Generation of Xanthomonas derived TALE proteins that inhibit gene transcription
Temiz, Şeyda Şaziye (2013) Generation of Xanthomonas derived TALE proteins that inhibit gene transcription. [Thesis]
In the first part of this study, we aimed to mutate several transcription factor binding sites in the IL-7R alpha gene locus by generating transcription activator like effector (TALE) nucleases (TALEN) targeting these sites. We designed, constructed and expressed 3 pairs of TALENs targeting the NF-kappaB, Notch, and glucocorticoid receptor (GR) binding sites in the IL7R gene enhancer. We generated cell lines with insertion and deletion (INDEL) mutations induced by these TALENs at these target sites and determined the effects of these mutations on IL-7R alpha gene expression. We assessed TALEN induced mutations in murine Neuro-2a and RLM11 cell lines by a modified restriction fragment length polymorphism (RFLP) assay and by DNA sequencing. We demonstrate that mutations induced by TALEN pairs targeting the IL7R enhancer NF-kappaB site reduce IL-7R alpha gene expression, while mutations in the Notch binding site did not change IL-7R expression. In the second part of this study, we aimed to inhibit the transcription activation function of the NF-kB protein by competitive binding of target sites with TALE proteins. We generated plasmids encoding TALE-dsRed fusion proteins that were designed to bind NF-kB binding sites in a reporter cell line. TNF-alpha treatment of this cell line results in NF-kB nuclear translocation and a resultant increase in GFP fluorescence. TALE-dsRed fusion proteins with increasing numbers of DNA binding repeats competed for NF-kB binding to this reporter and resulted in reduced GFP expression upon TNF treatment. Our experiments demonstrate that TALENs and TALEs can efficiently inhibit gene transcription.
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