Untersuchungen zur molekularen und biologischen Funktion des Transkriptionsfaktors Sp2
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Philipps-Universität Marburg
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Abstract
The transcription factor Sp2 belongs to the Sp/KLF family of transcription factors. This family is characterized by a C-terminal DNA-binding domain composed of three zinc fingers of the C2H2 type. The zinc finger domains mediate binding to GC-rich DNA elements, so called GC- or GT-boxes. These elements are found in a number of promoters, directing transcription of diverse genes, such as house keeping genes or genes that are regulated by specific stimuli.
When this work was initiated, little was known about the function of the Sp2 protein. DNA-binding of the full length protein was not observed, although the Sp2 zinc finger region showed DNA-binding in EMSAs. Also, activation of reporter genes was not detected. DNA-binding analyses of deletion mutants indicated, that an N-terminal region might inhibit DNA binding of full-length Sp2, possibly by mediating interactions with other proteins. A transgenic Sp2 knock-out mouse line had already been generated and it had been shown, that mice lacking Sp2 protein die during early embryonic development.
The aims of this work were (I) to analyze the DNA-binding properties of Sp2 in more detail, (II) to test if Sp2 interacts with other proteins and, if so, identify these interaction partners, and (III) to establish Sp2 deficient MEFs as well as a conditional Sp2 knock-out mouse line in order to allow further analysis of the biological function of Sp2.
To analyze the DNA-binding capacity of Sp2 in more detail, a series of deletion mutants was examined in EMSA in parallel with full-length Sp2. DNA-binding of full-length Sp2 was never observed under the conditions tested. In addition, it was shown that at least three regions in the Sp2 protein inhibit DNA binding, possibly by mediating protein-protein interactions.
Size exclusion chromatography indicated that Sp2 most likely interacts with other proteins, as Sp2 was found in several high molecular weight fractions. To identify these interacting proteins, a yeast-two-hybrid screen was performed and RNF197 and RACK1 were found to interact with Sp2. But these interactions could not be validated by co-immunoprecipitation experiments. Therefore, a second approach was employed for the identification of interaction partners. Co-immunoprecipitation experiments using Sp2-3Flag were performed followed by mass spectrometric analyses. In total, 279 proteins were found to co-elute with Sp2-3Flag. The potential interactions of E2F6 and RBBP4 with Sp2 were analyzed by co-immunoprecipitation experiments, but could not be validated.
To analyze the function of Sp2 in vivo, MEFs were isolated from E9.5 Sp2 knock-out embryos but these MEFs did not proliferate in culture. Therefore, a conditional Sp2 knock-out mouse line was generated and MEFs were isolated from E13.5 embryos for further analysis. Depletion of Sp2 from these MEFs lead to inhibition of proliferation. Therefore, Sp2 is not only essential for normal mouse embryonic development, but also for proliferation of cells in culture.
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Created: 2012Issued: 2012-07-11Updated: 2012-09-21
Faculty
Medizin
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
Transcription factor
DFG-subjects
Transkriptionsfaktor
DDC-Numbers
610
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Nau, Kerstin (1023317079): Untersuchungen zur molekularen und biologischen Funktion des Transkriptionsfaktors Sp2. : Philipps-Universität Marburg 2012-07-11. DOI: https://doi.org/10.17192/z2012.0599.
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