Co-operation between the PAI and RED subdomains of Pax-8 in the interaction with the thyroglobulin promoter

Pax proteins are transcription factors that play an important role in the d ifferentiation of several cell types. These proteins bind to specific DNA s equences through the paired domain. This evolutionarily conserved element i s composed of two subdomains (PAI and RED), located at the N- and C-termina ls, respectively. Due to the presence of these two subdomains, Pax proteins may recognize DNA in different modes, a possibility that has not been exha ustively explored yet. The C site of the thyroglobulin promoter is bound by the thyroid-specific transcription factor Pax-8. In this study we have cha racterized the mode by which the Pax-8 paired domain interacts with the C s ite. Results allow the identification of the respective positions of the PA I and RED subdomains when the full-length protein is bound to the C site. T he binding of the isolated PAI and RED subdomains to the C site and to seve ral related mutants was also evaluated. Both subdomains interact with DNA a s a monomer and display a lower binding affinity than the full-length prote in. Therefore, the Pax-8 paired domain-C site interaction occurs through a cooperation between the two subdomains. The binding properties of the PAI s ubdomain suggest that the co-operation between PAI and RED subdomains does not merely consist of the sum of contacts established by the single subdoma in: the presence of the RED subdomain is necessary for correct DNA recognit ion by the PAI subdomain, thus accounting for a sort of chronology of event s during DNA binding. Since the RED subdomain is much more variable than th e PAI subdomain among Pax proteins, these results could explain how distinc t Pax proteins may select different target genes.