Figure 8
Two CpG islands are present in the ACVRL1 promoter and treatment with the demethylating agent 5'-aza-2'-deoxycytidine increases ALK1 expression in endothelial cells. (A) Schematic representation of the ACVRL1 promoter comprising the region -1,035 to + 210 bp. CG sites are depicted by black bars. Two CpG islands near the transcriptional start site were detected using CpGplot software tool. CG content is shown as percentage the total number of G+C (top), and by the methylation-susceptible CG pairs, represented by the observed-versus-expected index (bottom). (B, C) ALK1 and Id1 transcript levels from endothelial (HUVEC, HMEC-1) versus non endothelial (HEK293T) cells prior and after treatment with the demethylating agent 5-aza-dC. Id1 mRNA levels were measured as a target gene of ALK1 signalling. (B) Cells were treated with 1 μM or 5 μM 5-aza-dC for one week. Treatment with 5 μM was cytotoxic in HUVEC. RNA was extracted and mRNA levels were measured by real time RT-PCR. Results are shown as the fold change respect to basal expression (2 -ΔΔCt). (C) Basal ALK1 and Id1 levels show the differences between ALK1 expression in endothelial cells HMEC-1 and HUVEC versus the HEK293T cells. (D) Effect of the demethylating agent 5-aza-dC on the ALK1 pathway specific reporter, p(BRE) 2-luc, in HMEC-1 cells. Results are shown as fold change of expression levels or luciferase activity (***p < 0.005).