Studies on the properties of transcriptional activation by the sea urchin stage -specific activator protein, SSAP
Benuck, Mitchel Leonard
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This thesis focuses on the mechanism of transcriptional activation by the sea urchin stage specific activator protein (SSAP). SSAP binds to three sites in a late stage specific enhancer that is responsible for the temporal activation of the late H1 gene. The predicted amino acid sequence of SSAP reveals that it represents a previously unrecognized class of transcription factors. At its N-terminal end, it contains a DNA binding domain that shares homology with the R&barbelow;NA r&barbelow;ecognition m&barbelow;otif (RRM) rather than typical DNA binding domains. Its C-terminal 202 amino acids form a glycine/glutamine rich domain (GQ domain) that, when fused to a heterologous DNA-binding domain, can activate transcription fivefold over VP16, the most potent activator yet characterized.;How does the GQ domain activate transcription so effectively? To address this question, I performed random mutagenesis on the GQ domain to identify amino acids or motifs critical for its ability to activate transcription. I observed that, unlike other glutamine-rich activation domains, SSAP can activate transcription to moderate levels in yeast. I utilized this activity to screen in yeast for intragenic mutations that enhance or inhibit the transcriptional activity of the GQ domain. I identified thirty-seven loss of function and twenty-three gain of function mutants. All thirty-seven loss of function mutants contained frameshifts at various locations in the GQ domain sequence, leading to premature truncation of the protein.;To further characterize SSAP as a transcriptional activator, I searched for human homologues of SSAP in an attempt to identify additional members of this family. Mobility shift and UV crosslinking assays, coupled with Western blot analysis and supershift assays, demonstrate that a human protein from HeLa cell nuclear extracts both binds with specificity to the USE IV element and cross-reacts with anti-SSAP antiserum. In a nucleic acid hybridization screen, I identified the TLS cDNA as a candidate SSAP homologue. TLS fails to account for the DNA binding and immunologic activity in HeLa cell extracts.;In a separate section, I turned my attention to the USE0 element of the late H1 gene. In sea urchins, the stage specific enhancer only functions in the context of a USE0-containing promoter. Thus, the identity of the USE0 binding protein would provide critical information about the mechanism of SSAP activity. Using a computer based search, I identified an Ikaros isoform as a transcription factor predicted to bind the late USE0 element. Mobility shift assays confirmed that Ikaros isoforms recognize the late USE0 element with high specificity. (Abstract shortened by UMI.).
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