Fig. 6.

Pitx2c is a direct transcriptional target of TEAD1 and promotes smooth muscle-specific gene activation in a CArG-dependent manner. a Schematic diagram of the mouse Pitx2c gene locus showing a conserved MCAT element within the proximal promoter region. A 2.5 kbp DNA fragment of the Pitx2c gene promoter containing a WT or mutated MCAT element (underscored) was cloned into the pGL2b luciferase vector. A set of primers spanning the MCAT element was used for qChIP assays in (b) as shown by arrows. b Chromatin was harvested from control embryonic VSMCs or heart and aortic tissues from E13.5 embryos and the chromatin was subjected to IP using either IgG or anti-TEAD1 antibodies. Immunoprecipitated DNA complexes were then analyzed by qPCR using the primer sets indicated in “A”. The degree of TEAD1 binding is expressed as relative to IgG control (set to 1). N = 3–4. *p < 0.05. c Pitx2c promoter-luciferase reporters (WT or MCAT mutant) were transfected into primary embryonic VSMCs in the absence or presence of TEAD1 expression plasmid and promoter activity was evaluated by a dual luciferase assay. Pitx2c promoter activity was normalized using the control vector (set to 1). N = 8. *p < 0.05. d Control or Tead1-null VSMCs were transduced with GFP or Pitx2c adenovirus and the expression of VSMC-specific genes analyzed by Western blot. e Densitometric analysis of protein expression in (d) normalized to the loading control VCL. *p < 0.05, relative to control cells infected with GFP (set to 1, red line); ^#p < 0.05, vs. KO cells infected with GFP. Over-expression of Pitx2c in Tead1-null VSMCs restores expression of VSMC-specific contractile proteins. f Tgfb1i1 or Lmod1 g gene promoter-luciferase reporters (WT or mutated CArG) were transfected into MEFs in the absence or presence of plasmids expressing Pitx2c and dual luciferase assays were performed. Baseline activity of the WT promoter in the absence of Pitx2c co-transfection was set to 1. N = 6. *P < 0.05