Identification of a portable repression domain and an E1A-responsive activation domain in Pax4: a possible role of Pax4 as a transcriptional repressor in the pancreas

Pax4 is a paired-domain (PD)-containing transcription factor which plays a crucial role in pancreatic beta/delta-cell development. In this study, we c haracterized the DNA-binding and transactivation properties of mouse Pax4. Repetitive rounds of PCR-based selection led to identification of the optim al DNA-binding sequences for the PD of Pax4. In agreement with the conserva tion of the optimal binding sequences among the Pax family transcription fa ctors, Pax4 could bind to the potential binding sites for Pax6, another mem ber of the Pax family also involved in endocrine pancreas development. The overexpression of Pax4 in HIT-T15 cells dose dependently inhibited the basa l transcriptional activity as well as Pax6-induced activity. Detailed domai n mapping analyses using GAL4-Pax4 chimeras revealed that the C-terminal re gion of Pax4 contains both activation and repression domains. The activatio n domain was active in the embryonic kidney-derived 293/293T cells and embr yonal carcinoma-derived F9 cells, containing adenoviral E1A protein or E1A- like activity, respectively but was inactive or very weakly active in other cells including those of pancreatic beta- and alpha-cell origin. Indeed, t he exogenous overexpression of type 13S E1A in heterologous cell types coul d convert the activation domain to an active one. On the other hand, the re pression domain was active regardless of the cell type. When the repression domain was linked to the transactivation domain of a heterologous transcri ption factor, PDX-1, it could completely abolish the transactivation potent ial of PDX-1. These observations suggest a primary role of Pax4 as a transc riptional repressor whose function may involve the competitive inhibition o f Pax6 function. The identification of the E1A-responsive transactivation d omain, however, indicates that the function of Pax4 is subject to posttrans lational regulation, presiding further support for the complexity of mechan isms that regulate pancreas development.