A zebrafish Ftz-F1 (Fushi Tarazu Factor 1) homologue requires multiple subdomains in the D and E regions for its transcriptional activity

A zebrafish Ftz-F1 homologue, zFF1A (zebrafish Ff1a or Nr5a2, a member of nuclear receptor superfamily) and its C-terminally truncated variant (zFF1B) were previously identified. Due to lack of the identity box (I-box) and activation function 2 (AF-2) domain, zFF1B lacks transactivation function and fails to synergize with estrogen receptor (ER) in regulating promoters. It was speculated that the I-box might be involved in the zFF1A/ER interaction. In the present study, the function of the I-box was examined. In the absence of the I-box or with an altered heptad 9, the AF-2 of zFF1A was not functional, either in the presence or absence of ER. The GST pull-down assay showed that zFF1A and its mutants exerted similar physical contacts with ER-LBD, suggesting that the 'dimerization' domain (I-box) is essential for the transcriptional activity of zFF1A. Moreover, nuclear receptor coactivator selectively activated zFF1 with the I-box but exerted no effect on zFF1B, indicating that the I-box is able to interact with the coactivators. By deletion study and analysis of the identified domains in GAL4-DNA binding domain, other regions of zFF1A critical for its AF were also delineated. Consistent with the mutation analysis, AF-2 was active only in the presence of the I-box. We also identified a novel AF domain (AF-3) located in the hinge region (amino acids 155-267), although the activity of AF-3 was inhibited by its flanking region. We suggest that the D and E regions of zFF1A possess both positive and negative transactivation functions, and interdomain 'cross-talk' may confer the full transcriptional activity of the protein.