The purification, characterization and cloning of an embryonic enhancer binding protein

Abstract

It has been previously demonstrated that a highly conserved segment of DNA between position {dollar}-{dollar}288 to {dollar}-{dollar}317 (USE IV) is responsible for the transcriptional activation of the sea urchin H1-{dollar}\beta{dollar} 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. A 43 kd polypeptide has been purified which binds to and footprints the USE IV enhancer element. This protein is referred to as Stage Specific Activator Protein 1 (SSAP-1). Immunoblot analysis demonstrates that SSAP-1 is present in low but detectable amounts early in development and then increases as development proceeds. Although SSAP-1 DNA binding activity is present in the embryo before the enhancer is activated, a change in the molecular weight of SSAP precisely parallels the increase in H1-{dollar}\beta{dollar} gene transcription. Early in development, SSAP appears as a 43 kd monomer but it undergoes a post-translational modification beginning at about 12 hr post-fertilization (early blastula). Modified SSAP has an apparent molecular weight of approximately 90-100 kd. Thus, it is the disappearance of the 43 kd species and the appearance of the 90-100 kd species which coincides with the H1-{dollar}\beta{dollar} gene activation. Partial purification of the modified form of SSAP suggests that it is a heterodimer of 43 kd (SSAP-1) and 48 kd (SSAP-2) subunits. The correlation between the presence of the SSAP dimer and the pattern of H1-{dollar}\beta{dollar} gene expression strongly suggests that this dimer, and therefore the appearance of the 48 kd subunit, is directly responsible for the blastula stage-specific transcriptional activation of the late H1 gene.;The gene which codes for SSAP-1 has been cloned and sequenced. The protein product for this gene has been expressed in vitro and is able to specifically bind to USE IV. SSAP does not contain any known DNA-binding motifs. Instead SSAP-1 contains two 90 amino acid repeats which show significant homology to ribonuclear (RNP) and helix destabilizing proteins. These RNP repeats are necessary and sufficient for sequence specific binding of SSAP-1 to USE IV and may represent a novel DNA binding motif.