Transcription factor Sp2: Molecular characterization and generation of Sp2 gene targeted mice
Summary The Sp family of transcription factors is characterised by its DNA-binding domain, an array of three conserved C2H2 zinc fingers. As a consequence of the conserved DNA binding motif, Sp members recognize GC (GGGGCGGGG) and GT (GGTGTGGGG) boxes with similar specificity and affinity. GC and...
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The Sp family of transcription factors is characterised by its DNA-binding domain, an array of three conserved C2H2 zinc fingers. As a consequence of the conserved DNA binding motif, Sp members recognize GC (GGGGCGGGG) and GT (GGTGTGGGG) boxes with similar specificity and affinity. GC and GT boxes are im-portant for the expression of many different ubiquitous as well as tissue-specific cellular and viral genes. To date, nine members of the Sp family (Sp1 - Sp9) have been identified. In addition to their DNA-binding domain, Sp1 - Sp4 also share other structural features like two glutamine-rich transactivation domains and two serine/threonine-rich regions. Molecular and functional properties have been de-scribed for Sp1, Sp3 and Sp4. Mouse deletion mutants, which have been gener-ated for these factors, demonstrate their manifold function and essential impor-tance for mammalian development. Since the cloning of Sp2, which is the less conserved factor among Sp1 - Sp4, no reports about its function, neither in vitro nor in vivo have been published. Therefore, the aim of this thesis work was to unravel Sp2 function by two parallel approaches: a functional molecular characterization (including expression, transactivation and DNA binding studies) and the generation of Sp2 gene targeted mice.
To study the Sp2 protein at the molecular level, Sp2-specific rabbit polyclonal anti-bodies were generated. Sp2 protein, which is exclusively localized to the nucleus, was detected in all analyzed cell lines and adult mouse tissues, although in differ-ent amounts. This favours at least a widely expression of the transcription factor Sp2. To explore Sp2 transactivation properties, reporter assays were performed with full-length Sp2 protein as well as various Sp2 deletion mutants using different GC- and GT-box-containing promoters. Unlike transcription factor Sp1, which is a strong activator, Sp2 proteins did not activate reporter gene expression. Also, when fusing Sp2 deletions to a heterologous Gal4 DNA binding domain, no activation was detectable. In addition, the DNA binding capacity and specificity of full-length Sp2 protein and a series of Sp2 deletion mutants were investigated by Electropho-retic Mobility Shift Assays. Full-length Sp2 protein was not able to bind to DNA, nei-ther to GC boxes (the “classical” Sp1 binding site) and GC box variants, nor to other DNA binding sequences like GT and CT boxes. However, when deleting the N-terminal amino acids 1-179, GC box binding was possible. These results suggest that the DNA binding activity is regulated in vivo. To unravel the physiological func-tion of transcription factor Sp2, targeted mice were generated. These mice are not viable; they die shortly before or after birth. Whereas Sp2-targeted embryos develop normal until day E12.5, day E18.5 embryos are characterized by a strongly reduced body size and weight, however with strong variations. These results demonstrate a fundamental role of the transcription factor Sp2 for normal mouse development.|
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