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 n uclear receptor superfamily) and its C-terminally truncated variant (zFF1B) were previously identified. Due to lack of the identity box (I-box) and ac tivation 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 interactio n. In the present study, the function of the I-hox was examined. In the abs ence of the I-box or with an altered heptad 9, the AF-2 of zFF1A was not fu nctional, either in the presence or absence of ER The GST pull-down assay s howed 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 s electively activated zFF1 with the I-box but exerted no effect on zFF1B, in dicating that the I-box is able to interact with the coactivators. By delet ion study and analysis of the identified domains in GAIA-DNA binding domain , other regions of zFF1A critical for its AF were also delineated. Consiste nt 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 reg ion (amino acids 155-267), although the activity of AF-3 was inhibited by i ts Banking region. We suggest that the D and E regions of zFF1A possess bot h positive and negative transactivation functions, and interdomain "cross-t alk" may confer the full transcriptional activity of the protein.