Temporal activation of the sea urchin late H1 gene requires stage-specificphosphorylation of the embryonic transcription factor SSAP

Stage-specific activator protein (SSAP) is a 41-kDa polypeptide that binds to embryonic enhancer elements of the sea urchin late H1 gene. These enhanc er elements mediate the transcriptional activation of the late H1 gene in a temporally specific manner at the mid-blastula stage of embryogenesis. Alt hough SSAP can transactivate the late H1 gene only at late stages of the de velopment, it resides in the sea urchin nucleus and maintains DNA binding a ctivity throughout early embryogenesis. In addition, it has been shown that SSAP undergoes a conversion from a 41-kDa monomer to a similar to 80- to 1 00-kDa dimer when the late H1 gene is activated. We have demonstrated that SSAP is differentially phosphorylated during embryogenesis. Serine 87, a cy clic AMP-dependent protein kinase consensus site located in the N-terminal DNA binding domain, is constitutively phosphorylated, At the mid-blastula s tage of embryogenesis, temporally correlated with SSAP dimer formation and late H1 gene activation, a threonine residue in the C-terminal transactivat ion domain is phosphorylated. This phosphorylation can be catalyzed by a br eak-ended double-stranded DNA-activated protein kinase activity from the se a urchin nucleus in vitro. Microinjection of synthetic SSAP mRNAs encoding either serine or threonine phosphorylation mutants results in the failure t o transactivate reporter genes that contain the enhancer element, suggestin g that both serine and threonine phosphorylation of SSAP are required for t he activation of the late H1 gene. Furthermore, SSAP can undergo blastula-s tage-specific homodimerization through its GQ-rich transactivation domain. The late-specific threonine phosphorylation in this domain is essential for the dimer assembly. These observations indicate that temporally regulated SSAP activation is promoted by threonine phosphorylation on its transactiva tion domain, which triggers the formation of a transcriptionally active SSA P homodimer.