Fig. 5
ERG binds to and transactivates the thrombomodulin promoter in vitro. a Putative ERG binding sites (black bars) are located within the TM promoter upstream and downstream of the transcription start site (black arrow); ERG ChIP-sequencing data show a significant ERG peak on TM proximal promoter. ENCODE sequence conservation between 100 vertebrates is shown across this gene. ENCODE ChIP-seq data profiles for H3K4Me3 (tri-methylation of lysine (K) 4 on histone 3), H3K27Ac (acetylation of lysine (K) 27 on histone 3) and RNA polymerase II (RNA pol II) in HUVEC indicate open chromatin and active transcription. Location of qPCR primers covering regions R1 and R2 (black bar) are indicated. b ChIP-qPCR using primers to region R1 and R2 on ERG-bound chromatin from siCtrl or siERG-treated HUVEC. Primers for a region within 5′UTR region of TM gene (TM neg con) were used as negative control. Data are shown as fold change over IgG (n = 3 independent experiments). Graphical data are mean ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test. c TM promoter luciferase reporter assay. ERG cDNA expression plasmid (ERG) or empty expression plasmid (pcDNA) were co-transfected with TM promoter-luciferase constructs (TM wild type (WT), TM mutant 1 or TM mutant 2) or a pGL4 empty vector in HUVEC, and luciferase activity was measured. Values represent the fold change in relative luciferase activity over the empty pGL4 vector alone (n = 4 independent experiments). Graphical data are mean ± s.e.m., **P < 0.01, ***P < 0.001, One-way ANOVA; ##P < 0.01 compared to pcDNA-TM WT condition, Student’s t-test. d ChIP-qPCR using primers to region R1 and R2 on H3K27Ac-bound chromatin from HUVEC treated with DMSO or p300 inhibitor (10 µM) for 1 h. Data are shown as fold change over IgG (n = 3 independent experiments). e–f ChIP-qPCR using primers to region R1 and R2 on e p300- (n = 3 independent experiments) or f H3K27Ac-bound (n = 4 independent experiments) chromatin from siCtrl or siERG-treated HUVEC. Data are shown as fold change over IgG. All graphical data for ChIP experiments are mean ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test. Source data are provided as a Source Data file
