Purification and characterization of the stage-specific embryonic enhancer-binding protein SSAP-1.

Abstract

We have demonstrated that a highly conserved segment of DNA between positions -288 and -317 (upstream sequence element IV [USE IV]) is largely responsible for the transcriptional activation of the sea urchin H1-1 histone gene during the blastula stage of embryogenesis. This sequence is capable of acting as an embryonic enhancer element, activating target genes in a stage-specific manner. Nuclear extracts prepared from developmentally-staged organisms before and after the gene is activated all contain a factor which specifically binds to the enhancer. We have purified a 43-kDa polypeptide which binds to and footprints the USE IV enhancer element. We refer to this protein as stage-specific activator protein 1 (SSAP-1). Early in development before the enhancer is active, SSAP appears as a 43-kDa monomer, but it undergoes a change in its molecular weight beginning at about 12 h postfertilization (early blastula) which precisely parallels the increase in H1-j3 gene expression. Modified SSAP has an apparent molecular mass of approximately 90 to 100 kDa and contains at least one 43-kDa SSAP polypeptide. Thus, it is the disappearance of the 43-kDa species and the appearance of the 90-to 100-kDa species which coincide with the H1-13 gene activation. The correlation between the change in molecular weight of SSAP and the stage-specific activation of H1-13 gene expression strongly suggests that this higher-molecular-weight form of SSAP is directly responsible for the blastula stage-specific transcriptional activation of the late Hi gene.

Temporal Activation of the Sea Urchin Late H1 Gene Requires Stage-Specific Phosphorylation of the Embryonic Transcription Factor SSAP

“…Threonine phosphorylation in the GQ domain mediates homodimerization of SSAP at mid-blastula stage of embryogenesis. Gel mobility-shift assays and Superose 12 gel filtration chromatography showed that SSAP is a 41-kDa species early in embryogenesis and changes to an 80-to ϳ100-kDa species coincident with the activation of late H1 gene transcription (12,13). However, no additional proteins were copurified with SSAP through protein-protein interaction, as determined by either affinity chromatography or immunoprecipitation under nondenaturing conditions (12, 14a).…”

“…Most of them have temporal profiles of expression that resemble those of their target genes, suggesting that genes encoding those transcription factors are themselves under the control of temporal regulatory mechanisms. Immunoblot analysis demonstrated that SSAPs are present in the sea urchin throughout embryogenesis (13). Microinjection experiments indicated that SSAP can transactivate the late H1 gene promoter through the USE IV enhancer sequence beginning at the mid-blastula stage even though its DNA binding activity is present in the embryos at earlier stages (12,13).…”

“…Immunoblot analysis demonstrated that SSAPs are present in the sea urchin throughout embryogenesis (13). Microinjection experiments indicated that SSAP can transactivate the late H1 gene promoter through the USE IV enhancer sequence beginning at the mid-blastula stage even though its DNA binding activity is present in the embryos at earlier stages (12,13). Mobility gel-shift assays have shown that during the early stages of development, when the late H1 gene is transcribed at basal levels, SSAP appears in the embryo and binds to the enhancer elements as a 41-kDa monomer.…”

“…However, an increase in molecular mass of enhancer binding protein from 41 kDa to 80 to 100 kDa begins at about 12 h postfertilization (early blastula), which coincides with the late H1 gene activation (12). The higher-molecular-mass complex is believed to be a dimer containing at least one 41-kDa SSAP molecule (13). The temporal correlation between the appearance of SSAP dimer and the late H1 gene expression strongly suggests that the blastula-stage-specific transcriptional activation is governed by posttranslational modifications which mediate the formation of this dimeric species.…”

“…Previous studies of the regulation of sea urchin late H1 gene expression led to the identification of a transcription factor called stage-specific activator protein (SSAP) (12,13,28,29).…”