Developmental regulation of sea urchin H1beta gene through phosphorylation of the embryonic transcription factor SSAP
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It is a prerequisite for normal development that genes be expressed in correct temporal and spatial patterns. Sea urchins contain two families of histone genes that are differentially expressed during embryogenesis. Expression of the early histone genes begins shortly after fertilization, peaks at the blastula stage and then undergoes an abrupt decline. The late histone genes, however, are transcribed at low basal levels shortly after fertilization and become transcriptionally activated at the blastula stage, ultimately replacing the early histone mRNA.;Stage Specific Activator Protein (SSAP) is a 41kDa polypeptide that binds to embryonic enhancer elements within the sea urchin late H1 gene. These enhancer elements mediate the transcriptional activation of the late HI gene in a temporally specific manner at the mid-blastula stage of embryogenesis. Although SSAP can transactivate the late H1 gene only at late stages, it resides in the sea urchin nucleus and maintains DNA-binding activity throughout embryogenesis. It has been previously shown that SSAP undergoes a conversion from a 41kDa monomer to a ∼80 to 100 kDa dimer when the late H1 gene is activated, indicating the involvement of a posttranslational modification event.;I have demonstrated that SSAP is differentially phosphorylated during embryogenesis. Serine 87, a cAMP consensus site located in the N-terminal DNA binding domain, is constitutively phosphorylated. At the mid-blastula stage of embryogenesis, temporally correlated with SSAP dimer formation and late H1 gene activation, a threonine residue in the C-terminal transactivation domain is phosphorylated. This phosphorylation can be catalyzed by a break-ended double-stranded DNA-activated protein kinase activity from the sea urchin nucleus in vitro. Microinjection of synthetic SSAP mRNAs encoding either serine or threonine phosphorylation mutants results in a failure to transactivate reporter genes that contain the enhancer element, suggesting that both serine and threonine phosphorylations of SSAP are required for activation of the late H1 gene. Furthermore, SSAP can undergo blastula stage specific homodimerization through its GQ-rich transactivation domain. The late-specific threonine phosphorylation in this domain is essential for dimer assembly. These observations indicate that SSAP activation is promoted by threonine phosphorylation on its transactivation domain, which triggers the formation of a transcriptionally active homodimer.