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DNA binding proteins and cell fate

Artuz, Crisbel Marie, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW

2015

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  • Title:
    DNA binding proteins and cell fate
  • Author/Creator/Curator: Artuz, Crisbel Marie, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW
  • Subjects: Megakaryopoiesis; ZNF217; DNA binding; Reprogramming; Transdifferentiation
  • Resource type: Thesis
  • Type of thesis: Ph.D.
  • Date: 2015
  • Supervisor: Crossley, Merlin, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW
  • Language: English
  • Additional Information: Thesis restricted until May 2017
  • Permissions: This work can be used in accordance with the Creative Commons BY-NC-ND license.
    Please see additional information at https://library.unsw.edu.au/copyright/for-researchers-and-creators/unsworks

  • Description: Sequence-specific DNA binding proteins, known as transcription factors, play a central role in the control of eukaryotic gene regulation. Understanding the mechanisms through which DNA binding domains recognise their target sequences will greatly improve our understanding of genetic diseases that result from mutations in DNA binding domains and gene promoters. Such information will also assist in the design of factors capable of artificially controlling gene expression. The zinc finger motif, commonly present in tandem arrays of three or more fingers, is the most prevalent DNA recognition structure found in eukaryotic transcription factors. The first project in this thesis aimed to better understand how zinc finger domains bind DNA by examining the two-zinc finger motif of the transcriptional regulator and oncogene ZNF217. By performing a comprehensive mutagenesis analysis, we were able to identify the amino acid residues that are essential for DNA recognition. Our findings indicate that ZNF217 binds to its preferred consensus site by a novel mechanism, an understanding of which may lead to a better appreciation of diseases that result from dysregulation of ZNF217 oncogenic function, and ultimately to the design of novel therapeutic strategies. In the second project, we examined the potential of DNA binding proteins to alter gene expression networks and hence cell fate, in the context of reprogramming fibroblasts towards the megakaryocytic lineage. Megakaryocytes are required for the production of platelets, which are essential for blood coagulation. Reduction in their numbers causes a life-threatening condition termed thrombocytopenia, which is currently treated by platelet transfusions. However, this treatment is restricted by the short storage life and limited supply of platelet concentrates. To investigate alternative approaches, we examined the potential of ectopic expression of combinations of transcription factors to direct fibroblasts towards the megakaryocyte lineage. We have discovered that over-expression of a combination of GATA1 or its mutant isoform, GATA1 short (GATA1s), FLI1 and TAL1 can drive phenotypic changes consistent with partial reprogramming of fibroblasts towards the megakaryocyte lineage, laying the foundation for follow up studies.

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