies are shown ± SEM.