Figure 9

Isl1 directly binds to Gata3 enhancer regions and regulates the Gata3 enhancer activity. (A) A schematic representation of the Gata3 gene surrounding the transcription start site. Putative Isl1 binding sequences (containing the ATTA/TAAT sequence) are shown as grey rectangles. (B) ChIP-PCR amplification was obtained using P1 to P10 primers which would amplify Isl1 consensus-containing fragments in the vicinity of the Gata3 transcription start site. ChIP with Isl1 antibody and amplification of fragments using the indicated primers (Additional file 2: Table S3) demonstrated binding of Isl1 to the Gata3 promoter regions in pylorus of wild-type mouse embryos at E14.5. A cell aliquot before precipitation was designated as the input sample. IgG was a negative control provided by the kit. (C) Fold change of enriched DNA fragment from ChIP detected by qPCR. (D) Effects of an Isl1 expression vector on the transiently transfected Gata3 gene enhancers (P1 and P6 regions) fused to luciferase reporter genes in 293FT cells. Data are mean ± SEM (n = 4). **P <0.01 (Student’s t-test). (E) EMSA were performed with in vitro translated pcDNA3.1-Isl1 and control vector respectively. Isl1 efficiently bound to oligonucleotides representing number 1 and 3 sites of the Gata3-P1 enhancer region. (F) Labeled ATTA number 1 and 3 probes of the P1 region were incubated with in vitro translated pcDNA3.1-Isl1 protein and assayed by EMSA. Specificity of protein-DNA binding was determined by competition with excess unlabeled wild-type or mutant competitor oligonucleotides. Additionally, Isl1 binding to oligonucleotide probes was blocked by antibodies to Isl1. bp, base pairs; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assays; IgG, immunoglobulin G; MT, mutant type; WT, wild type.