Cloning and characterization of SSAP, a novel embryonic transcription factor

Abstract

During early development, genes must be expressed in precise temporal and spatial patterns. Sea urchins contain several families of histone genes that are differentially expressed during embryogenesis. The late histone genes are transcribed at low, basal levels shortly after fertilization and become transcriptionally activated at the blastula stage of development. The late histone genes remain transcriptionally active thereafter and constitute the somatic set of histone genes.;Earlier studies of the Strongylocentrotus purpuratus late histone H1 promoter, the subject of this thesis, have shown that the blastula stage activation of this gene is controlled by a single enhancer element. This enhancer is composed of three binding sites (USE III, Site 2, and USE IV) for a 43 kDa protein, termed Stage Specific Activator Protein (SSAP). It has been previously demonstrated that SSAP DNA binding activity is present early in embryogenesis, prior to the activation of the late H1 gene. At the time that the late H1 gene becomes activated, SSAP undergoes a conversion from a 43 kDa monomer to a 90-100 kDa dimer. This has led to the hypothesis that SSAP dimer is the transcriptionally active form of SSAP and that it is the conversion from monomer to dimer that is responsible for the activation of the late H1 gene.;The SSAP cDNA clone encoding SSAP has been isolated and sequenced. SSAP exhibits striking homology with a large family of proteins involved in RNA processing. Results indicate that SSAP is a sequence specific DNA binding protein that recognizes both single and double stranded DNA. In addition, the remainder of the SSAP polypeptide constitutes an extremely potent bipartite transcription activation domain. This domain is composed of a large stretch of amino acids that contain glutamine-glycine rich and serine-threonine-basic amino acid rich regions. SSAP appears to stimulate transcription at least in part by interacting with several members of the basal transcription machinery, including TBP, TFIIB, TFIIF74, and dTAF{dollar}\rm\sb{lcub}II{rcub}{dollar} 110. Finally, characterization of the SSAP dimer suggests that it is in fact a homodimer.