Figure 2

Pax8 activates transcription from the Wnt4 5′-flanking region. A) Transcriptional activity of Wnt4 5′-flanking genomic region. The deletion reporter constructs were transfected into FRTL-5 and HeLa cells and the luciferase activity was determined. All the constructs have the highest activity in thyroid cells and only the 190Wnt4LUC construct shows a strongly reduced activity. Data are expressed as the mean ± SD (P < 0,0001); B) Pax8 activates transcription from the Wnt4 promoter region. HeLa cells were transiently transfected with 300Wnt4LUC and 190Wnt4LUC reporter constructs alone and in combination with increasing concentration (100 and 200 ng) of the expression vector encoding Pax8 (CMV5-Pax8). The transcriptional activity was determined 48 h after transfection as the firefly over renilla luciferase activity. Data are expressed as fold induction over the transcription obtained with 300Wnt4LUC and 190Wnt4LUC, whose values were set at 1. Data are from three independent experiments, each performed in duplicate and are expressed as the mean ± SD (P < 0,005); C) Pax8 binds the Wnt4 promoter region in vivo. Chromatin extracted from cross-linked FRTL-5 cells was immunoprecipitated with an unrelated antibody or for Pax8. The immunoprecipitates were analyzed by PCR with oligonucleotides able to amplify the region between -300 bp and -190 bp of the 5′flanking region of the Wnt4 gene.