Purification and characterization of the stage-specific embryonic enhancer-binding protein SSAP-1.
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-beta 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-beta 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-beta gene activation. The correlation between the change in molecular weight of SSAP and the stage-specific activation of H1-beta 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 H1 gene.
DeAngelo
D J
DJ
Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, New York 10461.
DeFalco
J
J
Childs
G
G
D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D001667:Binding, Competitive; D002467:Cell Nucleus / Q000737:chemistry; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002874:Chromosome Mapping; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D006657:Histones / Q000235:genetics*; D008845:Microinjections; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins; D012617:Sea Urchins / Q000196:embryology; D013997:Time Factors; D014158:Transcription, Genetic; D015053:Zygote
D004268:DNA-Binding Proteins; D006657:Histones; D009841:Oligonucleotides; D011994:Recombinant Proteins; C079872:SSAP protein, sea urchin; D015500:Chloramphenicol O-Acetyltransferase