Figure 7
Sp1 interacts with ACVRL1 promoter in HUVECs. (A) Scheme showing the primers used for Chromatin immunoprecipitation (ChIP). The whole sequence of ACVRL1 promoter is mapped along four regions of approximately 200-250 bp. (B) Sp1 ChIP on ACVRL1 promoter in HUVECs. The chromatin was digested obtaining a 150-300 bp fragments-enrichment. Anti-Histone 3 and a pool of rabbit-IgGs were used as positive and negative controls. Input DNA was loaded before (Inp) and after (PInp) a preclearing process. (C) As a negative control of a gene promoter that does not ChIP with Sp1, a fragment of erythropoietin (EPO) promoter was used [36]. (D) Sp1 binding to the different pALK1 regions from the ChIP experiment in B was measured by densitometry of the individual bands and values of the (Sp1-IgG)/PInput ratios were represented. (E) Scheme of the ACVRL1 promoter fragment used as probe for EMSA assays and competitor mutant probes generated. (F) Electrophoretic Mobility Shift Assays (EMSA) shows the binding of Sp1 to the -89/-56 bp region of ACVRL1 promoter. Two Sp1 sites and one KLF6 site are framed in this region. EMSAs were performed with 32P-labelled WT probe. Cold probes were: WT; Mut A, mutated at the -84/-78 site; Mut B, mutated at the -67/-62 site; and two irrelevant sequences, the -823/-795 region of ACVRL1 promoter (-823) and AATT. A positive control of Sp1 was included in lane 8 using a probe from ENG promoter as described in Methods. The retarded Sp1 band is indicated by the arrow. The asterisks indicate the supershifted band obtained by addition of the anti-Sp1 antibody. The insert on the right, includes an over-exposition of the supershift corresponding to lanes 14 and 15. As negative controls, anti-Sp3 and anti-NFκB antibodies were included.