SEARCH

SEARCH BY CITATION

Keywords:

  • PAX;
  • paired box;
  • proline-rich domain;
  • DNA binding

The mammalian paired box (Pax) genes encode a family of transcription factors involved in embryo-genesis. The murine and human Pax8 genes are expressed in developing and adult thyroid as well as in the developing secretory system and at the lower level in adult kidney. In the secretory system expression is localized to the induced, extensively differentiating parts that undergo a transition from mesenchyme to epithelium. The human PAX8 gene generates at least five different alternatively spliced transcripts encoding different PAX8 isoforms. These isoforms differ in their carboxy-terminal regions downstream of the paired domain that has been shown previously to be responsible for the DNA binding. The PAXSa isoform contains a 63 amino-acid serine-rich region that is absent in the isoform PAXSb whereas PAXSc reveals a novel 99-amino-acid proline-rich region. This proline-rich region arises due to an unusual reading-frame shift in the PAX8 transcript. RNAse protection and RT(reverse transcription)-PCR analysis show the expression of all three PAX8 transcripts in human thyroid, kidney and five Wilms' tumors. Band-shift assay indicates a greatly reduced binding affinity of the isoform PAXSc to a DNA sequence from the promoter of the thyroperoxidase gene compared to the binding of PAXSa and PAXSb to this sequence. Deletion analysis of murine PAXSa indicates that its activating domain resides at the carboxy terminus of the protein which is shared by isoforms PAXSa and PAXSb. In accordance with these data PAXSa and PAXSb activate transcription from a thyroglobulin promoter as well as from a cotransfected synthetic PAXS-specific promoter/chlorampericol acetyltransferase (CAT) reporter containing a PaxS-binding oligonucleotide in front of the basal herpes simplex virus thymidine kinase (HSV-TK) promoter (P11/12-TK-CAT). However if the basal HSV-TK promoter of this reporter is substituted by a minimal adenovirus Elb TATA element, PAXSa and PAXSb fail to activate transcription. Of the three chimaeric forms containing the GAL4 DNA-binding domain at the amino-terminal end fused to the corresponding carboxy-terminal regions of the PAX8 isoforms beginning immediately downstream of the paired domain only a GAL4-PAXSb fusion significantly activates transcription from a cotransfected GAL4-specific upstream-activating-sequence (UAS)-TK-CAT reporter. Substitution of the basal HSV-TK promoter in this reporter by the minimal Elb TATA element does not affect this activation. These results indicate that thePAX8 isoforms display different functional properties and may also function differently in vivo. Furthermore they indicate functional interactions between the paired domain and the carboxy-terminal domain(s) of thePAX8 protein.