DISTINCT FUNCTIONAL-PROPERTIES OF 3 HUMAN PAIRED-BOX-PROTEIN, PAX8, ISOFORMS GENERATED BY ALTERNATIVE SPLICING IN THYROID, KIDNEY AND WILMS-TUMORS

The mammalian paired box (Pax) genes encode a family of transcription factors involved in embryogenesis. The murine and human Pax8 genes are expressed in developing and adult thyroid as well as in the developin g secretory system and at the lower level in adult kidney. In the secr etory system expression is localized to the induced, extensively diffe rentiating parts that undergo a transition from mesenchyme to epitheli um. The human PAX8 gene generates at least five different alternativel y spliced transcripts encoding different PAX8 isoforms. These isoforms differ in their carboxy-terminal regions downstream of the paired dom ain that has been shown previously to be responsible for the DNA bindi ng. The PAX8a isoform contains a 63 amino-acid serine-rich region that is absent in the isoform PAX8b whereas PAX8c reveals a novel 99-amino -acid proline-rich region. This proline-rich region arises due to an u nusual reading-frame shift in the PAX8 transcript, RNAse protection an d RT(reverse transcription)-PCR analysis show the expression of all th ree PAX8 transcripts in human thyroid, kidney and five Wilms' tumors. Band-shift assay indicates a greatly reduced binding affinity of the i soform PAX8c to a DNA sequence from the promoter of the thyroperoxidas e gene compared to the binding of PAX8a and PAX8b to this sequence, De letion analysis of murine PAX8a indicates that its activating domain r esides at the carboxy terminus of the protein which is shared by isofo rms PAX8a acid PAX8b. In accordance with these data PAX8a and PAX8b ac tivate transcription from a thyroglobulin promoter as well as from a c otransfected synthetic PAX8-specific promoter/chlorampericol acetyltra nsferase (CAT) reporter containing a Pax8-binding oligonucleotide in f ront of the basal herpes simplex virus thymidine kinase (HSV-TK) promo ter (P11/12-TK-CAT). However if the basal HSV-TK promoter of this repo rter is substituted by a minimal adenovirus E1b TATA element, PAX8a an d PAX8b fail to activate transcription. Of the three chimaeric forms c ontaining the GAL4 DNA-binding domain at the amino-terminal end fused to the corresponding carboxy-terminal regions of the PAX8 isoforms beg inning immediately downstream of the paired domain only a GAL4-PAX8b f usion significantly activates transcription from a cotransfected GAL4- specific upstream-activating-sequence (UAS)-TK-CAT reporter. Substitut ion of the basal HSV-TK promoter in this reporter by the minimal E1b T ATA element does not affect this activation. These results indicate th at the PAX8 isoforms display different functional properties and may a lso function differently in vivo. Furthermore they indicate functional interactions between the paired domain and the carboxy-terminal domai n(s) of the PAX8 protein.