Figure 6
Demethylation of the Robo4 promoter by ETV2 and TET1/TET2. (A,B) Methylation patterns of Robo4 proximal promoter in fibroblasts expressing ETV2 and/or TET1/TET2. Fibroblasts were infected with adenovirus vectors at days 0 and 3 to express either GFP or ETV2 with or without TET1/TET2, and cultured for 3–7 days. Methylation of the Robo4 promoter was analyzed by bisulfite sequencing. Data are % methylated CpGs within 200 bp upstream of the transcription start site. (C) Effect of ETV2 and TET1/TET2 on Robo4 expression in fibroblasts. Fibroblasts were infected with adenovirus vectors to express GFP or ETV2 with or without TET1/TET2, and cultured for 3–7 days.. Expression of Robo4 was measured by real-time RT-PCR. Data are means ± S.D. (n = 3). *p < 0.05 by two-tailed Tukey-Kramer test. (D) Binding of ETV2 to endogenous Robo4 proximal promoter in fibroblasts. Chromatin immunoprecipitation was performed using anti-HA antibody and human dermal fibroblasts expressing HA-ETV2. Immunoprecipitated DNA was analyzed by real-time PCR targeting the Robo4 proximal promoter. Data are means ± S.D. (n = 5). *p < 0.05 by two-tailed Student’s t-test vs control IgG. (E) ETV2-dependent TET1/2 binding to the endogenous proximal promoter. Chromatin immunoprecipitation was performed using fibroblasts expressing HA-TET1 or HA-TET2 with or without ETV2. Data are means ± S.D. (n = 4 for HA-TET1, n = 3 for HA-TET2). *p < 0.05 by two-tailed Student’s t-test vs control IgG.
