Figure 3

GRHL2 binds to the proximal regions and regulates the activity of its target gene promoters. (a) PMP assay was performed with two different regions of each promoter denoted as R1 and R2, location of which is shown in the diagram with respect to the TSS (+1). (b) PMP was performed using 50 μg nuclear extracts of NHEK with R1 and R2 fragments of each promoter immobilized onto Dynabeads. With the protein samples that co-precipitated with the promoter fragments, western blotting was performed for GRHL2 and Sp1. (c) PMP was performed with the promoter fragments of GRHL1, GRHL3, and PCNA in the presence or absence of non-immobilized competitor DNA, and western blotting was done for GRHL2 and Sp1. Data illustrates the specificity of interaction between the proteins (GRHL2 and Sp1) and the promoter fragments. (d) Promoter activity was measured in NHEK/LXSN and NHEK/GRHL2 for LCE3D, IVL, SPRR2A, and hTERT, which were differentially regulated by GRHL2. Firefly luciferase reporter gene expression was driven by the promoters of EDC genes or hTERT, and normalized against Renilla luciferase driven by SV40 promoter